METHODS AND DEVICES FOR EVALUATING DRUG CANDIDATES

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/982,769, filed April 22, 2014, U.S. Provisional Application No. 62/022,956, filed July 10, 2014, and U.S. Provisional Application No. 62/149,450, filed April 17, 2015, which applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The cost of new drug development from discovery through phase III trials is estimated to be between $800 million and $1.7 billion and the process can take between eight and ten years. Despite success in classical anticancer models, many cancer therapeutics do not reach the clinic. Numerous drug candidates fail during clinical trials. It is estimated that more than 90% of cancer-related therapeutics will fail phase I or II clinical trial evaluation. The failure rate in phase III trials is almost 50%.

[0003] Since every person has a different genetic makeup, individuals experiencing the same disease or disorder can often have drastically different responses to treatment conditions. Current methods for testing the effects of biological agents in treating disease conditions are largely based on clinical screening in sample populations. While this may help assess the proper treatment for a majority of subjects, these effects may vary from one individual to another. In some instances, tissues extracted from the same subject may respond to an agent differently (e.g. based on disease state, drug desensitization, and/or local physiological conditions). There therefore exists a need to develop a tool that will allow for quick and reliable testing of large libraries of compounds directly on primary tissue samples.

[0004] Given the need for direct testing of biological agents in primary tissue samples, methods and devices that provide a quick and reliable means for evaluating different biological agents can be invaluable for selecting the proper agents for treatment and thereby enhance therapeutic efficacy and minimize unnecessary side effects.

SUMMARY OF THE INVENTION

[0005] The present disclosure is directed to methods and devices that can be used to evaluate drug candidates. In one aspect, the disclosure provides a method of screening a biological agent for treating a disease in a subject in need thereof. In some cases, the method comprises administering the biological agent to a tissue sample isolated from the subject using a device comprising a humidity chamber and at least two adapters, each configured to administer a different agent to a different location within the tissue. In some cases, the method further provides for assaying tissue response to the agent thereby determining the therapeutic effects and side effects of the biological agent. In another case, the method further comprises selecting the biological agent for clinical trial based on said therapeutic effects.

[0006] In another aspect, the disclosure provides for a method of predicting a clinical response of a subject to a biological agent for treating a disease. In some cases, the method comprises administering the biological agent to a tissue sample isolated from the subject using a device comprising a humidity chamber and at least two adapters, each configured to administer a different agent to a different location within the tissue. In some cases, the method further comprises assaying tissue response to the drug thereby predicting clinical response of the subject to the biological agent. In further cases, the tissue response to more than one drug is tested and compared to thereby determine which drug is most effective and/or safest to the subject.

[0007] In a further aspect, the methods disclosed herein provide for identifying optimal dosage of a biological agent for treating a disease in a subject in need thereof. In some cases, the method comprises administering a dose of the biological agent to a tissue sample isolated from the subject using a device comprising a humidity chamber and at least two adapters, each configured to administer a different agent to a different location within the tissue. In some cases, the method further comprises assaying a tissue response to the dose thereby identifying an optimal dosage of the biological agent for treating the disease. In further cases, the method comprises selecting an optimal dose based on the therapeutic effects and side effects. In some cases, the method further comprises correlating assay results to clinical response of the subject to the dose.

[0008] In yet another aspect, the disclosure provides for a method of identifying a standard of care drug for combination therapy with a biological agent for treating a disease in a subject in need thereof. In some cases, the method comprises administering a combination therapy comprising the biological agent and the standard of care drug to a tissue sample isolated from the subject. In some cases, the method further comprises assaying tissue response to the combination therapy thereby determining therapeutic effects and side effects of the standard of care drug on the combination therapy. In a further case, the method comprises selecting an optimal standard of care drug for the combination therapy based on the therapeutic effects and side effects. [0009] In one aspect, the disclosure provides for a method of identifying an optimal dose of a standard of care drug for combination therapy with a biological agent for treating a disease in a subject in need thereof. In some cases, the method comprises administering a combination therapy comprising the biological agent and a dose of the standard of care drug to a tissue sample isolated from the subject. In some cases, the method further comprises assaying tissue response to the dose of the standard of care drug in the combination therapy thereby determining therapeutic effects and side effects of the dose. In a further case, the method comprises selecting an optimal dose based on the therapeutic effects and side effects.

[0010] In another aspect, the disclosure provides for a method of selecting an optimal treatment regime for treating a disease in a subject in need thereof. In some cases, the method comprises conducting a treatment regime comprising at least one biological agent on a tissue sample isolated from the subject. In some cases, the method further comprises assaying tissue response to the treatment regime thereby determining therapeutic effects and side effects of the treatment regime. In further cases, the method comprises selecting an optimal treatment regime based on the therapeutic effects and side effects. In some cases, the treatment regime is selected from the group consisting of chemotherapy, radiation, antibody therapy, surgery, combination therapy, targeted therapy, and any combination thereof.

[0011] In another aspect, the disclosure provides for a method of identifying a subpopulation of subjects optimal for a treatment of a disease with a biological agent. In some cases, the method comprises administering the biological agent to a tissue sample isolated from each of the subjects using a device comprising a humidity chamber and at least two adapters, each configured to administer a different agent to a different location within the tissue. In some cases, the method further comprises assaying tissue response to the biological agent thereby determining therapeutic effects and side effects of the biological agent. In a further case, the method comprises selecting a subject subpopulation optimal for the treatment based on the therapeutic effects and side effects.

[0012] In some cases, the tissue sample of any of the methods of the disclosure is cultured ex vivo. In some cases, the methods of the disclosure comprise isolating a tissue sample from the subject. In some cases, the methods of the disclosure further comprise culturing the tissue sample ex vivo. In some cases, the biological agent is administered at one or more concentration. In further cases, a second biological agent is administered to the tissue sample. In some cases, a second tissue sample is isolated from a different location of the subject. In some instances, the methods further comprise administering a second biological agent to the tissue sample. In some cases, the disease is selected from the group consisting of hyperproliferative disorder, inflammatory disease, immune disease, nervous system disease, metabolic disease, angiogenic disease, ophthalmic disease, respiratory disease, and cardiac disease. In a specific case, the hyperproliferative disorder is cancer. In a further specific case, the tissue sample is a solid tumor. In some cases, the biological agent or standard of care drug is an anti-cancer agent. In some cases, more than one biological agent is administered to a specific location of the tissue sample. In some instances, the assaying comprises analyzing local cell toxicity. In some instances, the biological agent or standard of care drug is selected based on local cell toxicity and host toxicity. In further cases, the methods comprise reporting the assay results to a designated person or entity.

[0013] In some aspects, a device is provided for administering one or more biological agents to a tissue sample. In some cases, the device comprises a supporting means comprising a plurality of reservoirs, wherein each of the plurality of reservoirs can independently hold the one or more biological agents. In some cases, the device comprises a humidity chamber connected to the supporting means, wherein humidity in the humidity chamber can be adjusted between 10% and 100% relative humidity. In some cases, the tissue sample is placed between the supporting means and the humidity chamber, and made in contact with the one or more biological agents. In some cases, the tissue sample is isolated from a subject. In some cases, the tissue sample is cultured ex vivo.

[0014] In yet further aspects, a biological evaluation kit is provided. In some cases, the kit comprises a surgical excision device, wherein the device can be used to isolate a tissue sample from a subject. In some cases, the kit comprises a library of one or more biological agents. In some cases, the kit comprises a supporting means comprising a plurality of reservoirs. In some cases, the kit comprises a humidity chamber, wherein humidity in the humidity chamber can be adjusted between 10%> and 90%> relative humidity. In some cases, the tissue sample is cultured ex vivo. In further cases, the kit comprises an analytical device, wherein a biological activity of the one or more biological agents can be measured by the analytical device.

[0015] In another aspect, a device is provided for administering one or more biological agents to a tissue sample. In some cases, the device comprises the tissue sample, wherein the tissue sample is isolated from a subject. In some cases, the device comprises a plurality of microdialysis tubes inserted through the tissue sample, wherein culture media can pass through the plurality of microdialysis tubes and contact the tissue sample. In some cases, the one or more biological agents can be independently and optionally supplemented into the culture media passing through the plurality of microdialysis tubes. In some examples, the tissue sample is cultured ex vivo.

[0016] In another aspect, a biological evaluation kit is provided. In some cases, the kit comprises a surgical excision device, wherein the device can be used to isolate a tissue sample from a subject. In some cases, the kit comprises a library of one or more biological agents. In some cases, the kit comprises a reservoir of culture media. In some cases, the kit comprises a plurality of microdialysis probes. In some cases, the tissue sample is cultured ex vivo. In further cases, the kit comprises an analytical device, wherein a biological activity of the one or more biological agents can be measured by the analytical device.

[0017] In yet another aspect, the disclosure provides for a method of screening for an effective drug candidate for a disease. In some cases, the method comprises isolating a tissue sample from a subject experiencing the disease. In some cases, the method further comprises culturing the tissue sample ex vivo. In some cases, the method further comprises locally administering one or more agents to one or more specific locations on the tissue sample. In some cases, the method further comprises analyzing biological effects of the one or more agents on the one or more specific locations on the tissue sample. In further cases, the method comprises selecting the one or more agents for treating the subject based on the biological effects on the one or more specific locations on the tissue sample.

[0018] In one aspect, a device is provided. In some cases, the device comprises two or more needles. In some cases, the device comprises one or more agent. In some cases, the device comprises two or more reservoirs, wherein the reservoirs are in fluid communication with the two or more needles, and wherein a first reservoir of the two or more reservoirs comprises a first agent of the one or more agent, and a second reservoir of the two or more reservoirs comprises a second agent of the one or more agent. In some cases, the device comprises a channel. In some instances, the one or more agent of the two or more reservoirs is injected into an injection site in a tissue. In some instances, a pattern of coverage of the first agent in the tissue overlaps with a pattern of coverage of the second agent in the tissue. In some cases, the two or more needles comprise a needle array. In an example, the two or more needles comprise at least four needles. In some cases, the two or more needles are microinjection needles. In some instances, the first agent and the second agent are the same agent. In some cases, the first agent is a different concentration of the second agent. In some cases, the device is configured to deliver the one or more agent at an amount undetectable outside the tissue, or at a therapeutically effective amount. In some instances, the one or more agent is delivered at or below a systemically detectable concentration. In one example, the device is a microfluidic device. In another example, the channel is a microfluidic channel. In a further example, the channel comprises an inlet port in fluid communication with the channel. In some cases, the channel is in fluid communication with the two or more needles. In some instances, the one or more agent comprises an anti-cancer agent. In further instances, the one or more agent comprises a small molecule agent. In some cases, the one or more agent comprises a position marker. In some examples, the one or more agents comprises a negative control. In other examples, the one or more agent comprises a prodrug of the agent. In further examples, the content of the reservoir diffuses from the injection site, thereby forming a permeation perimeter. In a specific example, two or more of the permeation perimeter overlap thereby forming an overlapping area. In a further example, the overlapping area comprises two permeation perimeters. In another example, the overlapping area comprises three permeation perimeters. In yet another example, the overlapping area comprises four or more permeation perimeters. In some cases, the tissue is subcutaneous. In some cases, the tissue comprises a tumor. In a specific example, the tumor is a pediatric tumor. In another specific example, the tumor is a skin-related tumor. In some examples, the tumor comprises melanoma. In another example, the tumor comprises lymphoma. In some cases, the tumor is a benign tumor or a malignant tumor. In further cases, the tumor is selected from the group consisting of a primary tumor, an invasive tumor and a metastatic tumor. In yet further cases, the tumor comprises a cancer cell selected from the group consisting of a prostate cancer cell, a lymph node cell, a breast cancer cell, a colon cancer cell, a lung cancer cell, a brain cancer cell, a melanoma cell, a sarcoma cell, an ovarian cancer cell, and any combination thereof. In other examples, the tumor comprises a cancer cell selected from the group consisting of a lymphoma cell, a breast cancer cell, a melanoma cell, a sarcoma cell, and any combination thereof. In yet other examples, the tumor comprises a cancer selected from the group consisting of adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, chondrosarcoma, lymphoma, sarcoma, and fibrosarcoma. In some examples, the tissue is selected from the group consisting of brain, liver, lung, kidney, prostate, ovary, spleen, lymph node, thyroid, pancreas, heart, skeletal muscle, intestine, larynx, esophagus, skin and stomach. In some cases, the device attaches to the tissue. In particular examples, the device attaches to the tissue for more than 30 minutes. [0019] In some aspects, the disclosure provides for a method for delivering two or more agents to a tissue. In some cases, the method comprises contacting the tissue with a device. In some cases, the method comprises a device comprising two or more needles. In some cases, the method comprises a device comprising a first and a second agent. In some cases, the method comprises a device comprising two or more reservoirs, wherein the two or more reservoirs are in fluid communication with the two or more needles, and wherein a first reservoir of the two or more reservoirs comprises the first agent, and a second reservoir of the two or more reservoirs comprises the second agent. In some cases, the method comprises a device comprising a channel. In further cases, the method comprises injecting the first and the second agents into the tissue. In yet further cases, the first agent is injected into an injection site in the tissue and diffuses to form a first permeation perimeter. In further cases, the second agent is injected into the injection site and diffuses to form a second permeation perimeter. In even further cases, the first permeation perimeter overlaps with the second permeation perimeter to generate an overlapping area. In some examples, the two or more needles are part of a needle array. In some cases, the two or more needles are four needles. In some cases, the two or more needles are microinjection needles. In some examples, the first agent and the second agent are the same agent. In some examples, the first agent is a different concentration of the second agent. In some examples, the device is configured to deliver the first and the second agents at an amount undetectable outside the tissue, or at a therapeutically effective amount. In some cases, the first and the second agents are delivered at or below a systemically detectable concentration. In some cases, the device is a microfiuidic device. In some cases, the channel is a microfiuidic channel. In some cases, the channel comprises an inlet port in fluid communication with the channel. In some instances, the channel is in fluid communication with the two or more needles. In particular examples at least one of the first and the second agents comprise an anti-cancer agent. In some examples, at least one of the first and the second agents comprise a small molecular agent. In some instances, at least one of the first and the second agents comprise a position marker. In some instances, at least one of the first and the second agents comprise a negative control. In some instances, at least one of the first and the second agents comprise a prodrug of the agent. In some examples, the first and the second of the reservoir diffuses from the injection site, thereby forming a permeation perimeter. In a particular example, two or more of the permeation perimeter overlap thereby forming an overlapping area. In some cases, the overlapping area comprises two permeation perimeters. In other cases, the overlapping area comprises three permeation perimeters. In yet other cases, the overlapping area comprises four or more permeation perimeters. In some cases, the overlapping area is a site of a drug response. In a particular case of this example, the drug response is used to assess an efficacy of the first and the second agents. In some cases, the tissue is subcutaneous. In some cases, the tissue comprises a tumor. In particular examples, the tumor is a pediatric tumor. In some instances, the tumor is a skin-related tumor. In particular examples, the tumor comprises melanoma. In other particular examples, the tumor comprises lymphoma. In some examples, the tumor is a benign tumor or a malignant tumor. In more particular examples, the tumor is selected from the group consisting of a primary tumor, an invasive tumor and a metastatic tumor. In some examples, the tumor comprises at least one cancer cell selected from the group consisting of a prostate cancer cell, a lymph node cell, a breast cancer cell, a colon cancer cell, a lung cancer cell, a brain cancer cell, a melanoma cell, a sarcoma cell, an ovarian cancer cell, and any combination thereof. In other examples, the tumor comprises at least one cancer cell selected from the group consisting of a lymphoma cell, a breast cancer cell, a melanoma cell, a sarcoma cell, and any combination thereof. In yet other examples, the tumor comprises a cancer selected from the group consisting of adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell

undifferentiated carcinoma, chondrosarcoma, lymphoma, sarcoma, and fibrosarcoma. In some instances, the tissue is selected from the group consisting of brain, liver, lung, kidney, prostate, ovary, spleen, lymph node, thyroid, pancreas, heart, skeletal muscle, intestine, larynx, esophagus, skin and stomach. In some cases, the injecting comprises intermittent injection of the first and the second agents. In other cases, the injecting comprises continuous injection of the first and the second agents. In some cases, the injecting occurs at low flow rates. In some examples, the injecting is performed in vivo. In other examples, the injecting is performed in vitro. In some cases, the device attaches to the tissue. In particular examples, the device attaches to the tissue for more than 30 minutes. In some cases, the method further comprises removing the overlapping area. In some cases, the method further comprises evaluating the injection sites. In a particular example, the evaluating comprises imaging the tissue. In further examples, the imaging comprises radiographic imaging, magnetic resonance imaging, positron emission tomography, or biophotonic imaging. In some instances, the imaging occurs during, or after the injecting of the first and the second agents. In some instances, the evaluating comprises three-dimensional evaluation of the solid tissue. [0020] In further aspects, a composition is provided. In some cases, the composition comprises a hydrogel matrix comprising a plurality of peptides. In some instances, the said hydrogel matrix is formed by subjecting the plurality of peptides in a medium. In some cases, the composition comprises an agent captured in the hydrogel matrix. In some examples, the hydrogel matrix comprises a plurality of matrix pores with a diameter in the range of 5 to 1000 nanometers. In other examples, the hydrogel matrix comprises a plurality of matrix pores with a diameter in the range of 5 to 200 nanometers. In some examples, the hydrogel matrix comprises a plurality of matrix pores with a diameter in the range of 10 to 200 nanometers. In other examples, the hydrogel matrix comprises a plurality of matrix pores with a diameter in the range of 10 to 100 nanometers. In some examples, the peptides are subjected in the medium at a concentration between 0.01% and 20%. In other examples, the peptides are subjected in the medium at a concentration between 0.1% and 20%. In yet other examples, the peptides are subjected in the medium at a concentration between 0.1% and 10%. In still other examples, the peptides are subjected in the medium at a concentration between 1% and 10%>. In some examples, the peptides comprise a C-terminus domain comprising at least 5 amino acids. In some instances, the at least 10% of the C-terminus domains comprise a hydrophobic residue. In some examples, the hydrophobic residues are selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. In particular examples, the hydrophobic residues are unnatural amino acids. In some instances, at least 10% of the peptides are further conjugated to a lipid tail. In some examples, at least 10% of the peptides comprise a motif. In some cases, the medium is an aqueous medium. In some cases, the medium has an ionic strength less than 100 mM. In some cases, the medium has an ionic strength greater than 100 mM. In some examples, the agent is an anticancer agent. In some examples, the agent is a small molecule agent. In other examples, the agent is an enzyme inhibitor. In some examples, the agent comprises a tag. In further examples, the tag is a fluorescence tag. In some further examples, the composition comprises a vehicle. In particular examples, the vehicle is cyclodextrin. In some cases, the peptides are self-assembling peptides.

[0021] In further aspects, the disclosure provides for a method for delivering an agent to a site in a subject in need thereof. In some cases, the method comprises subjecting a plurality of peptides in a medium and thereby forming a hydrogel matrix. In some cases, the method comprises capturing the agent in the hydrogel matrix. In some cases, the method comprises contacting the hydrogel matrix with the site. In some instances, the agent is delivered to the site at a rate at least 2 times slower than without the hydrogel matrix. In particular examples, the hydrogel matrix comprises a plurality of matrix pores with a diameter in the range of 5 to 1000 nanometers. In some examples, the peptides are subjected in the medium at a concentration between 0.01% and 20%. In some instances, the peptides comprise a C- terminus domain comprising at least 5 amino acids. In further instances, the at least 10% of the C-terminus domains comprise a hydrophobic residue. In particular examples, the at least 10% of the peptides are further conjugated to a lipid tail. In further particular examples, the at least 10% of the peptides comprise a tag. In further particular examples, the medium is an aqueous medium. In some examples, the agent is an anticancer agent. In some examples, the agent is a small molecule agent. In some examples, the agent is an enzyme inhibitor. In some examples, the agent comprises a tag. In some cases, the method further comprises providing a vehicle. In some instances, the agent is delivered to at least a second site. In some examples, the peptides comprise self-assembling peptides.

[0022] In another aspect, a device is provided. In some cases, the device comprises two or more needles. In some cases, the device comprises one or more agents. In some cases, the device comprises two or more reservoirs. In some instances, the reservoirs are in fluid communication with the two or more needles. In further instances, a first reservoir of the two or more reservoirs comprises a first agent of the one or more agents. In further instances, a second reservoir of the two or more reservoirs comprises a second agent of the one or more agents. In some cases, the device further comprises a guide needle. In some cases, the guide needle is adapted to introduce a guide marker. In particular examples, the guide marker is selected from the group consisting of an anchor suture, a hookwire, and any combination thereof. In some examples, the guide needle has a needle gauge from 17 to 20. In some examples, the guide needle has a needle length from 7 centimeters to 12 centimeters. In some examples, the guide needle encloses the guide marker. In some examples, the guide marker is attached to a mandril wire. In particular examples, the mandril wire has a diameter of at least 0.018 inches. In some examples, the guide marker is adapted to maintaining tumor orientation. In some instances, the guide marker comprises a spring coil anchor. In particular instances, the spring coil anchor is at least 2 centimeters in length. In further instances, the spring coil anchor has a diameter of at least 0.035 inches. In some examples, the spring coil anchor comprises stainless steel. In some examples, the guide marker comprises a puncture needle. In further examples, the guide marker comprises a localization wire. In some examples, the puncture needle is from 18 gauge to 20 gauge. In some cases, the puncture needle has an insertion length from 8 centimeters to 10 centimeters. In some cases, the localization wire length is from 25 centimeters to 30 centimeters. In some cases, the guide marker is a single or double hookwire.

[0023] In one aspect, the disclosure provides for a method of orienting a tumor. In some cases, the method comprises inserting a guide needle into a first location in the tumor. In some cases, the method comprises contacting the guide needle with a microinjection device. In some cases, the method comprises injecting one or more agents through the microinjection device into one or more locations in the tumor. In some cases, the method comprises threading a guide marker through the guide needle into the first location. In some cases, the method comprises orienting the tumor, wherein the orienting comprises comparing the one or more locations of the one or more agents with the first location of the guide marker. In some instances, the microinjection device comprises two or more needles as part of a needle array. In particular examples, the two or more needles are four needles. In some examples, the two or more needles are microinjection needles. In some examples, the one or more agent comprises one or more chemotherapeutic agents. In some instances, the orienting comprises matching the one or more locations of the one or more agents with the first location of the guide marker. In some cases, the comparing comprises imaging tumor. In some cases, the comparing comprises imaging the guide marker and the one or more agents. In some examples, the guide needle is inserted into the tumor in a location distal to fat, large vessels, or necrotic tissue. In some examples, the imaging comprises a dye selected from the group consisting of DAPI, ICG, CC3, H&E, IDS, and any combination thereof. In some examples, the imaging comprises imaging a UV dye. In particular examples, the guide marker comprises poly(lactic-co-glycolic acid). In some cases, the guide marker is preloaded with an agent. In some cases, the guide marker is implanted into a tumor. In some cases, the guide marker releases the agent over the course of 12 days. In some instances, the guide marker is preloaded with an agent by electrospinning. In some instances, the agent comprises one or more chemotherapeutic agents.

[0024] In a further aspect, the disclosure provides a method of analyzing a tumor. In some cases, the method comprises positioning an imaging agent into the tumor. In some cases, the method comprises systemically administering a candidate agent to a patient with the tumor. In some cases, the method comprises imaging the tumor. In some cases, the method comprises visualizing the imaging agent to determine whether the candidate agent has an effect on the tumor. In some examples, the imaging agent is indicative of enzyme activity, genetic or gene expression changes, apoptotic cells, or tumor size. In particular examples, the enzyme activity comprises pro-apoptotic enzyme activity. In some instances, the patient is diagnosed with cancer. In some cases, the imaging agent is positioned into the tumor using a microinjection delivery device. In some instances, the microinjection delivery device comprises one or more needles configured to deliver the imaging agent into a solid tissue of the tumor along an axis of the one or more needles. In some cases, the microinjection delivery device comprises a needle. In some instances, the needle has a first end and a second end. In some cases, the microinjection delivery device comprises a reservoir. In some instances, the reservoir is in fluid communication with the first end of the needle. In some cases, the microinjection delivery device comprises a plunger. In some instances, the plunger is in fluid communication with the reservoir. In some cases, the microinjection delivery device comprises a controller. In some instances, the controller controls the rate of fluid delivery when the needle is withdrawn from the solid tissue. In some examples, the candidate agent is a chemotherapeutic. In some examples, the imaging agent of the tumor is by MRI, computed tomography, ultrasound, radiography, or fluorescent imaging. In further examples, the imaging agent positioned into the tumor is conjugated to an anchoring group. In some instances, the imaging agent and anchoring group are linked via a peptide linkage. In further instances, the peptide linkage contains a proteolytic site. In some instances, the imaging agent is modified with a lipid, a n-hydroxysuccinimidyl ester, a self-assembling peptide, or any combination thereof. In some cases, the anchoring group binds to a target enzyme. In particular cases, the target enzyme is selected from the group consisting of a caspase, a metalloprotease, a cysteine cathepsin, an esterase, a serine hydrolase, a histone deacetylase, and a deubiquinating enzyme. In some examples, the anchoring group covalently binds to the target enzyme. In further examples, the anchoring group comprises a chelator moiety or a photoinducible chemical crosslinker. In even further examples, the anchoring group targets the imaging agent to a zone of apoptotic cells. In some instances, the proteolytic site is cleaved in the presence of cleaved caspase-3 protease (CC3). In some cases, the imaging agent is indicative of the target enzyme activity. In some cases, the enzyme activity is indicative of the cleavage of a quenching molecule that suppresses the imaging agent when present. In some cases, the imaging agent positioned into the tumor is a contrast agent. In particular cases, the contrast agent is selected from the group consisting of gadobenic acid, gadobutrol, gadodiamide, gadofesveset, gadolinium, gadopentetic acid, gadoteric acid, gadoteridol, gadoversetamide, gadoxetic acid, ferric ammonium citrate, mangafodipir, ferumoxsil, ferristene, iron oxide, nanoparticles, and perflubron.

[0025] In one aspect, the disclosure provides for a method of determining tumor localization. In some cases, the method comprises administering two or more agents to the tumor. In some cases, at least one of the one or more agents is a candidate agent. In some cases, at least one of the one or more agents is a UV tattoo dye.

INCORPORATION BY REFERENCE

[0026] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative cases, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0028] FIG. 1 depicts a schematic of a device embodying principles of the present invention.

[0029] FIG. 2 illustrates a transfer assembly for loading a concentration gradient of one or more agents onto the organotypic tissue slice depicted in embodying principles of the present invention.

[0030] FIG. 3 illustrates a transfer assembly for loading a combination of one or more agents onto the organotypic tissue slice depicted in embodying principles of the present invention.

[0031] FIG. 4 A, B illustrates another exemplary case of the disclosure.

[0032] FIG. 5 depicts the various modes of analysis.

[0033] FIG. 6 illustrates another exemplary case of the disclosure.

[0034] FIG. 7 A, B illustrates a self-assembling hydrogel peptide depicted in embodying principles of the present invention.

[0035] FIG. 8 illustrates a self-assembling hydrogel peptide depicted in embodying principles of the present invention.

[0036] FIG. 9 A, B illustrates a self-assembling hydrogel peptide depicted in embodying principles of the present invention. [0037] FIG. 10 A, B depicts nanofibers of self-assembling peptides depicted in embodying principles of the present invention.

[0038] FIG. 11 illustrates another exemplary case of the disclosure.

[0039] FIG. 12 A-D illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0040] FIG. 13 A-C illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0041] FIG. 14 A-E illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0042] FIG. 15 A-D illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0043] FIG. 16 A-E illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0044] FIG. 17 A-D illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0045] FIG. 18 A-C illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0046] FIG. 19 A-C illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0047] FIG. 20 A-G illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0048] FIG. 21 A-H illustrates images of injection sites in tumor tissue depicted in embodying principles of the present invention.

[0049] FIG. 22 illustrates a flow diagram of injection and resection procedures depicted in embodying principles of the present invention.

[0050] FIG. 23 A-C illustrates an image and guide-needle procedure for injection into tumor tissue depicted in embodying principles of the present invention.

[0051] FIG. 24 A-C illustrates an image and guide-needle procedure for injection into tumor tissue depicted in embodying principles of the present invention.

[0052] FIG. 25 D-F illustrates an image and guide-needle procedure for injection into tumor tissue depicted in embodying principles of the present invention.

[0053] FIG. 26 A-E illustrates placement of a guide marker for maintaining tumor orientation depicted in embodying principles of the present invention. [0054] FIG. 27 illustrates a hookwire guide marker depicted in embodying principles of the present invention.

[0055] FIG. 28 illustrates various components of a guide marking procedure for maintaining tumor orientation depicted in embodying principles of the present invention.

[0056] FIG. 29 illustrates a microinjection device depicted in embodying principles of the present invention.

[0057] FIG. 30 illustrates a microinjection device depicted in embodying principles of the present invention.

[0058] FIG. 31 illustrates the center hole for placement of a guide needle into a

microinjection device depicted in embodying principles of the present invention.

[0059] FIG. 32 illustrates the inside of a microinjection device in which the arrow points out the center chamber through which the guide needle is inserted.

[0060] FIG. 33 illustrates a microinjection device depicted in embodying principles of the present invention.

[0061] FIG. 34 illustrates a microinjection device depicted in embodying principles of the present invention.

[0062] FIG. 35 illustrates a microinjection device depicted in embodying principles of the present invention.

[0063] FIG. 36 A-C illustrates a guide marker preloaded with an agent depicted in embodying principles of the present invention.

[0064] FIG. 37 illustrates a device for administering nanodoses of agents into a tumor tissue depicted in embodying principles of the present invention.

[0065] FIG. 38 A, B illustrates Venn Vivo analysis for analyzing drug combinations in tumor tissue depicted in embodying principles of the present invention.

[0066] FIG. 39 depicts an example of an analysis of a synergistic or antagonistic effect of two different drugs in tumor tissue depicted in embodying principles of the present invention.

[0067] FIG. 40 A-K depicts sequences of self-assembling peptides.

[0068] FIG. 41 illustrates MRI imaging data of a contrast agent microinjected into a tumor depicted in embodying principles of the present invention.

[0069] FIG. 42 depicts a system for software facilitation.

[0070] FIG. 43 A-H depicts an example of a device of the present disclosure.

[0071] FIG. 44 A-C depicts performance of a device of the present disclosure.

[0072] FIG. 45 A-C depicts performance of a device of the present disclosure. [0073] FIG. 46 A-C depicts performance of a device of the present disclosure.

[0074] FIG. 47 A-E depicts performance of a device of the present disclosure.

[0075] FIG. 48 A-C depicts performance of a device of the present disclosure.

[0076] FIG. 49 A-E depicts performance of a device of the present disclosure.

[0077] FIG. 50 A, B depicts performance of a device of the present disclosure.

[0078] FIG. 51 A, B depicts performance of a device of the present disclosure.

[0079] FIG. 52 depicts performance of a device of the present disclosure.

[0080] FIG. 53 depicts performance of a device of the present disclosure.

[0081] FIG. 54 A-C depicts performance of a device of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0082] While preferred cases of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such cases are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the cases of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0083] In one aspect, the invention provides methods, systems and/or devices for extracting a cancer sample from a subject for organotypic culture. In some non-limiting examples, the organotypic sample can be cultured using membrane interface culture methods, wherein the sample is maintained on a porous membrane filter at the interface between medium and a humidified atmosphere. The membrane filter containing the organotypic sample can be placed on an assay plate comprising a plurality of wells for carrying medium, wherein each well of medium may or may not contain an agent to be tested and be made in contact with the organotypic sample via capillary action. The therapeutic efficacy of the different agents can be evaluated based on the biological effects on the corresponding region of the organotypic sample. The therapeutic efficacy may be evaluated after a single administration or after a dosing regimen comprising multiple administrations, wherein the media is changed, supplemented with the same or a different agent, and again made in contact with the organotypic sample for one or more cycles.

[0084] In another aspect, the invention provides methods, systems and/or devices for directly culturing the organotypic sample in a humidity chamber. In some cases, microdialysis probes can be used. The microdialysis probes can be used to deliver medium to local sample sites. Each microdialysis probe can deliver an agent. The agent can be delivered via a suitable medium. The medium may contain an agent. The medium may not contain an agent (e.g. to serve as a negative control) to be tested and be made in contact with the organotypic sample. The therapeutic efficacy of the different agents can be evaluated based on the biological effects on the corresponding region of the organotypic sample. The therapeutic efficacy may be evaluated after a single administration or after a dosing regimen comprising multiple administrations, wherein the media is changed, supplemented with the same or another agent, and again made in contact with the organotypic sample for one or more cycles.

[0085] In a further aspect, the invention provides methods, systems and/or devices for comparing the therapeutic efficacies of individual test conditions, including but not limited to single administration of a single agent, multiple administrations of a single agent, single administrations of multiple agents, and multiple administrations of multiple agents. This can be useful for determining the optimal treatment condition including but not limited to parameters such as the composition, combination, and the concentration of the agent(s), the dosing regimen, the potential use of solubilizing and delivery vehicles, etc.

[0086] In a further aspect, the invention provides methods, systems and/or devices for maintaining tumor orientation. A guide needle can be used to mark the site of an injection of an agent into the tumor. The guide needle can be contacted with a microinjection device. The microinjection device can have a plurality of needles as part of a needle array. One or more agents can be injected into the tumor by the microinjection device. The one or more agents can be but is not limited to chemotherapeutic agents. The microinjector can be removed while the guide needle remains in place. A guide marker can be threaded through the guide needle. The guide marker can be but is not limited to an anchor suture or hookwire.

[0087] Certain Terminology

[0088] A term "agent" or "biologically active agent" can refer to a biological,

pharmaceutical, or chemical compound or other moiety. In some examples, the agent is a biological agent. Some non-limiting examples include a simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a vitamin derivative, a carbohydrate, a toxin, or a

chemotherapeutic compound. Various compounds can be synthesized, for example, small molecules and oligomers (e.g., oligopeptides and oligonucleotides), and synthetic organic compounds based on various core structures. In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.

[0089] In some examples, the agent is a candidate agent. A "candidate agent" is an agent that is tested for an effect on human tissue. In some cases, a candidate agent can comprise a biological agent. A candidate agent can comprise an agent that is selected from (a) a gene therapy agent; (b) a chemotherapy agent; (c) a small molecule; (d) an antibody; (e) a protein; (f) one of a small interfering R A and an encoding polynucleotide therefor; (g) one of an antisense RNA and an encoding polynucleotide therefor, (h) one of a ribozyme and an encoding polynucleotide therefor; (i) one of a therapeutic protein, polypeptide, and a peptidomimetic; j) and antibody-drug conjugates. In some cases the "candidate agent" can be a non-FDA approved agent or investigational agent.

[0090] In some examples, the agent is an imaging agent. An "imaging agent" is an agent that can be used to detect tissue. An imaging agent can be used to detect a chemical in a tissue. In one non-limiting example, an imaging agent is used to detect whether a candidate agent has an effect on a tissue. In another non-limiting example, an imaging agent is positioned into a tumor and used to determine whether a systemically administered candidate agent has an effect on said tumor.

[0091] In some examples, the agent is a contrast agent. A "contrast agent" can be an imaging agent. A contrast agent can be used to increase the visibility of internal cellular structures. In one non-limiting example, a contrast agent can be used to increase the visibility of internal cellular structures during magnetic resonance imaging. In one non-limiting example, a contrast agent can be gadolinium-based.

[0092] The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system or directly into a tumor, such that it enters the patient's system and, thus, is subject to

metabolism and other like processes, for example, subcutaneous administration.

[0093] The term "treatment", "treating", "palliating" or "ameliorating" are used

interchangeably herein and can refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit can mean eradication or amelioration of the underlying disorder being treated. A therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of, or destroying, cancerous cells or other diseased cells, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, palliating the pain resulting from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals. Treatment can include preventing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition prior to the induction of the disease; suppressing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition after the inductive event but prior to the clinical appearance or reappearance of the disease; inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a protective composition after their initial appearance; preventing re-occurrence of the disease and/or relieving the disease, that is, causing the regression of clinical symptoms by administration of a protective composition after their initial appearance.

[0094] The term "anti-cancer agent", "anti-tumor agent" or "chemotherapeutic agent" can refer to any agent useful in the treatment of a cancer or a tumor. One class of anti-cancer agents can comprise chemotherapeutic agents. "Chemotherapy" can refer to the

administration of one or more chemotherapeutic drugs and/or other agents to a cancer patient by various methods, including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal, or inhalation or in the form of a suppository.

[0095] The term "co-administration," "administered in combination with," or their grammatical equivalents, as used herein, can encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present. [0096] The term "in vivo" can refer to an event that takes place in a subject's body.

[0097] The term "ex vivo" can refer to an event that takes place outside a subject's body.

[0098] The term "electrospinning" can refer to the technique in which a charge is applied to a polymer drug-mixture that has been dissolved in a volatile organic solvent, and this charge causes the solution to be ejected as a thin stream to a ground collector upon which polymer microfibers are deposited.

[0099] The term "cancer" or "cancerous" can refer to a neoplastic condition suffered by a subject. Non- limiting examples of such conditions include, but are not limited, to Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia,

Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site,

Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor,

Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor,

Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia,Esophageal cancer,

Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer,

Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma,

Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma,

Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma,

Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma,

Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma,

Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplasia Disease, Myelodysplasia Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma,

Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer,

Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation,

Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma,

Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, or any combination thereof. [00100] Agents

[00101] In some cases, the agents comprise an agent that is selected from (a) a gene therapy agent; (b) a chemotherapy agent; (c) a small molecule; (d) an antibody; (e) a protein; (f) one of a small interfering RNA and an encoding polynucleotide; (g) one of an antisense RNA and an encoding polynucleotide; (h) one of a ribozyme and an encoding polynucleotide; (i) a detectable label; (j) one of a therapeutic protein, a peptide, polypeptide, and a peptidomimetic; (k) an anti-angio genie agent; (1) an epigenetic modifier; (m) an antibody-drug conjugates; (n) a kinase inhibitor; and (o) an inhibitor of metabolic pathway targets that are preferentially expressed in cancer cells. In some cases, the agent can be a biological agent.

[00102] Some non-limiting examples of detectable label are selected from a radiolabel, a radio-opaque label, a fluorescent label, a colorimetric label, a dye, an enzymatic label, a GCMS tag, avidin, a DNA sequence tag, and biotin.

[00103] In certain cases, the agents are selected from (i) a gene therapy agent that comprises at least one operably linked promoter, (ii) a small interfering RNA-encoding polynucleotide that comprises at least one operably linked promoter; (iii) an antisense RNA encoding polynucleotide that comprises at least one operably linked promoter; and (iv) a ribozyme-encoding polynucleotide that comprises at least one operably linked promoter. In certain further cases, the operably linked promoter is selected from a constitutive promoter and a regulatable promoter. In certain still further cases, the regulatable promoter is selected from an inducible promoter, a tightly regulated promoter and a tissue-specific promoter. Examples of anti-angiogenic agents include, but are not limited to, bevacizumab and others in development. Examples of epigenetic modifiers include, but are not limited to, azacitididne and decitabine and others in development. The agent may be a small molecule agent with significant cytotoxicity. The agent may include ubiquitin activating enzyme inhibitors and proteasome inhibitors such as, bortezomib and ixazomib citrate.

[00104] Agents may be dissolved or suspended in an aqueous solution as a mixture or colloid that may be delivered to a solid tissue. When used to refer to agent delivered through capillary action or microdialysis, the term agent is to be read broadly on any substance capable of being delivered through the capillary membrane or microdialysis tube, including liquids, gases, colloids, suspended solids, etc.

[00105] In some cases, the agents can be active forms of a prodrug. Prodrugs can be converted to their active form normally by natural metabolic processes. Prodrugs can be classified as Type I or Type II. Type I prodrugs are activated intracellulary. Type I prodrugs can include nucleoside analogs, idoxurine, 5-flurouracil, 5-fluorocytosine, ganciclovir, Acyclovir, trifluorothymidine, adenine arabinoside bromovinyldeoxyuridine, penciclovir, diethylstilbestrol diphosphate, cyclophosphamide, L-dopa, 6-mercaptopurine, mitomycin C, zidovudine, Carbamazepine, captopril, carisoprodol, heroin, molsidomine, paliperidone, phenacetin, primidone, psilocybin, sulindac, and fursultiamine, ΜΤΧ-α-peptide. Type II prodrugs are activated extracellularly. Type II prodrugs can include Lisdexamfetamine, loperamide oxide, oxyphenisatin, sulfasalazine, Acetylsalicylate, bacampicillin, bambuterol, chloramphenicol succinate, dihydropyridine pralidoxime, dipivefrin, and fosphenytoin.

Chemotherapeutic prodrug therapy can include antibody-directed enzyme prodrug therapy (ADEPT), virus-directed enzyme prodrug therapy (VDEPT), gene-directed enzyme prodrug therapy (GDEPT), Clostridial-directed enzyme prodrug therapy (CDEPT). Prodrugs can be linked to nanoparticles or liposomes.

[00106] In various cases, the agents are marketed anti-cancer drugs. Marketed anti-cancer drugs include, but are not limited to, Lomustine, Carmustine, Streptozocin, Mechlorethamine, Melphalan, Uracil Nitrogen Mustard, Chlorambucil, Cyclophosphamide, Iphosphamide, Cisplatin, Carboplatin, Mitomycin, Thiotepa, Dacarbazin, Procarbazine, Hexamethyl Melamine, Triethylene Melamine, Busulfan, Pipobroman, Mitotane,

Methotrexate, Trimetrexate, Pentostatin, Cytarabine, Ara-CMP, Fludarabine phosphate, Hydroxyurea, Fluorouracil, Floxuridine, Chlorodeoxyadenosine, Gemcitabine, Thioguanine, 6-Mercaptopurine, Bleomycin, Topotecan, Ibrutinib, Irinotecan, Camptothecin sodium salt, Daunorubicin, Doxorubicin, Idarubicin, Idelalisib, Mitoxantrone, Teniposide, Etoposide, Dactinomycin, Mithramycin, Vinblastine, Vincristine, Navelbine, Paclitaxel, Prednisone, Lenalidomide, Revlimid, Pomalidomide, Pomalyst, Velcade, Bortezomib, Docetaxel, Taxotere, Everolimus, Romidepsin, ABT-199, ABT-263, and Palbociclib. In some cases, the agents are candidate oncology agents. Selection of candidate oncology agents is understood and determinable by one skilled in the relevant arts. Candidate oncology agents can be selected from resources that disclose listings of investigational therapeutics, for instance, the National Institutes of Health (Bethesda, MD) which maintains a database of ongoing and planned clinical trials at its "ClinicalTrials.gov" website.

[00107] In some examples, the agent can be an agent that enhances the activity of an anti-cancer agent. In this example, the agent can be co-administered with the anti-cancer agent. Non-limiting examples of suitable agents can include inhibitors of autophagy (e.g., hydroxychloroquine, PS- 1001) and agents that promote the activity of the immune system (e.g., VTX-2337).

[00108] Agents for use in screening methods and in methods of rating for development into therapeutic agents can be provided as "libraries" or collections of compounds, compositions or molecules. Such molecules typically include compounds known in the art as "small molecules" and having molecular weights less than 10 ⁵ daltons, less than 10 ⁴ daltons, or less than 10 ³ daltons.

[00109] For example, a plurality of members of a library of test compounds can be introduced as agents to a region of a solid tumor of known tumor type in each one or a plurality of subjects having a tumor of the known tumor type, by distributing each of the agents to a plurality of positions along an axis within the region of the tumor isolated from each subject, and after a selected period of time (e.g., a range of time, a minimum time period or a specific time period) the region of solid tumor in which the agents have been introduced can be compared by detecting an effect (if any) of each agent on the respective position within the region, for instance, by determining whether an altered physiologic state is present as provided herein, or the presence or absence of a biomarker, relative to positions in the region that are treated with control agents as provided herein, which would either produce no effect (negative control) or a readily detectable effect (positive control).

[00110] Agents further can be provided as members of a combinatorial library, which can include synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels. For example, various starting compounds can be prepared employing one or more of solid-phase synthesis, recorded random mix methodologies and recorded reaction split techniques that permit a given constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions. The resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures, such as a synthetic combinatorial library of peptides. Those having ordinary skill in the art will appreciate that a diverse assortment of such libraries can be prepared according to established procedures, and tested for their influence on an indicator of altered mitochondrial function, according to the present disclosure. Other agents can be proteins (including therapeutic proteins), peptides, peptidomimetics, polypeptides, and gene therapy agents (e.g., plasmids, viral vectors, artificial chromosomes and the like containing therapeutic genes or polynucleotides encoding therapeutic products, including coding sequences for small interfering R A (siRNA), ribozymes and antisense RNA) which in certain further cases can comprise an operably linked promoter such as a constitutive promoter or a regulatable promoter, such as an inducible promoter (e.g., IPTG inducible), a tightly regulated promoter (e.g., a promoter that permits little or no detectable transcription in the absence of its cognate inducer or depressor) or a tissue-specific promoter. Methodologies for preparing, testing and using these and related agents are known in the art.

[00111] In some cases, the agent is a small molecule agent. As used herein, the term "small molecule agent" means an agent with a molecule weight less than about 1000 daltons, less than about 800 daltons, or less than about 500 daltons. In some further cases, the small molecule agent is an anti-cancer agent. The anti-cancer agent may be an approved anticancer drug currently on the market, an anti-cancer drug currently in clinical trials, an anticancer drug withdrawn from clinical trials or market due to toxicity or lack of efficacy, or an early stage anti-cancer drug in the development.

[00112] Other agents can be antibodies, including naturally occurring, immunologically elicited, chimeric, humanized, recombinant, and other engineered antigen- specific immunoglobulins and artificially generated antigen-binding fragments and derivatives thereof, such as single-chain antibodies, minibodies, Fab fragments, bi-specific antibodies and the like.

[00113] Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences. Mack Publishing Co. (A.R. Gennaro edit. 1985). Some non-limiting examples can include dimethyl sulfoxide (DMSO), sterile saline, and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes and other ancillary agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives. In addition, antioxidants and suspending agents can be used. "Pharmaceutically acceptable salt" refers to salts of drug compounds derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The agents, including drugs, contemplated for use herein can be used in either the free base or salt forms, with both forms being considered as being within the scope of the certain present invention cases.

[00114] The pharmaceutical compositions that contain one or more agents can be in any form which allows for the composition to be administered to a subject. The composition can be in liquid form and the route of administration will comprise administration to a solid tissue as described herein. The term parenteral as used herein includes transcutaneous or subcutaneous injections, and intramuscular, intramedullar and intrastemal techniques.

[00115] The pharmaceutical composition can be formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the

composition to a subject such as a human subject. Compositions that will be administered to a subject can take the form of one or more doses or dosage units, where for example, a pre- measured fluid volume can comprise a single dosage unit, and a container of one or more compositions (e.g., drugs) in liquid form can hold a plurality of dosage units. A dose of an agent includes all or a portion of a therapeutically effective amount of a particular agent that is to be administered in a manner and over a time sufficient to attain or maintain a desired concentration range of the agent, for instance, a desired concentration range of the agent in the immediate vicinity of a delivery needle in a solid tissue, and where the absolute amount of the agent that comprises a dose will vary according to the agent, the subject, the solid tissue and other criteria with which the skilled practitioner will be familiar in view of the state of the medical and pharmaceutical and related arts. A therapeutically effective concentration may be determined by distributing the agent to a plurality of positions within the solid tissue and observing an altered physiological state. In certain cases, at least two doses of a same agent can be administered, and in certain other cases 3, 4, 5, 6, 7, 8, 9, 10 or more doses of the same agent can be administered. In certain further cases, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the doses are administered at different concentrations.

[00116] A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, can include one or more of the following adjuvants: sterile diluents such as water for injection, physiological saline, Ringer's solution, saline solution (e.g., normal saline, or isotonic, hypotonic or hypertonic sodium chloride), fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some cases, physiological saline is the adjuvant. An injectable pharmaceutical composition can be sterile. It can also be desirable to include other components in the preparation, such as delivery vehicles including but not limited to aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, hydrogels, and liposomes.

[00117] While any suitable carrier known to those of ordinary skill in the art can be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration and whether a conventional sustained drug release is also desired. For parenteral administration, such as supplemental injection of drug, the carrier can comprise water, saline, alcohol, a fat, a wax or a buffer. Biodegradable microspheres (e.g., polylactic galactide) can also be employed as carriers for the

pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. In some cases, the microsphere can be larger than approximately 25 microns, while other cases are not so limited and contemplate other dimensions.

[00118] Pharmaceutical compositions can also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. In some cases, an agent (e.g., a therapeutic drug or a candidate oncology drug) is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.

[00119] Candidate Agents

[00120] In some examples, the agents as disclosed herein can be candidate agents. The candidate agents in various cases may comprise an anti-cancer agent, a chemotherapeutic, an anti-inflammatory agent, an anti-infective agent, a regenerative agent, a relaxing agent, an apoptosis-inhibiting agent, an apoptosis-inducing agent, an anti-coagulatory agent, a dermatological agent, a growth-stimulating agent, an immunomodulating agent, a modified T-cell therapy, a vasodilating agent, a vasorestricting agent, an analgesic agent, an antiallergic agent, a gene modulating agent, an RNAi molecule, or a combination thereof. In a further case, the candidate agents comprise an anti-cancer agent.

[00121] In some cases, the candidate agent can comprise an agent that is selected from

(a) a gene therapy agent; (b) a chemotherapy agent; (c) a small molecule; (d) an antibody; (e) a protein; (f) one of a small interfering RNA and an encoding polynucleotide therefor; (g) one of an antisense RNA and an encoding polynucleotide therefor, (h) one of a ribozyme and an encoding polynucleotide therefor; (i) one of a therapeutic protein, polypeptide, and a peptidomimetic; (j) and antibody-drug conjugates. In certain cases, the agents are selected from (i) a gene therapy agent that comprises at least one operably linked promoter, (ii) a small interfering RNA-encoding polynucleotide that comprises at least one operably linked promoter; (iii) an antisense RNA encoding polynucleotide that comprises at least one operably linked promoter; and (iv) a ribozyme-encoding polynucleotide that comprises at least one operably linked promoter. In certain further cases, the operably linked promoter is selected from a constitutive promoter and a regulatable promoter. In certain still further cases, the regulatable promoter is selected from an inducible promoter, a tightly regulated promoter and a tissue-specific promoter.

[00122] Selection of candidate oncology agents is understood and determinable by one skilled in the relevant arts (see, e.g., Berkowet al, eds., The Merck Manual, 16 ^th edition, Merck and Co., Rahway; N.J., 1992; Goodman et al, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10 ^th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001); De Vita, Hellman, and Rosenberg's Cancer: Principles and Practice of Oncology (2008, Lippincott, Williams and Wilkins, Philadelphia/ Ovid, New York); Pizzo and Poplack, Principles and Practice of Pediatric Oncology (Fourth edition, 2001, Lippincott, Williams and Wilkins, Philadelphia/ Ovid, New York); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, MD. (1987), Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci al, eds., Remington's Pharmaceutical Sciences, 18 ^th edition, Mack Publishing Co., Easton, PA (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, CT (1992)). Candidate agents can be selected from resources that disclose listings of investigational therapeutics, for instance, the National Institutes of Health (Bethesda, MD) which maintains a database of ongoing and planned clinical trials at its "ClinicalTrials.gov" website.

[00123] Candidate agents can be provided as "libraries" or collections of compounds, compositions or molecules. Such molecules typically include compounds known in the art as "small molecules" and having molecular weights less than 10 ⁵ daltons, less than 10 ⁴ daltons, or less than 10 daltons.

[00124] Candidate agents can be proteins (including therapeutic proteins), peptides, peptidomimetics, polypeptides, and gene therapy agents {e.g., plasmids, viral vectors, artificial chromosomes and the like containing therapeutic genes or polynucleotides encoding therapeutic products, including coding sequences for small interfering R A (siRNA), ribozymes and antisense RNA) which in certain further cases can comprise an operably linked promoter such as a constitutive promoter or a regulatable promoter, such as an inducible promoter (e.g., IPTG-inducible), a tightly regulated promoter (e.g., a promoter that permits little or no detectable transcription in the absence of its cognate inducer or derepressor) or a tissue-specific promoter. Methodologies for preparing, testing and using these and related agents are known in the art. See, e.g., Ausubel (Ed.), Current Protocols in Molecular Biology (2007 John Wiley & Sons, NY); Rosenzweig and Nabel (Eds), Current Protocols in Human Genetics (esp. Ch. 13 therein, "Delivery Systems for Gene Therapy", 2008 John Wiley & Sons, NY); Abell, Advances in Amino Acid Mimetics and

Peptidomimetics, 1997 Elsevier, NY.

[00125] Other candidate agents can be antibodies, including naturally occurring, immunologically elicited, chimeric, humanized, recombinant, and other engineered antigen- specific immunoglobulins and artificially generated antigen-binding fragments and derivatives thereof, such as single-chain antibodies, minibodies, Fab fragments, bi-specific antibodies and the like. See, e.g., Coligan et al. (Eds.), Current Protocols in Immunology (2007 John Wiley & Sons, NY); Harlow and Lane, Antibodies: A Laboratory Manual (1988 Cold Spring Harbor Press, Cold Spring Harbor, NY); Harlow and Lane, Using Antibodies (1999 Cold Spring Harbor Press, Cold Spring Harbor, NY). Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remingtons Pharmaceutical Sciences. Mack Publishing Co. (A.R. Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes and other ancillary agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid can be added as preservatives. In addition, antioxidants and suspending agents can be used. "Pharmaceutically acceptable salt" refers to salts of drug compounds derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The agents, including drugs, contemplated for use herein can be used in either the free base or salt forms, with both forms being considered as being within the scope of the certain present invention cases.

[00126] The pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject such as a human subject. Compositions that will be administered to a subject can take the form of one or more doses or dosage units, where for example, a pre-measured fluid volume can comprise a single dosage unit, and a container of one or more compositions (e.g. , drugs) in liquid form can hold a plurality of dosage units. In certain cases, at least two doses of the agent can be administered, and in certain other cases 3, 4, 5, 6, 7, 8, 9, 10 or more doses can be administered.

[00127] A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, can include one or more of the following adjuvants: sterile diluents such as water for injection, physiological saline, Ringer's solution, saline solution (e.g., normal saline, or isotonic, hypotonic or hypertonic sodium chloride), fixed oils such as synthetic mono or digylcerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some cases, physiological saline is the adjuvant. An injectable pharmaceutical composition can be sterile. It can also be desirable to include other components in the preparation, such as delivery vehicles including but not limited to aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, hydrogels, and liposomes.

[00128] While any suitable carrier known to those of ordinary skill in the art can be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration and whether a conventional sustained drug release is also desired. For parenteral administration, such as supplemental injection of drug, the carrier can comprise water, saline, alcohol, a fat, a wax or a buffer. Biodegradable microspheres (e.g. , polylactic galactide) can also be employed as carriers for the

pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075, 109. In some cases, the microspheres can be larger than approximately 25 microns, while other cases are not so limited and contemplate other dimensions.

[00129] Pharmaceutical compositions can also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. In some cases, an agent (e.g., a therapeutic drug or a candidate drug) is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.

[00130] In some cases the candidate agent is administered systemically. In some cases the candidate agent is administered by a suitable route of administration, including, but not limited to, parenteral (intravenous, subcutaneous, intraperitoneal, intramuscular,

intravascular, intrathecal, intravitreal, or infusiom), oral, or nasal administration.

[00131] Imaging Agents

[00132] In some examples, the agent can be an imaging agent.

[00133] In some cases the imaging agent is peptide based.

[00134] In some cases the imaging agent is a contrast agent that is used in MRI. In some cases the contrast agent can be gadolinium-based. In some cases the gadolinium-based contrast agent is gadobenic acid, gadobutrol, gadodiamide, gadofesveset, gadolinium, gadopentetic acid, gadoteric acid, gadoteridol, gadoversetamide, gadoxetic acid. In some cases the contrast agent is ferric ammonium citrate, mangafodipir, ferumoxsil, ferristene, iron oxide, nanoparticles, or perflubron.

[00135] In some cases the imaging agent is a contrast agent used in X-ray and CT. In some cases the contrast agent can be diatrizoic acid, metrizoic acid, iodamide, iotalamic acid, ioxitalamic acid, ioglicic acid, acetrizoic acid, iocarmic acid, methiodal, diodone,

metrizamide, iohexol, ioxaglic acid, iopamidol, iopromide, iotrolan, ioversol, iopentol, iodixanol, iomeprol, iobitridol, ioxilan, iodoxamic acid, iotroxic acid, ioglycamic acid, adipidone, iobenzamic acid, iopanoic acid, iocetamic acid, sodium iopodate, tyropanoic acid, calcium iopodate, ethyl esters of iodised fatty acids, iopydol, propyliodone, iofendylate, lipiodol, or barium sulfate.

[00136] In some cases the imaging agent can be a contrast agent used in ultrasound. In some cases the contrast agent can be microspheres of human albumin, microparticles of galactose, peflenapent, microspheres of phospholipids, or sulfur hexafluoride.

[00137] In some cases the imaging agent can be a fluorescent dye. In some cases the fluorescent dye can be a fluorescein dye, a rhodamine dye and a cyanine dye. In some cases the fluorescent dye can be 5-carboxyfluorescein, fluorescein-5-isothiocyanate, fluorescein-6- isothiocyanate, 6-carboxyfluorescein, tetramethylrhodamine-6-isothiocyanate, 5- carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyl and tetraethyl rhodamine, diphenyldimethyl and diphenyldiethyl rhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, IRDYE680, Alexa Fluor 750, IRDye800CW, or ICG.

[00138] In some cases the imaging agent can be a radiographic probe that is used with a radiographic system. In some cases the radiographic probe can be 21 lAt, 1311, 1251, 90Y, 186Re, 188Re, 153Sm, 212Bi, 32P, 64Cu, a radioactive isotope of Lu, or any combinations thereof.

[00139] In some cases an imaging agent is coupled to an anchoring group. In some cases the anchoring group localizes the imaging agent to a target enzyme within the target tumor. In some cases the target enzyme is an enzyme that is active during apoptosis. In some cases the target enzyme is an enzyme that is inactive during apoptosis. In some cases the target enzyme is a protease. In some cases the protease is a caspase.

[00140] In some cases, the target enzyme is an enzyme that is implicated in cancer progression and tumori genesis. In some cases the target enzyme is a metalloprotease, cysteine cathepsin, esterase, serine hydrolase, histone deacetylase, or a deubiquinating enzyme.

[00141] In some cases, the anchoring group covalently binds to the catalytic site of the target enzyme. In some cases the anchoring group comprises a chelator moiety. In some cases the anchoring group comprises a photoinducible chemical crosslinker. In some cases the anchoring group reacts with only the functionally active form of the enzyme. In some cases, the anchoring group can only bind to the inactive form of the enzyme.

[00142] In some cases, the imaging agent is linked to the anchoring group through various linkers. In some cases the linker group is a carboxyl bead surface that is coupled via an amide linkage. In some cases the imaging agent is modified with a lipid, a n- hydroxysuccinimidyl ester, self-assembling peptide, or any combination thereof. In some cases the linker group localizes the contrast agent reporter directly within a zone of apoptotic cells.

[00143] Many anchoring groups that can bind to target enzymes are known in the art, including, Paulick and Bogyu, Application of activity-based probes to the study of enzymes involved in cancer progression. Genetics & Development, 18: 97-106, (2008).

[00144] In some cases the imaging agent is pre-coupled to the anchoring group via a substrate proteolytic site. The loss of the imaging agent moiety directly reflects the protease activity in the local spatial area since sites are liberated only in the presence of a targeted protease enzyme. In preferred cases, the peptide bond is cleaved in the presence of cleaved caspase-3 protease (CC3) via DEV/D bond cleavage (Lee et al., Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radiolabeled multimodality molecular probe. J. Biomed Opt. 2009 July-Aug: 14(4): 040507) which releases the imaging agent thereby allowing it to diffuse out of tumor injection site. Higher concentrations of the CC3 enzyme result in faster cleavage of the imaging agent and washout of the agent from the tumor injection site. Thus, the rate at which a chemotherapy treatment induces the release of CC3 from tumor cells (an indicator of treatment effect), is

quantitatively indicated by a change in imaging at the injection site over time. An alternative to cleaving the agent from the anchoring system is to include a cleavable quenching molecule that suppresses the imaging agent signal when present. Cleaving of the quenching molecule in the presence of the imaging target leads to increasing signal of the imaging agent.

[00145] In some cases the imaging agent-anchoring group is fluorescently quenched when unbound to the target enzyme and free in solution. In some cases the fluorescent quencher moiety is released upon covalent binding to the target enzyme.

[00146] In some cases the imaging agent-anchoring group linkage provides an indirect measure of enzyme activity.

[00147] In some cases the imaging-anchoring group conjugate is positioned at a single site by a single hypodermic needle. In some cases the imaging-anchoring group conjugate uses a microinjection system that permits multiplexed injection of multiple columns of the agent in precise amounts. Use of one or more simultaneous injections enables the tumor microenvironment to be sampled at distinct locations thereby differentiating tumors as complete responders (all sites respond), partial responders (some sites respond) and non- responders (no sites respond). The microinjection platform also enables additional injections of fiducial markers at one or more injections sites that allow the sites to be localized on images. In some cases, the same molecular anchoring system and imaging agent, without a cleavable substrate is used as a loading control fiducial. In addition to serving to localize the injection, this strategy allows measurement of the baseline elimination of the agent in the absence of the molecular target for comparison.

[00148] Indications

[00149] The subject methods are useful for testing the therapeutic efficacy of biological agents in many disease conditions. For example, the methods can be used for testing the therapeutic efficacy of biological agents in diseases for which current treatment regimens result in adverse events, limited tolerability, or patient non-compliance. In some cases, the disease condition can be a proliferative disorder including but not limited to cancer. In other cases, the disorder can be diabetes. In still other cases, the disorder can be an autoimmune disorder.

[00150] In some cases, the subject methods can be useful for testing the therapeutic efficacy of biological agents in a proliferative disorder, such as a neoplastic condition. Non- limiting examples of such conditions include but are not limited to Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS- Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer,

Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site,

Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor,

Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor,

Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia,Esophageal cancer,

Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer,

Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplasia Disease, Myelodysplasia Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer,

Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma,

Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T- lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on

Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's

transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma,

Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer,

Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macro globulinemia, Warthin's tumor, Wilms' tumor, or any combination thereof.

[00151] The subject methods can also be useful for testing the therapeutic efficacy of biological agents in solid tumors. Solid tumors include malignancies (e.g., sarcomas, adenocarcinomas, and carcinomas) of the various organ systems, such as those of lung, breast, lymphoid, gastrointestinal (e.g., colon), and genitourinary (e.g., renal, urothelial, or testicular tumors) tracts, pharynx, prostate, and ovary. Exemplary adenocarcinomas include colorectal cancers, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, and cancer of the small intestine. Additional exemplary solid tumors include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastrointestinal system carcinomas, colon carcinoma, pancreatic cancer, breast cancer, genitourinary system carcinomas, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadeno carcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, endocrine system

carcinomas, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[00152] In some examples, the subject methods can be useful for testing the therapeutic efficacy of biological agents in heart conditions including atherosclerosis, heart hypertrophy, cardiac myocyte dysfunction, elevated blood pressure and vasoconstriction.

[00153] In other examples, the subject methods can be useful for testing the

therapeutic efficacy of biological agents in a disease selected from the group consisting of tumor angiogenesis, chronic inflammatory disease such as rheumatoid arthritis,

atherosclerosis, inflammatory bowel disease, skin diseases such as psoriasis, eczema, and scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma, melanoma, sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer.

[00154] In yet other examples, the subject methods can be useful for testing the therapeutic efficacy of biological agents in a disease condition related to an undesirable, overactive, harmful or deleterious immune response in a mammal, collectively termed

"autoimmune disease." Autoimmune disorders include, but are not limited to, Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis, juvenile arthritis and ankylosing spondilitis, Other non-limiting examples of autoimmune disorders include autoimmune diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), rheumatoid spondylitis, gouty arthritis, allergy, autoimmune uveitis, nephrotic syndrome, multisystem autoimmune diseases, autoimmune hearing loss, adult respiratory distress syndrome, shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis, silicosis, idiopathic interstitial lung disease, chronic obstructive pulmonary disease, asthma, restenosis, spondyloarthropathies, Reiter's syndrome, autoimmune hepatitis, inflammatory skin disorders, vasculitis oflarge vessels, medium vessels or small vessels, endometriosis, prostatitis and Sjogren's syndrome. Undesirable immune response can also be associated with or result in, e.g., asthma, emphysema, bronchitis, psoriasis, allergy, anaphylaxsis, autoimmune diseases, rhuematoid arthritis, graft versus host disease, transplantation rejection, lung injuries, and lupus erythematosus. The pharmaceutical compositions of the present invention can be used to treat other respiratory diseases including but not limited to diseases affecting the lobes of lung, pleural cavity, bronchial tubes, trachea, upper respiratory tract, or the nerves and muscle for breathing. The methods of the invention can be further used to treat multiorgan failure.

[00155] The invention also provides methods that can be useful for testing the therapeutic efficacy of biological agents in liver diseases (including diabetes), pancreatitis or kidney disease (including proliferative glomerulonephritis and diabetes- induced renal disease) or pain in a mammal.

[00156] The invention further provides methods that can be useful for testing the therapeutic efficacy of biological agents in neurological or neurodegenerative diseases including, but not limited to, Alzheimer's disease, Huntington's disease, central nervous system trauma, and stroke.

[00157] The invention also provides methods that can be useful for testing the therapeutic efficacy of biological agents in a disease related to vasculogenesis or

angio genesis in a mammal which can manifest as tumor angio genesis, chronic inflammatory disease such as rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, skin diseases such as psoriasis, eczema, and scleroderma, diabetes, diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, hemangioma, glioma, melanoma, sarcoma and ovarian, breast, lung, pancreatic, prostate, colon and epidermoid cancer. [00158] The invention further provides methods that can be useful for testing the therapeutic efficacy of biological agents in disorders involving platelet aggregation or platelet adhesion, including but not limited to Bernard-Soulier syndrome, Glanzmann's

thrombasthenia, Scott's syndrome, von Willebrand disease, Hermansky-Pudlak Syndrome, and Gray platelet syndrome.

[00159] In further examples, the subject methods can be useful for testing the therapeutic efficacy of biological agents in a disease which is skeletal muscle atrophy, skeletal muscle hypertrophy, leukocyte recruitment in cancer tissue, invasion metastasis, melanoma, sarcoma, acute and chronic bacterial and viral infections, sepsis, glomerulo sclerosis, glomerulo, nephritis, or progressive renal fibrosis.

[00160] Target Tissues

[00161] In some cases, the present disclosure exemplifies a method for screening agents in a solid tissue. Solid tissues are well known to the medical arts and may include any cohesive, spatially discrete non-fluid defined anatomic compartment that is substantially the product of multicellular, intercellular, tissue and/or organ architecture, such as a three-dimensionally defined compartment that may comprise or derive its structural integrity from associated connective tissue and may be separated from other body areas by a thin membrane (e.g., meningeal membrane, pericardial membrane, pleural membrane, mucosal membrane, basement membrane, omentum, organ-encapsulating membrane, or the like). Non-limiting exemplary solid tissues may include brain, liver, lung, kidney, prostate, ovary, spleen, lymph node (including tonsil), thyroid, pancreas, heart, skeletal muscle, intestine, larynx, esophagus and stomach. Anatomical locations, morphological properties, histological characterization, and invasive and/or non-invasive access to these and other solid tissues are all well known to those familiar with the relevant arts. In some cases, the tissue is, or is suspected of being, cancerous, inflamed, infected, atrophied, numb, in seizure, or coagulated. In some cases, the tissue is, or is suspected of being, cancerous. In some cases, the tissue is cancerous.

[00162] In some cases, the present method is directed to cancer, and the target tissue comprises a tumor, which may be benign or malignant, and comprises at least one cancer cell selected from the group consisting of a prostate cancer cell, a breast cancer cell, a colon cancer cell, a lung cancer cell, a brain cancer cell, and an ovarian cancer cell. In certain cases, the tumor comprises a cancer selected from adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, chondrosarcoma and fibrosarcoma. Art-accepted clinical diagnostic criteria have been established for these and other cancer types, such as those promulgated by the U.S. National Cancer Institute (Bethesda, MD, USA). In certain cases the selected region of tissue is a portion of a tumor in a subject, and in certain further cases the subject is one of a preclinical model, a companion animal, a dog, a cat, or a human patient.

[00163] Some cases contemplate a subject or biological source that is a human subject such as a patient that has been diagnosed as having or being at risk for developing or acquiring cancer according to art-accepted clinical diagnostic criteria, such as those of the U.S.

National Cancer Institute (Bethesda, MD, USA); certain cases contemplate a human subject that is known to be free of a risk for having, developing or acquiring cancer by such criteria.

[00164] Certain cases contemplate a non-human subject or biological source, for example a non-human primate such as a macaque, chimpanzee, gorilla, vervet, orangutan, baboon or other non-human primate, including such non-human subjects that may be known to the art as preclinical models, including preclinical models for solid tumors and/or other cancers.

Certain other cases contemplate a non-human subject that is a mammal, for example, a mouse, rat, cat, dog, rabbit, pig, sheep, horse, bovine, goat, gerbil, hamster, guinea pig or other mammal; many such mammals may be subjects that are known to the art as preclinical models for certain diseases or disorders, including solid tumors and/or other cancers. The range of cases is not intended to be so limited, however, such that there are also contemplated other cases in which the subject or biological source may be a non-mammalian vertebrate, for example, another higher vertebrate, or an avian, amphibian or reptilian species, or another subject or biological source. A transgenic animal is a non-human animal in which one or more of the cells of the animal includes a nucleic acid that is non-endogenous (i.e., heterologous) and is present as an extrachromosomal element in a portion of its cell or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells). In certain cases of the present invention, the tissue of a transgenic animal may be targeted.

[00165] Methods of the current invention are suitable for administering agents to a variety of animal tissues; thus the methods have medical and veterinary uses. In some cases, the animal tissue is soft tissue. Non-limiting examples of soft tissue include muscle, adipose, skin, tendons, ligaments, blood, and nervous tissue. In some cases, the animal is a reptile, an amphibian, an aves, or a mammal. In further cases, the animal is a mammal. In some examples, the animal is a mouse. In other examples, the animal is a human. In various cases, the animal is a pet, a companion, a guardian, a working animal, a breeding animal, a service animal, a racing animal, a farm animal, a herded animal, or a laboratory animal. [00166] In some examples, the solid tissue does not exhibit features of a disease, but may be used to assess the response of an individual tissue to one or more compounds. In some cases, one or more agents may be administered to produce an altered physiologic state or a detectable biomarker within a tissue. An altered physiologic state can be any detectable parameter that directly relates to a condition, process, pathway, dynamic structure, state or other activity in a solid tissue (and in some cases in a solid tumor) including in a region or a biological sample that permits detection of an altered (e.g., measurably changed in a statistically significant manner relative to an appropriate control) structure or function in a biological sample from a subject or biological source. The methods of the present invention thus pertain in part to such correlation where an indicator of altered physiologic state can be, for example, a cellular or biochemical activity, including as further non-limiting examples, cell viability, cell proliferation, cell death, apoptosis, cellular resistance to anti-growth signals, cell motility, cellular expression or elaboration of connective tissue-degrading enzymes, cellular recruitment of angiogenesis, or other criteria as provided herein.

[00167] The solid tissue may be a tumor. In some cases, the tumor is resistant to a therapy, for example, a chemotherapy. The tumor may respond to the therapy initially but develop resistance suddenly or gradually. There may be a variety of reasons for the development of drug resistance, including:

(1) Cell mutation. Cells that are not killed by the chemotherapy may mutate and become resistant to the drug. Their multiplication may produce more resistant cells than cells that are sensitive to the chemotherapy; (2) Gene amplification. Cancer cells may produce hundreds of copies of a particular gene. This gene may trigger an overproduction of protein that render the anticancer drug ineffective; (3) P-gp mediated efflux. Cancer cells may pump the drug out of the cell using a molecule called p-glycoprotein; and (4) Transporter inhibition. Cancer cells may stop taking in the drugs because the protein that transports the drug across the cell wall stops working.

[00168] Altered physiologic state can further refer to any condition or function where any structure or activity that is directly or indirectly related to a solid tissue function has been changed in a statistically significant manner relative to a control or standard, and can have its origin in direct or indirect interactions between a solid tissue constituent and an introduced agent, or in structural or functional changes that occur as the result of interactions between intermediates that can be formed as the result of such interactions, including metabolites, catabolites, substrates, precursors, cofactors and the like. Additionally, altered physiologic state can include altered signal transduction, respiratory, metabolic, genetic, biosynthetic or other biochemical or biophysical activity in some or all cells or tissues of a subject or biological source. As non-limiting examples, altered biological signal transduction, cell viability, cell proliferation, apoptosis, cellular resistance to anti-growth signals, cell motility, cellular expression or elaboration of connective tissue-degrading enzymes, cellular recruitment of angiogenesis, or other criteria including induction of apoptotic pathways and formation of atypical chemical and biochemical crosslinked species within a cell, whether by enzymatic or non-enzymatic mechanisms, can all be regarded as indicative of altered physiologic state.

[00169] Biomarkers

[00170] The present disclosure exemplifies a method in which an imaging agent can be microdosed into a tumor to detect whether a tumor is responding to candidate agent treatment administered systemically. An imaging agent can detect a response to treatment by indicating changes in enzyme activity, genetic or gene expression, presence of apoptotic cells, or tumor size.

[00171] The present disclosure further exemplifies a method for evaluating changes in the physiological status of tumor cells or tumorigenic cells by measuring the biomarkers secreted, expressed, synthesized, or produced by the cells. These biomarkers can be indicative of whether the tumor is responding to candidate agent treatment. Cells may communicate and respond to physiological cues by secreting the biomarkers that can be soluble factors including autocrines, paracrines, or endocrines. Tumor cells or tumorigenic cells may secrete a plurality of biomarkers that are known in the medical arts before, during or after a change of the physiological status. The biomarkers can be proteins, peptides, amino acids, RNA, DNA, nucleic acids, proteoglycans, lipids, small organic molecules, small inorganic molecules, or ions. In some cases, the biomarkers can be measured in

transcriptional levels as gene expressions or in protein levels. By measuring and detecting the biomarkers described herein over time, and relating the measurement to the biomarkers known in the medical art, thereby the physiological status or the changes in the physiological status of the tumor cells or tumorigenic cells, such as cell death, cell proliferation, cell signaling process or cellular responses, can be determined.

[00172] The death of tumor cells or tumorigenic cells can be via apoptosis or necrosis. Apoptosis is a process of programmed cell death, and may be activated via either the death receptor-mediated extrinsic pathway or the mitochondria-directed intrinsic pathway. Non-limiting examples of biomarkers of apoptosis that can be measured in gene expressions or protein levels include: activated caspase family such as caspases 2, 3, 7, 8, 9 and 10; tumor protein 53 (p53), phosphor-p53, p73, cyclin-dependent kinase inhibitor 1 (p21-wafl), and phosphor-H2AX/Ser 139 (pH2AX); B-cell lymphoma 2 (Bcl-2) family members such as Bcl- 2, B-cell lymphoma-extra large (Bcl-XL), Bcl-xs, Bcl-W, and induced myeloid leukemia cell differentiation protein (Mcl-1); pro-apoptotic protein family such as Bcl-2-associated X protein (Bax), and Bcl-2 homologous antagonist/killer (Bak); Bcl-2 homology (BH) domain family such as BH1, BH2, BH3, BH4, Bcl-2-associated death promoter (Bad), p53 upregulated modulator of apoptosis (PUMA), NOXA, Bcl-2 modifying factor (Bmf), Bcl-2 interacting killer (Bik), Bcl-2 -related ovarian killer (Bok), Bcl-2 interacting mediator of cell death (Bim), and BH3 interacting-domain death agonist (Bid); modulators of apoptosis proteins such as apoptotic protease activating factor 1 (APAF-1), apoptosis inducing factor (AIF), inhibitors of apoptosis (IAP) such as cIAPl, cIAP2, Cp-IAP, Op-IAP, XIAP, NAIP, survivin, and second mitochondria-derived activator of caspases (SMAC); markers to measure extent of DNA oxidative damage such as 8-hydroxy-2-deoxyguanosine and 3- nitrotyrosine; other biomarkers related to apoptosis such as cytochrome c, N-hydroxy-L- arginine (NOHA), 14-3-3 protein, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), reactive oxygen species (ROS), externalized phosphatidylserine, cytokeratins, poly(ADP-ribose) polymerase, nucleosomal DNA, apoptosis antigen 1 (Apo-1), TNF receptor superfamily, member 6 (Fas), Fas ligand (FasL), Fas-associated death domain protein (FADD), phosphorylated-FADD, glutathione-S-transferase-isoenzyme π (Gst-π), β- galactosidase, phosphorylated retinoblastoma suppressor protein and the like.

[00173] Necrosis is a premature death of cells or tissues, and may be caused by factors external to the cells or tissues. Other physiological events such as inflammatory responses of the cells may be triggered with necrosis. Non- limiting examples of biomarkers related to necrosis of tumor cells or tumorigenic cells that can be measured in gene expressions or protein levels include tumor necrosis factor (TNF), cachexin, cachectin, lymphotoxin, cyclophilin A, interleukin-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17, alpha 1 -antitrypsin, copeptin, myeloperoxidase, FLICE-like inhibitory protein (FLIP), transducer and activator of transcription (STAT), tumor necrosis factor receptor superfamily, member 19 (TROY), cyclooxygenase (COX)-l, COX-2, cell death factors, macrophage inflammatory proteins, macrophage activating factors, macrophage migration inhibitory factors, neuroleukin, immunologic suppressor factors, transfer factors, oncostatin, osteopontin, interferon type I, interferon gamma, interleukin 1 receptor antagonist protein, CD70, CD30, CD40, 4-1BB ligand, ectodysplasins, B-cell activating factor, receptor activator of nuclear factor kappa-B ligand (RANKL), lymphotoxin and the like.

[00174] In addition to measuring the biomarkers that can be related to cell death, the current disclosure further provides a method to measure biomarkers that can be measured in gene expressions or protein levels to relate to the proliferation/growth or mitotic activities of tumor cells or tumorigenic cells. Non- limiting examples of biomarkers described herein include Akt protein kinase B, Wilms tumor marker, retinoblastoma (Rb), Ki-67, proliferating cell nuclear antigen (PCNA), serine/threonine kinase, mammalian target of rapamycin (mTOR), neurotrophin, protein Mis 18 beta, myostatin, cyclin dependent kinases (Cdk) 1, 2, 4, and 6, cyclin dependent kinase comples 2 (Cdc2 p34), cyclin Dl, cyclin D2, cyclin D3, cyclin E, cyclin A, growth differentiation factors 1 , 2, 3, 5, 6, 9, 10 and 15 and the like.

[00175] The physiological status of a cell may be heavily modulated by a plurality of signal transduction pathways. Signal transduction occurs when an extracellular signaling molecule or a ligand binds to and further activates a cell surface receptor, thereby altering intracellular molecules creating a response. In some preferred aspects, the biomarkers related to signal transduction changes of tumor cells or tumorigenic cells can be measured in gene expressions or protein levels. The biomarkers described herein can participate in the signaling pathways as growth factors, enzymes, signaling factors, ligands, intermediate molecules generated in biological pathways, hormones, nutrients, transmembrane proteins, extracellular matrix proteins, intracellular components, downstream factors of protein phosphorylation and the like. Non-limiting examples of signal transduction biomarkers include human epidermal growth factor receptor (HER) family molecules such as HER1, 3, and 4; phosphatidylinositol 3-kinases (PI3K) / protein kinase B (Akt) signaling pathway molecules as PI3K/AKT, microtubule-associated protein kinase (MAPK) / extracellular signal-regulated kinase (ERK) pathway molecules such as MAPK, mitogen-activated protein kinase (MEK), Ras, proto-oncogene serine/threonine-protein kinase (RAF), ERKl and 2; hedgehog pathway proteins such as sonic hedgehog, desert hedgehog, indian hedgehog, hedgehog-interacting protein, smoothened protein (SMO), Gli-1, Gli-2, Gli-3, and forkhead box O (FoxO)-l; Wnt signal transduction pathway modulators such as Wntl, 2, 2B, 3, 3A, 4, 5A, 5B, 6, 7A, 7B, 8A, 8B, 9A, 9B, 10A, 10B, 11, 16, Wntl -inducible-signaling pathway protein 1 (Wisp-1), Wisp-2, and beta-catenin; parathyroid hormone -related proteins such as hypercalcemic hormone of malignancy, parathyroid hormone like tumor factor; phosphatase and tensin homolog (PTEN), serine/threonine-protein kinase (SGK3), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), tymidine kinase, growth hormone, pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1), citrate, nitride oxide, P70S6 kinase, glycogen synthase kinase 3 (GSK-3), Src homology 2 domain containing (SHC)- transforming protein 1, CD117, platelet-derived growth factor receptor (PDGFR)-alpha, PDGFR-beta, vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP)-l, CD9, keratin 7, p27,

parafibromin, BMI1 polycomb ring finger oncogene (Bmi-1), 14-3-3σ, cystatin-SA, epididymal secretory protein E4, whey acidic protein (WAP) four-disulfide core domain protein 2 (WFDC2), adiponectin, leptin, resistin, agouti signaling protein, agouti-related protein, angiopoietins, angiostatic proteins, cysteine-rich protein 61, nephroblastoma overexpressed protein, peptide PHI, peptide YY, insulin, glucose, pituitary hormones, placental hormones, relaxin, secretin, urocortins, urotensins, vasoactive intestinal peptide, autocrine motility factor, beta-thromboglobulin, leukemia inhibitory factor, leukocyte migration-inhibitory factors, lymphotoxin-alpha, endothelin, enphrin, bradykinin, kininogens, tachykinins, chemokines such as chemokine C, CC, CXC, CX3C and the like.

[00176] In certain aspects, the biomarkers capable of triggering a signal transduction pathway, in turn altering a cellular response can be a growth factor. Non- limiting examples of growth factors that can be measured in gene expressions or protein levels to relate tumor cells or tumorigenic cells to a physiological status include

erythropoietin (EPO), angiopoietin (Ang), stem cell factor (SCF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), nerve growth factor (NGF), hematopoietic cell growth factor, hepatocyte growth factor, hepatoma-derived growth factor, migration- stimulating factor, autocrine motility factor, epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), transforming growth factor (TGF), cartilage growth factor (CGF), keratinocyte growth factor (KGF), skeletal growth factor (SGF), osteoblast-derived growth factor (BDGF), cytoline growth factor (CGF), colony stimulating factor (CSF), integrin modulating factor (IMF), platelet-derived growth factor (PDGF), calmodulin, bone morphogenic proteins (BMP), tissue inhibitor matrix metalloproteinase (TIMP), pHH3, ubiquitin, c-MYC, and the like.

[00177] In certain cases, the biomarkers are immunohistochemistry (IHC) markers. Non- limiting examples of IHC markers that can be measured include hematopoetic markers, breast markers, carcinoma or mesothelial markers, colon markers, central nervous system markers, infectious disease markers, keratin or epithelial markers, lung markers, melanocytic markers, neuroendocrine markers/other hormones, other organ-related markers, prognostic other markers, prostate markers, stromal markers or tumor markers.

Hematopoetic markers include, but not limited to: annexin Al, BCL2 follicular lymphoma marker, BCL6 follicle center B cell marker, CD10, CD20, CD23, CD79a, cyclin Dl, hairy cell leukemia marker, multiple myeloma oncogene 1, PAX-g B cell transcriptional factor, ZAP 70, CD34, CD68, CD99, CD117, glycophorin-A, myeloperoxidase, terminal deoxynucleotidyl transferase, von willebrand factor VIII, anaplastic lymphoma kinase- 1, CD15, CD30, fascin, CD45, CD138, kappa immunoglobulin light chains, lambda

immunoglobulin light chains, plasma cell p63, CDla, CD2, CD3, CD4, CD5, CD7, CD8, CD43, CD56, CD57 and granzyme B. Breast markers include, but not limited to: Akt protein kinase, cytokeratin 5, p63, epithelial antigen, cathepsin D, cytokeratin 8, HMW cytokeratin high molecule weight, cytokeratin 5/6, cytokeratin 7, cytokeratin 19, cytokeratin 20, E- cadherin, estrogen receptor, HER2/neu, Ki67 cell proliferation marker, p53 tumor suppressor gene protein, progesterone receptor and smooth muscle actin. Carcinoma or medothelial markers include, but not limited to: BER-EP4 epithelial antigen, calretinin, ERA epithelial related antigen, cervical or gynecological markers, pl6 tumor suppressor gene protein, ProEx C biomarker, TAG72 and wilms tumor marker. Colon markers include, but not limited to: epidermal growth factor receptor, CDX2, microsatellite instability marker such as MLH1, MSH2, MSH6, PMS2 and p53. CNS markers include, but not limited to: human glial fibrillary acidic protein and neurofilament. Infectious disease markers include, but not limited to: cytomegalovirus, herpes simplex virus type I, II, pylori H and varicella zoster virus. Keratin and epithelian markers include, but not limited to: cytokeratin 5/6, cytokeratin 7, cytokeratin 8/18, cytokeratin 19, cytokeratin 20, cytokeratin high molecular weight, caldesmon smooth muscle, p63, collagen 9, smooth muscle myosin, cytokeratin cocktail and epithelial membrane antigen. Lung markers include, but not limited to: 34BE12, HMW cytokeratin high molecular weight, excision repair cross complementing polypeptide, synaptophysin and thyroid transcription factor- 1. Melanocytic markers include, but not limited to: HMB melanoma associated marker 45, melanoma cocktail, melanoma associated marker 1, si 00 protein and tyrosinase. Neuroendocrine markers and other hormones include, but not limited to: androgen receptor, calcitonin, chromogranin A, G cell antral pyloric mucosa, neuron-specific enolase, somatostatin and synaptophysin. Other organ-related markers include, but not limited to: CEA carcinoembryonic antigen, calectin-3, gross cyctic disease fluid protein 15, hepatocyte antigen, adrenal cortical inhibin and renal cell carcinoma marker. Prostate markers include, but not limited to: ΡΓΝ2 cocktail, ΡΓΝ4 cocktail, prostate specific antigen, prostatic acid phosphorase and p504s gene product. Stromal markers include, but not limited to: CD31, podoplanin, DOGl derived from GISTl, desmin filament protein, factor XHIa fibrohistocytic, human herpesvirus type 8, muscle specific actin, myogenin muscle marker, myoglobin cardiac and skeletal marker, si 00 protein, smooth muscle actin, smooth muscle myosin and vimentin. Tumor markers indluce, but not limited to: alpha detoprotein, Ca 19-9 CI, Ca-125 epitheliod malign marker and survivin.

[00178] In some cases, the biomarkers that can be measured in gene expressions or protein levels are metabolites or metabolic biomarkers. Non-limiting examples of metabolites or metabolic biomarkers include: adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), Guanosine-5 '-triphosphate (GTP), Guanosine-5 '-diphosphate (GDP), Guanosine-5'- monophosphate (GMP), nicotinamide adenine dinucleotide phosphate (NADP), NADPH, nicotinamide adenine dinucleotide (NAD), NADH, proliferating cell nuclear antigen, glucose, glucose-6-phosphate, fructose-6-phosphate, fructose 1,6-b phosphate, ribose-5- phosphate, erythrose-4-phosphate, xylulose 5 -phosphate, glyceraldehyde-3 -phosphate, sedoheptulose 7-phosphate, 3 ribulose-5 -phosphate, 1 ribose-5 -phosphate,

phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate, 1, 3-phosphoglycerate, dihydroxyacetone phosphate, malate, oxaloacetate, ketoglutarate, lactate, glutamine, alanine, glutamate, pyruvate, fatty acids, acetyl-coA, citrate, glycerol, uric acid, cholesterols, eicosanoids, glycolipids, phospholipids, shpingolipids, steoid, triacylglycerols, albumin, insulin, diols, Ros, NO, bilirubin, phosphor-creatine, ketone bodies, L-ornithine,

argininosuccinate, fumarate, L-arginine, urea, carbamoyl phosphate, ornithine, citrulline, histidine, isoleucine, leucine, lysine, methionine, phenylanine, threonine, tryptophan, valine, asparagines, aspartic acid, cysteine, glutamic acid, glycine, proline, selenocysteine, serine, taurine, tyrosine, citric acid and the like.

[00179] In some cases, the biomarkers can be ions. Non-limiting examples include hydrogen, potassium, sodium, calcium, chloride, magnesium, bicarbonate, phosphate, hydroxyl, iodine, copper, iron, zinc, sulfate and the like.

[00180] Methods of Delivering Agents

[00181] The present disclosure provides methods for delivering agents to a tissue. [00182] A method for delivering two or more agents to a tissue can comprise contacting the tissue with a device and injecting the agents into the tissue. The device can comprise two or more needles, a first and a second agent, two or more reservoirs, and a channel.

[00183] The reservoirs can be in fluid communication with the needles. A first reservoir can comprise the first agent and a second reservoir can comprise the second agent.

[00184] The first and the second agent can be injected into the tissue. The first agent can be injected into an injection site in the tissue and diffuse to form a first permeation perimeter, and the second agent can be injected into the injection site and diffuse to form a second permeation perimeter. The first permeation perimeter can overlap with the second permeation perimeter.

[00185] The tissue can be an epithelial tissue. Alternatively, a tissue can be a connective tissue, a muscle tissue, or a nervous tissue. Further, the tissue can be a

subcutaneous tissue.

[00186] The device can be a microfluidic device. The channel can be a microf uidic channel. The channel can also comprise an inlet port in fluid communication with the channel. Further, the channel can be in fluid communication with the one or more needles.

[00187] In some cases, the device can comprise at least 2, at least 3, at least 4, at least

5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, or at least 500 needles. In other cases, the device can comprise about 2 to 500, about 4 to 400, about 6 to 300, about 8 to 200, about 10 to 100, or about 20 to 80 needles.

[00188] A needle can be a microinjection needle. A needle can also be a porous needle. In some cases, a needle can comprise a luer lock mechanism.

[00189] The two or more needles can comprise a needle array. A needle array can be in a variety of configurations. For example, a needle array can be linear. Alternatively, a needle array can be in a block or matrix configuration.

[00190] The first agent and the second agent can be the same agent. Alternatively, the first agent and the second agent can be different agents. Further, the first agent can be a different concentration of the second agent.

[00191] In some cases, the one or more agents can be delivered at an amount undetectable outside the tissue. In other cases, a therapeutically effective amount of the one or more agents can be delivered to the tissue. In some examples, the one or more agents can be delivered to the tissue at a systemically detectable concentration. In other examples, the one or more agents can be delivered to the tissue below a systemically detectable

concentration.

[00192] The one or more agents can comprise an anti-cancer agent. The one or more agents can also comprise a small molecule agent.

[00193] The permeation perimeter can have a diameter. In some cases, the diameter can be less than about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 5.0 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, 10.0 mm, 15.0 mm, 20.0 mm, 30.0 mm, 40.0 mm, or 50.0 mm. Alternatively, the diameter can be between the about 0.1 mm to 50.0 mm, about 0.2 mm to 40.0 mm, about 0.5 mm to 20.0 mm, about 1.0 mm to 10.0 mm, or about 2.0 mm to 8.0 mm.

[00194] In some cases, two or more of the permeation perimeters can overlap to form an overlapping area. In some examples, the overlapping area can comprise 2 permeation perimeters. In other examples, the overlapping area can comprise 3 permeation perimeters. In further examples, the overlapping area can comprise 4 or more permeation perimeters, such as 5, 6, 7, 8, 9 or 10 permeation perimeters.

[00195] The overlapping area can be greater than about 0.1 mm 2 , 0.2 mm 2 , 0.3 mm 2 ,

0.4 mm 2 , 0.5 mm2 , 0.6 mm2 , 0.7 mm2 , 0.8 mm2 , 0.9 mm2 , 1.0 mm2 , 1.2 mm2 , 1,4 mm 2 , 1.6 mm 2 , 1.8 mm2 , 2.0 mm2 , 3.0 mm2 , 3.5 mm2 , 4.0 mm2 , 4.5 mm2 , 5.0 mm2 , 6.0 mm2 , 7.0 mm2 , 8.0 mm ² , 9.0 mm ² , 10.0 mm ² , 20 mm ² , 30 mm ² , 40 mm ² , 50 mm ² , 100 mm ² , 200 mm ² , 300

2 2 2

mm , 400 mm , or 500 mm . Alternatively, the overlapping area can be less than about 0.1 mm 2 , 0.2 mm2 , 0.3 mm2 , 0.4 mm2 , 0.5 mm2 , 0.6 mm2 , 0.7 mm2 , 0.8 mm2 , 0.9 mm2 , 1.0 mm2 ,

1.2 mm 2 , 1,4 mm 2 , 1.6 mm2 , 1.8 mm2 , 2.0 mm2 , 3.0 mm2 , 3.5 mm2 , 4.0 mm2 , 4.5 mm2 , 5.0 mm 2 , 6.0 mm 2 , 7.0 mm2 , 8.0 mm 2 , 9.0 mm2 , 10.0 mm 2 , 20 mm2 , 30 mm2 , 40 mm2 , 50 mm2 , 100 mm ² , 200 mm ² , 300 mm ² , 400 mm ² , or 500 mm ² .

[00196] In some instances, the overlapping area can be at least about 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 % of one permeation perimeter. In some instances, the overlapping area can be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 % of one permeation perimeter.

[00197] The injecting can comprise intermittent injection of the one or more agents.

Alternately, the injecting can comprise continuous injection of the one or more agents. In some cases, one or more agents are injected intermittently and one or more agents are injected continuously. In other cases, the injecting can comprise intermittent injection of a plurality of agents.

[00198] The injecting can occur at low flow rates. Alternately, the injecting can occur at high flow rates. In some cases, the injecting can occur at flow rates less than 10 μί/ηιίη, 20 μί/ηώι, 30 μί/ηιίη, 40 μί/ηιίη, 50 μί/ηιίη, 60 μί/ηιίη, 70 μί/ηιίη, 80 μί/ηιίη, 90 μί/ηιίη, 100 μί/ηιίη, 200 μί/ηιίη, 300 μί/ηιίη, 400 μί/ηιίη, 500 μί/ηιίη, 600 μί/ηιίη, 700 μί/ηιίη, 800 μί/ηιίη, 900 μί/ηιίη, 1 mL/min, 2 mL/min, 3 mL/min, 4 mL/min, 5 mL/min, 6 mL/min, 7 mL/min, 8 mL/min, 9 mL/min, 10 mL/min, 20 mL/min, 30 mL/min, 40 mL/min or 50 mL/min. In some cases, the injecting can occur at flow rates more than 10 μί/ηιίη, 20 μί/ηιίη, 30 μί/ηιίη, 40 μί/ηιίη, 50 μί/ηιίη, 60 μί/ηιίη, 70 μί/ηιίη, 80 μί/ηιίη, 90 μί/ηιίη, 100 μί/ηώι, 200 μί/ηιίη, 300 μί/ηώι, 400 μί/ηιίη, 500 μί/ηώι, 600 μί/ηώι, 700 μί/ηιίη, 800 μί/ιηίη, 900 μί/ηιίη, 1 mL/min, 2 mL/min, 3 mL/min, 4 mL/min, 5 mL/min, 6 mL/min, 7 mL/min, 8 mL/min, 9 mL/min, 10 mL/min, 20 mL/min, 30 mL/min, 40 mL/min or 50 mL/min.

[00199] The injecting can be performed in vivo. Alternately, the injecting can be performed in vitro. Further, the injecting can be performed ex vivo.

[00200] In some cases, the device can further attach to the tissue. For example, the device can attach to the tissue for more than 1 minute. In certain cases, the device can attach to the tissue for more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 120, 180, 240, or 360 minutes. In some instances, the device can be attached to a tissue for at least about 1, 2, 3, 4, 5, 6 or more days. In some instances, the device can be attached to a tissue for at least about 1, 2, 3,4 ,5, 6, or more weeks. In some instances, the device can be attached to a tissue for at least about 1, 2, 3, 4, 5, 6 or more months. In some cases, the device can attach to the tissue for at most about 1 minute. In certain cases, the device can attach to the tissue for at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 120, 180, 240, or 360 minutes. In some instances, the device can be attached to a tissue for at most about 1, 2, 3, 4, 5, 6 or more days. In some instances, the device can be attached to a tissue for at most about 1, 2, 3,4 ,5, 6, or more weeks. In some instances, the device can be attached to a tissue for at most about 1, 2, 3, 4, 5, 6 or more months.

[00201] The method can further comprise removing the overlapping area. The method can also further comprise evaluating the injection sites. For example, the evaluating can comprise imaging the tissue. Alternatively, the tissue can be evaluated by measuring the levels of biomarkers. The evaluating can also comprise three dimensional evaluation of the tissue.

[00202] The imaging can comprise radiographic imaging, magnetic resonance imaging, positron emission tomography, or biophotonic imaging. The imaging can occur during or after the injecting of the agents.

[00203] A method for delivering an agent to a site in a subject in need thereof can comprise subjecting a plurality of peptides in a medium and thereby forming a hydrogel matrix, capturing the agent in the hydrogel matrix, and contacting the hydrogel matrix with the site.

[00204] In some cases, the agent can be delivered to the site at a rate at least about 2, 3,

4, 5, 6, 7, 8, 9, 10, 20, 50 or 100 times slower than without the hydrogel matrix.

[00205] The peptides can be subjected in the medium at a concentration between about

0.01 % and 50%, between about 0.01% and 20%, between about 0.1% and 20%, between about 0.1%) and 10%>, or between about 1% and 10%>. Alternatively, the peptides can be subjected in the medium at a concentration greater than about 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0 %, 2.2 %, 2.4 %, 2.6%, 2.8 %, 3.0%, 3.2, 3.4%, 3.6%, 3.8%, 4.0%, 4.2, 4.4%, 4.6%, 4.8%, 5.0%, 5.5%, 6.0% ,6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 10%, 15%, 20%, 30%, 40%, or 50%.

[00206] The peptides can comprise a C-terminus domain comprising at least 1, 2, 3, 4,

5, 6, 7, 8, 9, or 10 amino acids. In some cases, at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%), 60%), 70%), 80%), 90%) or 100% of the C-terminus domains can comprise a hydrophobic residue. The hydrophobic residue can be selected from the group consisting of alanine, valine, isoleucine, methionine, phenylalanine, tyrosine and tryptophan. The hydrophobic residue can also be an unnatural amino acid.

[00207] The peptides can be further conjugated to a lipid tail. In some cases, at least

1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the peptides can be conjugated to a lipid tail. The lipid tail can comprise at least 5, 10, 15, 20, 30, 40 or 50 carbon atoms.

[00208] Further, the peptides can comprise a motif. The motif can be a motif used for imaging, including but not limited to a fluorescent motif or an isotopically labeled motif. The motif can also used for isolation and/or recognition, including but not limited to a biotin group, a chemoselective ligation motif, an alkyne group, or an azide group. [00209] The medium can be an aqueous medium. The aqueous medium can have an ionic strength less than about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1 M. Alternatively, the aqueous medium can have an ionic strength greater than The aqueous medium can have an ionic strength below about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1 M.

[00210] The agent can comprise a tag. The tag can be a tag used for imaging, including but not limited to a fluorescent tag or an isotopically labeled tag. The tag can also used for isolation and/or recognition, including but not limited to a biotin group, a chemoselective ligation tag, an alkyne group, or an azide group.

[00211] The method can further comprise providing a vehicle. For example, the vehicle can be cyclodextrin. The agent can also be delivered to at least a second site.

[00212] In one aspect, the invention provides methods for delivery of biological agents to solid tissues or portions thereof ex vivo. In particular cases, the invention provides methods for delivery of biological agents to solid tumors or portions thereof ex vivo and evaluation of the biological activity of the agents.

[00213] The methods described herein can use the relative configuration of agent administration to simultaneously deliver and compare the relative therapeutic efficacies and/or toxicities of a large number of candidate therapeutic agents. For example, at least one positional marker in one or more known locations of a device can be provided to permit ready identification of the effects at a particular location, if any, of the contents released from a particular reservoir at the tissue location. Such applications can find uses in drug screening and drug discovery, such as in preclinical animal models to identify and functionally characterize potential new therapeutics. For instance, a plurality of siRNAs can be administered to a tumor tissue and their relative abilities to knock down expression of a desired target gene can be compared. Additionally, a plurality of small molecule anti-cancer agents can be administered to the tumor tissue and their relative ability to inhibit tumor cell growth can be compared. Other similar cases can find uses in clinical contexts, for example, to "deselect", or eliminate from consideration, known therapeutic agents that have no effect in a particular tumor, or in other cases to prioritize known therapeutic agents based on the response in a particular tumor, thereby advantageously advancing the therapeutic management of a subject by avoiding the loss of time and the undesirable side-effects that can be associated with administering an ineffectual treatment regimen. In some cases, multiple agents can be administered to evaluate agents for their synergistic efficacy.

[00214] The invention can provide a method of screening a biological agent for treating a disease in a subject in need thereof. The method can comprise administering the biological agent to a tissue sample isolated from the subject and assaying the response of tissue. The effect of the agent can be determined. The therapeutic effect can be determined. Side effects of the agent on the tissue can be analyzed. In certain examples, the method can further comprise selecting the biological for clinical trial based on said therapeutic effects and side effects. In some examples, the biological agent can be selected for providing desirable therapeutic effects. In various examples, the biological agent can be selected for minimizing side effects.

[00215] In other cases, the invention provides a method of predicting response of an individual to a biological agent for treating a disease. In some cases, the method can comprise administering the biological agent to a tissue sample isolated from the individual and assaying tissue response to the biological agent thereby predicting clinical response of the individual to the biological agent. In some examples, the tissue response to more than one biological agent can be assayed and compared to thereby determine which biological agent is most effective and/or safest to the individual.

[00216] In yet other cases, the invention provides a method of identifying optimal dosage of a biological agent for treating a disease in a subject in need thereof. In some examples, the method can comprise administering a dose of the biological agent to a tissue sample isolated from the subject and assaying tissue response to the dose thereby determining therapeutic effects and side effects of the dose. In some examples, the method can further comprise selecting an optimal dose based on the therapeutic effects and side effects. In further examples, the method can further comprise correlating assay results to clinical response of the individual to the dose.

[00217] In some examples, the dose can be about 0.1 mg to about 10 g. In some cases, the dose can be about 0.2 mg to 5 g. In some instances, the dose can be about 0.5 mg to 1 g. In some cases, the dose can be about 1 mg to 500 mg. In some further examples, the dose can be about 5 mg to 200 mg. In some further cases, the dose can be about 10 mg to 100 mg.

[00218] In further cases, the invention provides a method of identifying a standard of care drug for combination therapy with a biological agent for treating a disease in a subject in need thereof. In some cases, the method can comprise administering a

combination therapy comprising the biological agent and the standard of care drug to a tissue sample isolated from the subject and assaying tissue response to the combination therapy thereby determining therapeutic effects and side effects of the standard of care drug on the combination therapy. In some examples, the method can further comprise selecting an optimal standard of care drug for the combination therapy based on the therapeutic effects and side effects.

[00219] In some cases, the invention provides a method of identifying an optimal dosage of a standard of care drug for combination therapy with a biological agent for treating a disease in a subject in need thereof. In some examples, the method can comprise administering a combination therapy comprising the biological agent and a dose of the standard of care drug to a tissue sample isolated from the subject and assaying tissue response to the dose of the standard of care drug in the combination therapy thereby determining therapeutic effects and side effects of the dose. In some examples, the method can further comprise selecting an optimal dose based on the therapeutic effects and side effects.

[00220] The standard of care drug can be an anti-cancer agent. Alternatively, the standard of care drug can be an antiviral agent. The standard of care drug can also be an antimicrobial agent. For example, the antimicrobial agent can be an anti-bacterial agent. The antimicrobial agent can also be an anti-fungal agent.

[00221] In other cases, the invention provides a method of selecting an optimal treatment regime for treating a disease in a subject in need thereof. In some cases, the method can comprise conducting a treatment regime on a tissue sample isolated from the subject and assaying tissue response to the treatment regime thereby determining therapeutic effects and side effects of the treatment regime. In some examples, the method can further comprise selecting an optimal treatment regime based on the therapeutic effects and side effects. In further examples, the treatment regime can be selected from the group consisting of chemotherapy, radiation, antibody therapy, surgery, combination therapy, targeted therapy, or a combination thereof.

[00222] In yet other cases, the invention provides a method of identifying a subpopulation of patients optimal for a treatment of a biological agent. In some examples, the method can comprise administering the biological agent to a tissue sample isolated from the patients and assaying tissue response to the biological agent thereby determining therapeutic effects and side effects of the biological agent. In some cases, the method can further comprise selecting a patient subpopulation optimal for the treatment based on the therapeutic effects and side effects.

[00223] In further cases, the invention provides a method of screening for an effective candidate agent for a disease. In certain cases, the method can comprise: (a) isolating a tissue sample from a subject experiencing said disease; (b) culturing said tissue sample ex vivo; (c) locally administering one or more agents to one or more specific locations on said tissue sample; (d) analyzing the biological effects of said one or more agents on said one or more specific locations on said tissue sample; and (e) selecting said one or more agents for treating said subject based on said biological effects on said one or more specific locations on said tissue sample.

[00224] In some examples, the method can further comprise isolating a tissue sample from the subject. In further examples, the methods can further comprise culturing the tissue sample ex vivo.

[00225] In some examples, the biological agent can be administered at one or more concentration. In various examples, a second biological agent can be administered to the tissue sample. In certain examples, a second tissue sample can be isolated from a different location of said subject. In further examples, the method can further comprise administering a second biological agent to said tissue sample.

[00226] The disease can be selected from the group consisting of

hyperproliferative disorder, inflammatory disease, immune disease, nervous system disease, metabolic disease, angiogenic disease, ophthalmic disease, respiratory disease, and cardiac disease. In some examples, the hyperproliferative disorder is cancer.

[00227] The tissue sample can be a solid tumor. The biological agent or standard of care drug can be an anti-cancer agent. In some examples, more than one biological agent can be administered to a specific location of the tissue sample.

[00228] The analyzed biological effect can be local cell toxicity. In certain examples, the biological agent or standard of care drug can be selected based on local cell toxicity and host toxicity. Additionally, the method can further comprise reporting the assay results to a designated person or entity.

[00229] In accordance with some cases, FIG. 1 illustrates a general outline for the method utilized in the delivery of biological agents to solid tissues ex vivo and evaluation of the biological activity of the agents. A section of tissue slice 100 can be first isolated from a subject in need thereof to yield tissue samples 102. In some examples, the subject in need thereof can be suffering from cancer, and the tissue slice 100 is a tumor slice. Tissue samples 102 can be submitted to device 104, wherein the tissue samples 102 can be cultured ex vivo and subjected to a library of biological agents. The effects of the biological agents can be evaluated by an analyzer 106. The analysis performed can be based on measurements of efficacy, toxicity, bioavailability, solubility, and/or other genotypic, phenotypic, or morphological effects and biomarkers induced by the agent onto the tissue sample 102. The results of the analysis can be utilized by a caregiver 108, who can administer one or more agents to the subject based on the analysis results.

[00230] Devices for Delivering Agents

[00231] In some cases, a delivery device 200 for delivery of at least one agent to a tissue membrane is provided. As depicted in FIG. 2, the device can comprise a tissue membrane 202 that can be cultured in between a humidity chamber 212 and a supporting means 204 that can comprise one or more reservoirs 206. The one or more reservoirs 206 can be loaded with one or more concentrations of an agent 208. The one or more reservoirs 206 can be loaded with one or more agents 208. The one or more concentrations of the one or more agents 208 can be locally administered to the tissue membrane 202 by direct contacting the tissue membrane 202. The one or more concentrations of the one or more agents 208 can be locally administered to the tissue membrane 202 via capillary action. In a further example, the agents 208 can be locally administered to the tissue membrane 202 via capillary action through one or more delivery conduits 210.

[00232] In some cases, a method for delivering an agent to a tissue is provided.

As exemplified in FIG. 3, tissue slices 308 can be isolated from an animal and cultured on tissue membrane 302. The one or more reservoirs 306 can be loaded with one or more concentrations of one or more agents 310. By contacting the tissue membrane 302 with the supporting means 304 comprising the reservoirs 306, the agents 308 can be locally administered to the tissue membrane 302 at the specific locations corresponding to the agents loaded in the reservoirs and the effects of the agents can be assessed according to the corresponding locations on the tissue membrane.

[00233] In a further case, illustrated in FIG. 4, the supporting means can be loaded with one or more agents 412/414 in the reservoirs and made in contact with the tissue membrane 402 comprising the tissue slices 408. Following a first cycle of contact, wherein the agents 412/414 are locally administered to the tissue membrane 406, the orientation of the supporting means 404 can be rotated relative to the tissue membrane 402 and again made in contact with the tissue membrane 402. Under these conditions, the individual tissue regions 416/418 of the tissue slice 408 can undergo more than one cycle of agent administration. For example, tissue region 416 is first administered with 412 and again administered with 412, whereas tissue region 418 is first administered with 414 and then administered with 412. By using the relative geometric orientations of the tissue membrane and the reservoirs, the effects of more than one cycle of agent administration can be efficiently evaluated by assessing the effects on the corresponding locations of the tissue membrane.

[00234] An aspect of the present disclosure provides devices for administering agents to a tissue. A device for administering agents to a tissue can comprise two or more needles, one or more agents, two or more reservoirs, and a channel. In some cases, the reservoirs can be in fluid communication with the needles. In some examples, a first reservoir of said two or more reservoirs can comprise a first agent of said one or more agents. In further examples, a second reservoir of said two or more reservoirs can comprise a second agent of said one or more agents.

[00235] The tissue can be an epithelial tissue. Alternatively, a tissue can be a connective tissue, a muscle tissue, or a nervous tissue. Further, the tissue can be a

subcutaneous tissue.

[00236] A content of the reservoir can be injected into an injection site in the tissue. In some cases, a pattern of coverage of a first agent in the first solid tissue can overlap with a pattern of coverage of a second agent in the tissue.

[00237] In some cases, the device can comprise at least 2, at least 3, at least 4, at least

5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, or at least 500 or more needles. In other cases, the device can comprise about 2 to 500, about 4 to 400, about 6 to 300, about 8 to 200, about 10 to 100, or about 20 to 80 needles.

[00238] A needle can be a microinjection needle. A needle can also be a porous needle. In some cases, a needle can comprise a luer lock mechanism. A needle can be a biopsy needle.

[00239] The two or more needles can comprise a needle array. A needle array can be in a variety of configurations. For example, a needle array can be linear. Alternatively, a needle array can be in a block or matrix configuration. [00240] The first agent and the second agent can be the same agent. Alternatively, the first agent and the second agent can be different agents. Further, the first agent can be a different concentration of the second agent.

[00241] In some cases, the device can be configured to deliver the one or more agents at an amount undetectable outside the tissue. In other cases, the device can be configured to deliver a therapeutically effective amount of the one or more agents. In some examples, the device can be configured to deliver the one or more agents at a systemically detectable concentration. In other examples, the device can be configured to deliver the one or more agents below a systemically detectable concentration.

[00242] The device can be a microfluidic device. The channel can be a microfluidic channel. The channel can also comprise an inlet port in fluid communication with the channel. Further, the channel can be in fluid communication with the one or more needles.

[00243] The one or more agents can comprise an anti-cancer agent. The one or more agents can also comprise a small molecule agent.

[00244] The content of the reservoir can diffuse from the injection site and thereby form a permeation perimeter. The permeation perimeter can have a diameter. In some cases, the diameter can be less than about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 5.0 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, 10.0 mm, 15.0 mm, 20.0 mm, 30.0 mm, 40.0 mm, or 50.0 mm. Alternatively, the diameter can be between the about 0.1 mm to 50.0 mm, about 0.2 mm to 40.0 mm, about 0.5 mm to 20.0 mm, about 1.0 mm to 10.0 mm, or about 2.0 mm to 8.0 mm. In some cases, the diameter can be more than about 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, 4.5 mm, 5.0 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, 10.0 mm, 15.0 mm, 20.0 mm, 30.0 mm, 40.0 mm, or 50.0 mm.

[00245] In some cases, two or more of the permeation perimeters can overlap to form an overlapping area. In some examples, the overlapping area can comprise 2 permeation perimeters. In other examples, the overlapping area can comprise 3 permeation perimeters. In further examples, the overlapping area can comprise 4 or more permeation perimeters, such as 5, 6, 7, 8, 9 or 10 permeation perimeters. FIG. 1 illustrates overlapping permeation perimeters of agents injected into a tissue. A needle is injected into an injection site 104. The injection site can be surrounded by a permeation perimeter 105/110/115/120 of the one or more agents injected into the injection site. The needles of the injection device can be spaced such that the permeation perimeters 105/110/115/120 can overlap. An overlapping area 125 can comprise the permeation perimeters of two injection sites. An overlapping area 130 can comprise the permeation perimeters of three injection sites. An overlapping area 140 can comprise the permeation perimeters of four injection sites. The overlapping areas can be evaluated for a physiological effect on said solid tissue.

[00246] The device can attach to the tissue. In some cases, the device can attach to the tissue for more than 1 minute. In certain cases, the device can attach to the tissue for more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 120, 180, 240, or 360 minutes. In some instances, the device can be attached to a tissue for at least about 1, 2, 3, 4, 5, 6 or more days. In some instances, the device can be attached to a tissue for at least about 1, 2, 3, 4, 5, 6, or more weeks. In some instances, the device can be attached to a tissue for at least about 1, 2, 3, 4, 5, 6 or more months. In some cases, the device can attach to the tissue for at most about 1 minute. In certain cases, the device can attach to the tissue for at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 120, 180, 240, or 360 minutes. In some instances, the device can be attached to a tissue for at most about 1, 2, 3, 4, 5, 6 or more days. In some instances, the device can be attached to a tissue for at most about 1, 2, 3, 4, 5, 6, or more weeks. In some instances, the device can be attached to a tissue for at most about 1, 2, 3, 4, 5, 6 or more months.

[00247] Position Markers

[00248] Certain cases contemplate direct delivery of multiple agents, candidate drugs, imaging agents, positional markers, indicators of efficacy and appropriate control

compositions to a plurality of spatially defined locations along parallel axes in a solid tissue, such as a solid tumor, followed, after a desired time interval, by excision of the treated tissue and evaluation or analysis of the tissue for effects of the treatments. Indicators of efficacy can be, for example, detectable indicator compounds, nanoparticles, nanostructures or other compositions that comprise a reporter molecule which provides a detectable signal indicating the physiological status of a cell, such as a vital dye (e.g., Trypan blue), a colorimetric pH indicator, a fluorescent compound that can exhibit distinct fluorescence as a function of any

2_ _| _

of a number of cellular physiological parameters (e.g., pH, intracellular Ca or other physiologically relevant ion concentration, mitochondrial membrane potential, plasma membrane potential, etc.), an enzyme substrate, a specific oligonucleotide probe, a reporter gene, or the like. Control compositions can be, for example, negative controls that have been previously demonstrated to cause no statistically significant alteration of physiological state, such as sham injection, saline, DMSO or other vehicle or buffer control, inactive

enantiomers, scrambled peptides or nucleotides, etc.; and positive controls that have been previously demonstrated to cause a statistically significant alteration of physiological state, such as an FDA-approved therapeutic compound.

[00249] In some cases, a pharmaceutical formulation further comprises a dye. The dye can be imaged after administration of the pharmaceutical composition to an animal tissue to observe the distribution and activity of an agent present in the same pharmaceutical composition. In some cases, the dye is a fluorescent dye. In some cases, the dye is a radioactive dye, such as an iodide-based radioactive dye. In some cases, the dye is a radiocontrast agent. In some cases, the dye is a UV dye. In some cases the UV dye is SPOT ink. In some cases, the dye can be visualized with a black light. In some cases the dye can be visualized by human eye. In some cases, the UV dye is a tattoo dye. In some cases, the UV dye is sterile. In further cases, the UV dye is pyrogen-free. In some examples, the UV dye can be rose fluorescent pigment or yellow fluorescent pigment. The UV dye can further comprise Lumikol magenta dispersion. UV dyes suitable for use in the present disclosure are commercially available, for example, from United Mineral and Chemical Corporation. The UV dye can further comprise, without limitation, an astringent (e.g., Witch hazel), an emmolient (e.g., glycerin), and/or a solvent (e.g., propylene glycol).

[00250] Devices/Methods for Maintaining Tumor Orientation

[00251] After injection of an agent into a tumor, the orientation of the tumor may change, making it difficult to visualize the response of the tumor to the injected agent. The tumor orientation can be marked by introducing a guide marker. A guide marker includes but is not limited to an anchor suture or hookwire.

[00252] An anchor suture can be preloaded into an introducer needle. The anchor suture can be attached to a straight wire guide. The straight wire guide can be a mandril wire guide. The straight wire guide can be stainless and 75 centimeters in length. At the end of the anchor suture can be a spring coil anchor. The spring coil anchor can be 2 centimeters in length. The spring coil anchor can have a diameter of at least 0.35 inches.

[00253] A hookwire can comprise a puncture needle and a localization wire. The localization wire can be flexible which reduces the risk of breakage or unwanted dislocation. The hookwire can be made of but is not limited to titanium alloy which allows for clear visibility during MR imaging. The hookwire can also be a double hookwire. The double hookwire can be used to increase fixation in very soft tissues. The hookwire can have a double facet bevel tip which provides straight, clean tissue penetration. The hookwire can have laser etched markings for each centimeter. The hookwire can have a sliding stopper to ensure precise insertion depth control. The hookwire can be reinforced two centimeters behind hook deployment to ensure accurate hook placement. The guide needle gauges can be 18 gauge or 20 gauge. The insertion length can be 8 centimeters to 10 centimeter. The wire length can be 25 centimeters to 30 centimeters. The distal one centimeter of the hookwire can be hooked.

[00254] The placement of the guide marker can be visualized through the use of imaging methods. Such imaging methods include, but are not limited to radiographic imaging, magnetic resonance imaging, positron emission tomography, ultrasound imaging, or biophotonic imaging. The insertion location of the guide marker can be marked on the skin of the patient through the use of a radiopaque skin marker.

[00255] Reverse anchored drug loaded PLGA sutures

[00256] A guide marker can be an anchor suture. An anchor suture can be made of, but not limited to poly(lactic-co-glycolic acid) (PLGA). The anchor suture can be pre-loaded with an agent, including, but not limited to a chemotherapeutic agent (FIG. 36A). The anchor suture can be inserted into a tumor tissue to mark an injection site (FIG. 36B). The anchor suture can release the agent payload in a controlled release manner ranging from 0 to 24 hours, 1 to 2 days, 1 to 3 days, 1 to 4 days, 1 to 5 days, 1 to 6 days, 1 to 7 days, 1 to 8 days, 1 to 9 days, 1 to 10 days, 1 to 11 days, 1 to 12 days, 1 to 13 days, 1 to 14 days, 1 to 15 days, 1 to 16 days, 1 to 17 days, 1 to 18 days, 1 to 19 days, 1 to 20 days (FIG. 36C). The anchor suture can be preloaded with the agent by electrospinning.

[00257] Microinjection Devices

[00258] Microinjection systems are described in U.S. Patent Nos. 8,672,887,

8,657,786, 8,475,412, 8,349,554, U.S. Patent Application Publication No. 2014/0155861, and U.S. Patent Application No. 14/127,763.

[00259] In some cases the microinjection system comprises a device for delivery of a fluid to a solid tissue, comprising: one or more needles; one or more reservoirs, each in fluid communication with a respective of the one or more needles; and one or more actuators operatively coupled to respective one or more reservoirs and configured to control a fluid pressure within the reservoir. In certain cases each of the one or more actuators comprises one or more plungers, a first end of each of the one or more plungers being received in a respective one of the one or more reservoirs, and in certain further cases the one or more plungers are operatively coupled together at respective second ends so as to be

simultaneously depressable. Certain still further cases comprise a plunger driver configured to depress all of the one or more plungers at a selectively variable rate. In other cases each of the one or more actuators comprises one of a one or more fluid transmission lines having first and second ends, a first end of each of the one or more fluid transmission lines being coupled to a respective one of the one or more reservoirs. In other cases the device comprises a fluid pressure source, and each of the one or more actuators comprises a fluid coupling between the fluid pressure source and a respective one of the one or more reservoirs. In further cases the fluid pressure source comprises at least one of a compressor, a vacuum accumulator, a peristaltic pump, a master cylinder, a microfluidic pump, and a valve. In another case, each of the one or more needles comprises one or more ports distributed along its length.

[00260] In certain other cases the plunger driver comprises a driver shaft coupled to the plunger and having a threaded region, the plunger driver configured such that rotation of the driver shaft in a first direction depresses the plunger a distance corresponding to a thread pitch of the threaded region and a number of revolutions of the driver shaft. In certain further cases the device comprises a motor having a rotor coupled to the driver shaft of the plunger driver such that the rotor and the driver shaft are rotationally fixed with respect to each other, the motor being controllable to rotate the rotor at a selectably variable rate. In certain other further cases the device comprises a motor having a rotor coupled to the driver shaft of the plunger driver such that the rotor and the driver shaft are rotationally fixed with respect to each other, the motor being controllable to rotate the rotor to a selectable angle of rotation. Certain further cases comprise a controller coupled to the motor, the controller being programmable to control direction and speed of rotation of the rotor and to control a number of degrees from a start of rotation to an end of rotation. In other cases of the above described device, the dispenser comprises a dispenser cylinder; a first portion of the dispenser cylinder defines the reservoir; and a second portion of the dispenser cylinder defines the needle. In another case the one or more ports are sized and positioned along the length of the needle so as to deliver a substantially equal amount of fluid at any given location along the length of the needle. In another case the one or more ports is evenly distributed along a portion of the length of the needle.

[00261] In certain cases a size of each of the one or more ports is inversely related to a distance of the respective port from a tip-end of the needle. In certain other cases a distribution density of the one or more ports is inversely related to a distance of the respective port from a tip-end of the needle. In certain other cases the one or more ports is distributed in a spiral pattern along the length of the needle. In certain other cases the one or more ports is arranged in pairs of ports on opposite sides of the needle, with each pair of ports rotated 90 degrees with respect to adjacent pairs of ports along the length of the needle.

[00262] According to certain other cases disclosed herein, there is provided a method, comprising placing an agent in a reservoir of each the one or more dispenser needles;

inserting each of the one or more dispenser needles into a selected region of solid tissue; and introducing the agent in the reservoirs into the selected region of solid tissue by

simultaneously overpressurizing each of the one or more dispenser needles. In certain further cases the introducing comprises introducing the agent in the reservoirs into the selected region of solid tissue from one or more apertures along each of the one or more dispenser needles. Certain other further cases comprise at least one of imaging the solid tissue prior to the inserting, imaging the solid tissue concurrently with the inserting, and imaging the solid tissue after the inserting. In certain other further cases the inserting comprises inserting one or more introducer needles into a subject; inserting each of the one or more dispenser needles into a respective one of the one or more of introducer needles; and extending a tip-end of each of the one or more dispenser needles beyond a tip end of the respective one of the one or more of introducer needles and into the selected region of tissue. Certain further cases comprise removing stylets from the one or more introducer needles prior to inserting the one or more dispenser needles.

[00263] In certain cases the selected region of tissue is a portion of a tumor in a subject, and in certain further cases the subject is one of a preclinical model and a human patient. In certain other cases the method comprises excising at least the portion of the tumor after the introducing. Certain further cases comprise at least one of imaging the tumor prior to the excising, imaging the tumor concurrently with the excising, and imaging the tumor after the excising. In certain other cases the excising comprises excising at least the portion of the tumor at a time that is a selected period of time after introducing the agent. In certain further cases the selected period of time is one of a range of time, a minimum period of time for excising, and a specific period of time for excising. In certain cases the selected period of time is a period exceeding 48 hours. In certain cases the selected period of time is a period of time less than 24 hours. In certain cases the selected period of time is a range of between about 72 and about 96 hours. In certain cases the selected period of time is a period exceeding one week.

[00264] Turning to another case there is provided a fluid agent-delivering device comprising (i) one or more needles, each of said needles having, independently, one or more ports distributed along its length wherein at least one needle has said one or more ports, (ii) one or more reservoirs containing the fluid agent, each of said reservoirs being in fluid communication with a respective one of the one or more needles, and (iii) one or more plungers, a first end of each plunger being received in a respective one of the one or more reservoirs and a second end of each plunger being depressable such that depressing each plunger results in injection of the fluid agent through the respective one of the one or more needles.

[00265] In another case of the presently disclosed invention there is provided a method for selective delivery of a fluid agent to a solid tissue, comprising (a) introducing one or more needles of a fluid agent-delivering device into the solid tissue; and (b) administering the fluid agent into the solid tissue by injection through said needles. In certain further cases the solid tissue has been removed from a subject. In certain other further cases the solid tissue is in a subject. In certain further cases the agent is delivered to the solid tissue in a therapeutically effective amount. In certain still further cases, outside the solid tissue, the agent is either (i) undetectable, or (ii) if detectable outside the solid tissue, the agent is present at less than a minimal dose. In certain cases the solid tissue comprises a tumor. In certain further cases the tumor is selected from a benign tumor and a malignant tumor. In certain other further cases the tumor is selected from a primary tumor, an invasive tumor and a metastatic tumor. In certain other further cases the tumor comprises at least one cancer cell selected from a prostate cancer cell, a breast cancer cell, a colon cancer cell, a lung cancer cell, a brain cancer cell, and an ovarian cancer cell. In certain other further cases the tumor comprises a cancer selected from adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, chondrosarcoma and

fibrosarcoma. In certain other cases the solid tissue is selected from brain, liver, lung, kidney, prostate, ovary, spleen, lymph node, thyroid, pancreas, heart, skeletal muscle, intestine, larynx, esophagus and stomach.

[00266] In some examples, the needle can further function as a biopsy needle. In this example, the agent can be delivered by the needle directly to the tumor and upon retraction of the needle, the adjacent tissue is removed. In this method, the tissue will be maintained in the spatial orientation relative to the device to allow for assessment of efficacy, dose dependency and therapeutic response.

[00267] Microdialvsis Probes

[00268] In other examples, a method for delivering at least one agent to a solid tissue is provided. As described in FIG. 5, the method can comprise isolating a tissue slice 500 from an animal and inserting one or more microdialysis tubes 502 into the tissue slice 500. The tissue slice 500 can be cultured ex vivo by supplying media 504 through the microdialysis tubes 502. In a further example, one or more agents 506/508/510/512/514 can be provided along with the media 504 through the one or more microdialysis tubes 502 and administered to the tissue slice 500.

[00269] The effects of the agents can be analyzed using the methods illustrated in FIG.

6. Following passage through the microdialysis tubes 602 inserted in the tissue slice 600, the eluates (e.g. 624, 626, 628, 630, 632, and 634) from the corresponding tubes can be collected for biomarker analysis to determine the effects of agents (e.g. 604, 606, 608, 610, 612, and 614). Further, the tissue slice 600 can be subject to cross-sectional analysis (e.g. 616) or parallel-plane analysis (e.g. 618). The phenotypical and/or morphological features of the local tissue slice can be evaluated. The evaluation can be used to assess the effects of the agents contacted to the tissue. For example, if the local areas surrounding the microdialysis tubes (e.g. 620 and 622) exhibited cell death in both the cross-sectional analysis 616 and the parallel-plane analysis 618, that may indicate the cytotoxic effects of the corresponding agents 606 and 610 on the tissue.

[00270] In some cases the microinjection device can comprise a microdialysis probe.

Microdialysis probes are described in International Application No. PCT/US 12/62313. The present invention provides methods for the administration of an agent to a solid tissue through the use of one or more microdialysis probes. In some cases, the microdialysis probe has an inlet-tubing, an outlet-tubing and a membrane region. The solution in the inlet-tubing is termed "perfusate" while the solution in the outlet tubing is termed "dialysate". The inlet- and outlet-tubings may be made of a material suitable for microdialysis application. In some cases, the material is fused silica. In some other cases, the microdialysis probe has an inlet- tubing and a membrane region without an outlet-tubing. In this design, an imaging agent may be actively pumped across the membrane region.

[00271] The inventors have recognized the advantages of using microdialysis probes as a delivery tool, which include: (1) microdialysis probes are an enclosed system, not dependent upon delivery of a liquid volume, thus eliminating many of the microfluidic engineering hurdles; (2) the semi-permeable membrane surrounding the probe allows liquid to be filled and distributed evenly along probe membrane when injecting into a solid tissue; (3) initial delivery and biodistribution of agents are highly restricted and dependent upon passive diffusion forces, not deposition/delivery of a liquid; (4) true "microdosing" of agents can be achieved by controlling time, flow rate and concentration of perfusate; (5) multiple or timed dosing over an extended periods of time can be achieved by leaving probes in the solid tissue; (6) the amount of agents delivered can be accurately determined by analyzing the amount of agent in perfusate and dialysate; (7) the length of the probe/semi-permeable membrane can be customized to target various size tumors or length of targeting zone within a tumor; (8) one or more linear microdialysis probes can be designed to target the

proliferating zone in solid tumor xenografts, as well as avoiding the central regions necrosis; (9) better sampling of multiple zones, including the entire dimension of a solid tumor, to look for efficacy differences using linear probe arrays can be achieved; and (10) collection and analysis of dialysate at various time points following dosing may allow development and analysis of markers of tumor cell death, cell signal changes, or proliferation/mitotic changes. In addition, microdialysis probes can be used to coax contact-inhibited cells into cycling in order to kill them using checkpoint inhibition/DNA damage, or activate cell signal pathways that have been shut down in non-pro liferative zones.

[00272] A microdialysis probe may be suitable for containing, administering, delivering and transporting contents. The contents may be an aqueous solution comprising an imaging agent. The agents within a single microdialysis probe may be the same or a mixture of different types of imaging agents. Within one or more microdialysis probes, each microdialysis probe may contain the same agent as another probe, or different imaging agents as another probe. In some cases, every microdialysis probe contains agents that are unique from the agents contained in other microdialysis probes.

[00273] A microdialysis probe may have different shapes. In some cases, the microdialysis probe has a "Y" shape. In some other cases, the microdialysis probe has a linear shape. The linear shape may allow the microdialysis probe to penetrate across different sections of a tumor.

[00274] The membrane of a microdialysis probe may be semi-permeable. The membrane may permit the transport of some but not all solutes. In some cases, the membrane permits the transport of solutes with a molecule weight of less than 1 million Daltons. In a further cases, the membrane permits the transport of solutes with a molecule weight in the range of 5,000 Daltons to 1 million Daltons. In another further case, the membrane permits the transport of solutes with a molecule weight of less than 1,000 Daltons.

[00275] The movement of an imaging agent from one side to another side of a membrane may be driven by concentration gradient. In some cases, the movement of an imaging agent from one side to another side of a membrane is driven only by concentration gradient. An imaging agent may move from an area of higher concentration to an area of lower concentration through the semi-permeable membrane. In some cases, the agent diffuses from a microdialysis probe into a solid tissue. In some other cases, a solute in a solid tissue diffuses into a microdialysis probe. The solute can be collected and/or analyzed from dialysate. Alternatively, the movement of an imaging agent may be driven by active transporter, irrespective of concentration gradient. For example, in nature, some cells use active transporter to accumulate molecules, such as ions, glucose and amino acids.

Alternatively, the movement of an imaging agent may be driven by solubility difference. The imaging agent may have a higher solubility on one side of the membrane than the solubility on the other side. In some cases, the imaging agent moves from a higher concentration side to a lower concentration side. In some cases, the imaging agent moves from a lower

concentration side to a higher concentration side. In some cases, the movement of an imaging agent from one side to another side of a membrane is driven by a combination of any one of concentration gradient, active transportation, and solubility difference.

[00276] The membrane may be biocompatible. The membrane may be essentially physiologically inactive or does not trigger physiological events. In some cases, the membrane may not cause inflammation, immune response, infection, or any other sort of rejections within a solid tissue.

[00277] The membrane may be flexible. The flexibility of the membrane will permit the insertion of the membrane section into the solid tissue with minimal damage to the tissue. Yet, the membrane may have certain strength to maintain its integrity before, during or after the insertion. In some cases, the membrane is both flexible and durable.

[00278] The membrane material may be polymeric or co-polymeric. The polymeric or co-polymeric material may be linear or cross-linked. Non-limiting examples of membrane materials include PE (polyethylene), Kevlar, cuprophane, polyethersulfone, polyamine, polyamide, polycarbonate, polycarbamate, polyurethane, polyester, polyether, polyolefm, polysilicon oxide, cellulose acetate, and polyaromatic materials. [00279] The membrane material may be porous. In some cases, the average pore size is less than about 1, 5, 10, 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000, or 10000

nanometers. In some other cases, the average pore size is more than about 1, 5, 10, 20, 30, 40, 50, 100, 200, or 500 nanometers. In some other cases, the average pore size is in a range of 1-10, 1-40, 1- 100, 1-200, or 1-500 nanometers. In some other cases, all pores of a membrane has a substantially similar pore size.

[00280] The pore size may control the rate of diffusion. The pore size may be modulated to control the rate of diffusion. A membrane may be made with a selected average pore size for the purpose of controlling the rate of diffusion. Different imaging agents can diffuse through the membrane at varying rates, controlled in part by the physical and chemical properties of the pharmaceutical compositions, agents, and membrane materials. In some cases, the selected pore size permits the transport of solutes with a molecule weight of less than 1 million Daltons. In a further cases, the selected pore size permits the transport of solutes with a molecule weight in the range of 5,000 Daltons to 1 million Daltons. In another further case, the selected pore size permits the transport of solutes with a molecule weight of less than 1,000 Daltons. In addition, membranes with varying average pore sizes can be made and tested experimentally to find a pore size that provides a desirable diffusion rate for a specific imaging agent.

[00281] An imaging agent may be delivered to a microdialysis probe by using a pump, such as a peristaltic pump or syringe pump. The use of a pump can lead to controlled delivery. For example, the imaging agent can be delivered through a microdialysis probe in a continuous fashion. Alternatively, the imaging agent can be delivered in several doses. The time interval between any two doses can be controlled. Furthermore, the flow rate may be individually controlled for each microdialysis probe. The flow rate may be in a range of about 0.1 to about 5 microliter/min. The flow rate may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or about 5 microliter/min.

[00282] A microdialysis probe may be inserted into a solid tissue directly or indirectly. The indirect insertion may comprise the steps of: (1) insertion of a microdialysis probe into a needle; (2) insertion of the needle into a solid tissue; and (3) withdrawal of the needle from the solid tissue, therefore leaving the microdialysis probe in the solid tissue. In some cases, a one or more microdialysis probes are inserted into a solid tissue. One or more needles holds one of the one or more microdialysis probes. [00283] In addition, the present invention provides a microdialysis probe which has an inlet-tubing without an outlet-tubing. In some cases, the terminal end of the probe is surrounded by a semi-permeable membrane. In this design, the microdialysis probe may act as a diffuser in which liquid and small molecules are actively pumped across the semipermeable membrane.

[00284] The insertion of a microdialysis probe may be guided. In some cases, the insertion of a microdialysis probe is guided by a fixed guide to direct the insertion of a microdialysis probe into a selected region of a solid tissue. In some cases, the insertion of a microdialysis probe is guided by an arthroscopic device.

[00285] In some cases an implanted receptacle is used to deliver an agent to a solid tissue. In some cases the receptacle has a membrane that covers the opening of the tube. In some cases the membrane stretches across the top of the opening. In some cases the membrane controls the rate of release of the imaging agent after implantation.

[00286] Extrusion Devices

[00287] In some cases the microinjection device can comprise extrusion devices.

Extrusion devices are described in International Application No. PCT/US 13/54206. In some cases are methods of injecting an imaging agent into a solid tissue, comprising: (a) inserting at least one needle into the solid tissue; and (b) simultaneously injecting at least one imaging agent into and withdrawing the needles from the solid tissue. The methods described herein can be referred to as an "extrusion method". The term "standard injection method" used herein can generally refer to an injection method wherein in there is no substantial retraction of needles during injection of an agent.

[00288] In some cases a device, comprising a top block having one or more holes sized to allow a needle to pass through the top block and a bottom block having one or more holes sized to allow a needle to pass through the bottom block, wherein the top and bottom blocks are in a substantially parallel arrangement and wherein the top and bottom holes are positioned so as to allow one or more needles to pass through a hole in the top block and the bottom block in a path substantially vertical to the plane of both blocks. In some cases, the device further comprises at least one adjustable leg, wherein the at least one adjustable leg is attached to the bottom block. In some cases, there are four adjustable legs. In some cases, the at least one leg is vertically and horizontally adjustable. In some cases, the bottom block is stationary. In some cases, the top block moves vertically relative to the bottom block. In some cases, the top block moves along guide rods attached to the bottom block. In some cases, the device further comprises a system to control vertical movement of the top block. In some cases, the top and bottom holes are arranged in substantially parallel rows. In some cases, the device further comprises at least one needle. In some cases, a control attachment is attached to the at least one needle. In some cases, the control attachment stops the insertion of the at least one needle, thereby controlling depth of needle insertion into the solid tissue. In some cases, the device further comprises at least one spring, wherein the at least one spring is in substantial contact with the adjustable leg and the bottom block. In some cases, the device further comprises a guiding rod that penetrates the bottom block. In some cases, the guiding rod is inserted into a solid tissue. In some cases, the guiding rod positions the needles on a solid tissue.

[00289] In one aspect, the disclosure provides for a device comprising a top block having one or more holes sized to allow a needle to pass through the top block and a bottom block having one or more holes sized to allow a needle to pass through the bottom block, wherein the top and bottom blocks are in a substantially parallel arrangement and wherein the holes are positioned so as to allow one or more needles to pass through a hole in the top block and the bottom block in a path substantially vertical to the plane of both blocks.

[00290] In one aspect, the disclosure provides for a method of operating a device comprising a top block having one or more holes sized to allow a needle to pass through the top block and a bottom block having one or more holes sized to allow a needle to pass through the bottom block, wherein the top and bottom blocks are in a substantially parallel arrangement and wherein the top and bottom holes are positioned so as to allow one or more needles to pass through a hole in the top block and the bottom block in a path substantially vertical to the plane of both blocks, comprising: inserting one or more needles through the top block and the bottom block into a solid tissue, wherein at least one needle has a control attachment, and simultaneously move the top block away from the bottom block and injecting at least one agent into the solid tissue. In some cases, the at least one needle is inserted into the solid tissue in vivo. In some cases, the solid tissue is a tumor. In some cases, the control attachment controls the depth of needle insertion. In some cases, the at least one agent is positioned in a column. In some cases, the length of the column is from 1-10 millimeters. In some cases, the one or more needles comprises at least two needles. In some cases, the one or more needles comprises at least five needles.

[00291] In one aspect, the disclosure provides for a method of evaluating at least one agent in a solid tissue, comprising placing at least a portion of the solid tissue under the device comprising a top block having one or more holes sized to allow a needle to pass through the top block and a bottom block having one or more holes sized to allow a needle to pass through the bottom block, wherein the top and bottom blocks are in a substantially parallel arrangement and wherein the top and bottom holes are positioned so as to allow one or more needles to pass through a hole in the top block and the bottom block in a path substantially vertical to the plane of both blocks, inserting one or more needles through the top block and the bottom block into the solid tissue, wherein at least one needle has a control attachment, simultaneously move the top block away from the bottom block, injecting at least one imaging agent into the solid tissue, and evaluating an effect of a candidate agent administered systemically.

[00292] In some cases, the rate of injecting at least one agent is at least 0.1 μΐ/min. In some cases, the rate of injecting at least one agent is in the range of about 0.4 to about 4 μΐ/min. In some cases, the rate of the movement of the top block is at least 0.1 mm/min. In some cases, the rate of the movement of the top block is in the range of about 0.5 to about 5 mm/min. In some cases, the solid tissue is a tumor.

[00293] Implantable Microinjection Device

[00294] In some cases the microinjection device is an implantable microinjection device. The microinjection device can be wirelessly controlled for microdosing. The imaging agent can be administered as a bolus, extended bolus, or through continuous infusion. The implantable device comprises multiplexed integrated one or more micropumps and one or more micro-syringes.

[00295] In some cases the microinjection device includes a fiber optic bundle that is typically long enough to extend from the site of device implantation to a point outside of the patient's body, such that the fiber optic bundle remains externally accessible after implantation of device. In some cases the fiber optic bundle may be interfaced with an external energy source to trigger release of the imaging agent. In some cases the fiber optic bundles provide real time sensing of the effect of the candidate agent on a tumor. The fiber optic bundle can have means for remote accessing through a WiFi connection. In some cases optical fibers can be integrated into a cylindrical device.

[00296] In some instances, the implantable device can be a thread. In some cases, the thread can be a suture for use in e.g., surgical procedures. In some cases, the thread can be made of silk. Silk is a versatile material that is biocompatible, biodegradable, and mechanically strong. Silk is made of self-assembling proteins. In this example, silk can be pre-loaded with an agent of the present disclosure by mixing the agent with an aqueous silk solution prior to self-assembly of the silk proteins. In this case, the agent can become trapped in the structure and released upon degradation of the silk structure. The silk structure can be, without limitation, a thread, a tube, a scaffold, a film, a nanofiber, a nanoparticle, a microsphere, a microneedle, or a coating. In some instances, the silk proteins can be recombinant. In other instances, the silk proteins can be isolated from an insect. The silk proteins can be derived from any insect that naturally produces silk proteins. These can include, without limitation, a spider and a silkworm. In some examples, the silk protein is a silk fibroin. In some cases, the agent is delivered to the tissue in a controlled manner.

Hydrogel Compositions

[00297] Another aspect of the present disclosure provides compositions for delivering agents to a tissue. A composition can comprise a hydrogel matrix and an agent. The hydrogel matrix can comprise a plurality of peptides. The hydrogel matrix can be formed by subjecting the plurality of the peptides in a medium. The agent can be captured in the hydrogel matrix.

[00298] The hydrogel matrix can comprise a plurality of matrix pores. The matrix pores can have a diameter in the range of about 1 to 10000, about 5 to 10000, about 5 to 1000, about 5 to 200, about 10 to 200, or about 10 to 100 nanometers.

[00299] The peptides can be subjected in the medium at a concentration between about 0.01 % and 50%, between about 0.01% and 20%, between about 0.1% and 20%, between about 0.1%) and 10%>, or between about 1% and 10%>. Alternatively, the peptides can be subjected in the medium at a concentration greater than about 0.01%, 0.05%>, 0.1 %, 0.2%>, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0 %, 2.2 %, 2.4 %, 2.6%, 2.8 %, 3.0%, 3.2, 3.4%, 3.6%, 3.8%, 4.0%, 4.2, 4.4%, 4.6%, 4.8%, 5.0%, 5.5%, 6.0% ,6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 10%, 15%, 20%, 30%, 40%, or 50%.

[00300] The peptides can comprise a C-terminus domain comprising at least 1, 2, 3, 4,

5, 6, 7, 8, 9, or 10 amino acids. In some cases, at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%), 60%), 70%), 80%), 90%) or 100% of the C-terminus domains can comprise a hydrophobic residue. The hydrophobic residue can be selected from the group consisting of alanine, valine, isoleucine, methionine, phenylalanine, tyrosine and tryptophan. The hydrophobic residue can also be an unnatural amino acid.

[00301] The peptides can be further conjugated to a lipid tail. In some cases, at least

1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the peptides can be conjugated to a lipid tail. The lipid tail can comprise at least 5, 10, 15, 20, 30, 40 or 50 carbon atoms.

[00302] The peptides can be self-assembling peptides. The self-assembling peptides can undergo self-assembly into well-ordered nanofibers. The pore size of the hydrogel can be adjusted by changing the peptide hydrophobicity. The peptide hydrophobicity can be adjusted by introducing lysine mutations into the peptide (FIGS. 40 B-E). The self- assembling peptide can have one reactive NHS or aldehyde fixable residue in the self- assembly domain. The self-assembling peptide can have two reactive NHS or aldehyde fixable residues in the self-assembly domain. The self-assembling peptide can have three reactive NHS or aldehyde fixable residues in the self-assembly domain. The self-assembling peptide can have four reactive NHS or aldehyde fixable residues in the self-assembly domain. The self-assembling peptides can be pre-labeled with a lysine of the self-assembling peptide with a VivoTag 680 reactive NHS ester. The self-assembling peptide can have a

hydrophobic c-terminal modification that can bind to hydrophobic micelle drug formulations (FIGS. 40 F-I). The self-assembling peptides can be visualized by attaching a hapten tag which includes but is not limited to a biotinylated hapten, a dinitrophenol hapten, or a digoxigenin hapten tag. The hapten is conjugated to the self-assembling peptides through various linkages (FIGS. 40J-K).

[00303] Further, the peptides can comprise a motif. The motif can be a motif used for imaging, including but not limited to a fluorescent motif or an isotopically labeled motif. The motif can also used for isolation and/or recognition, including but not limited to a biotin group, a chemoselective ligation motif, an alkyne group, or an azide group.

[00304] The medium can be an aqueous medium. The aqueous medium can have an ionic strength less than about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1 M. Alternatively, the aqueous medium can have an ionic strength greater than The aqueous medium can have an ionic strength below about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1 M.

[00305] The agent can comprise a tag. The tag can be a tag used for imaging, including but not limited to a fluorescent tag or an isotopically labeled tag. The tag can also be used for isolation and/or recognition, including but not limited to a biotin group, a chemoselective ligation tag, an alkyne group, or an azide group.

[00306] The composition can further comprise a vehicle. For example, the vehicle can be cyclodextrin.

[00307] Devices and Kits

[00308] Another aspect of the present disclosure provides devices and kits for delivering biological agents to solid tumors ex vivo and evaluating the biological activity of the agents.

[00309] In some cases, a device for administering one or more biological agents to a tissue sample can comprise a supporting means comprising a plurality of reservoirs and a humidity chamber connected to the supporting means. In some cases, each of the reservoirs can independently hold one or more biological agents. In some cases, the humidity in the humidity chamber can be adjusted between 10% and 100% relative humidity. In some examples, the tissue sample can be placed between the supporting means and the humidity chamber, and made in contact with the one or more biological agents.

[00310] The tissue sample can be isolated from a subject. In some examples, the subject can be a mammal. In further examples, the subject can be a human subject. The tissue sample can also be cultured ex vivo.

[00311] The supporting means can be a microtiter plate. In some examples, the supporting means can comprise of polymeric material. In further examples, the polymeric material can be selected from the group consisting of polystyrene, polypropylene, polycarbonate, and polymers of cyclo-olefms.

[00312] In some examples, the supporting means can comprise at least 5 reservoirs. In other examples, the supporting means can comprise at least 20 reservoirs. In yet other examples, the supporting means can comprise at least 80 reservoirs. In further examples, the supporting means can comprise at least 320 reservoirs. In yet further examples, the supporting means can comprise at least 1280 reservoirs. In some examples, the supporting means can comprise at least 3200 reservoirs.

[00313] In some examples, the humidity in the humidity chamber can be adjusted between about 30% and 100% relative humidity. In other examples, the humidity in the humidity chamber can be adjusted between about 30% and 99% relative humidity. In further examples, the humidity in the humidity chamber can be adjusted between about 50% and 99% relative humidity. In yet further examples, the humidity in the humidity chamber can be adjusted between about 50% and 95% relative humidity.

[00314] In other cases, a device for administering one or more biological agents to a tissue sample can comprise a tissue sample isolated from a subject and a plurality of microdialysis tubes inserted through the tissue sample. In some examples, the culture media can pass through the microdialysis tubes and contact the tissue sample. In some cases, the one or more biological agents can be independently and optionally supplemented into the culture media passing through the plurality of microdialysis tubes.

[00315] The tissue sample can be isolated from a subject. In some examples, the subject can be a mammal. In further examples, the subject can be a human subject. The tissue sample can also be cultured ex vivo.

[00316] The tissue sample can be an epithelial tissue, a connective tissue, a muscle tissue or a nervous tissue. In some cases, the tissue sample can be a cancerous tissue.

[00317] In some examples, a microdialysis tube can comprise a permeable membrane. In other examples, a microdialysis tube can comprise a semipermeable membrane.

[00318] A microdialysis tube can comprise a polymeric material. In some examples, the polymeric material can be selected from the group consisting of polystyrene, polypropylene, polycarbonate, polymers of cyclo-olefins, polyarylethersulfone,

polyethersulfone, polyimide and polyurethane.

[00319] A microdialysis tube can have an inner diameter less than about 10 mm.

In some examples, a microdialysis tube can have an inner diameter less than about 5 mm. In other examples, a microdialysis tube can have an inner diameter less than about 1 mm. In yet other examples, a microdialysis tube can have an inner diameter less than about 0.5 mm. In further examples, a microdialysis tube can have an inner diameter less than about 0.2 mm. In yet further examples, a microdialysis tube can have an inner diameter less than about 0.1 mm.

[00320] In some examples, a microdialysis tube can have a length between about

1 centimeter and 1000 centimeters. In other examples, a microdialysis tube can have a length between about 2 cm and 500 centimeters. In yet other examples, a microdialysis tube can have a length between about 5 cm and 200 centimeters. In further examples, a microdialysis tube can have a length between about 10 centimeters and 100 centimeters.

[00321] In some cases, a biological evaluation kit can comprise a surgical excision device, a library of one or more biological agents, a supporting means comprising a plurality of reservoirs; and a humidity chamber. In some examples, the surgical excision device can be used to isolate a tissue sample from a subject. In some examples, the humidity in said humidity chamber can be adjusted between 10% and 100% relative humidity.

[00322] In other cases, a biological evaluation kit can comprise a surgical excision device, a library of one or more biological agents, a reservoir of culture media, and a plurality of microdialysis probes. In some examples, the surgical excision device can be used to isolate a tissue sample from a subject. In some examples, the microdialysis proves can be used to deliver the culture media to the tissue sample. In some examples, the culture media can comprise the biological agents.

[00323] In some cases of the biological evaluation kit, the tissue sample can be cultured ex vivo. In some cases, the biological evaluation kit can further comprise an analytical device. In some examples, the analytical device can be used to measure the biological activity of the one or more biological agents.

[00324] While preferred cases of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such cases are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the cases of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[00325] Imaging System

[00326] In some cases, an MRI system is used to perform imaging and a gadolinium chelate to serve as the contrast generating mechanism. Gadolinium induces a local reduction in the longitudinal relaxation time constant (Tl) of tissue magnetization that can be measured by MRI (O'Connor et al. DCE-MRI biomarkers in the clinical evaluation of anti-angiogenic and vascualr disruption agents. Br. J. Cancer. 2007 Jan 29: 96(2): 189-195)). This method can be used to assess the relative concentration of the contrast agent and elimination rate, thereby obtaining a quantitative in vivo kinetic measurement of chemotherapeutic drug efficacy. Alternatives to MRI include use of a fluorescent probe with a fluorescent imaging system, or a radiolabeled probe with a radiographic system (e.g. PET). In a possible clinical scenario, the invention would be used to inject the patient one day prior to the start of chemotherapy. Then, imaging sessions shortly after injection and immediately prior to chemotherapy would establish the baseline rate of agent elimination. One or more imaging sessions in the hours or days after chemotherapy would then be used to measure the difference in agent elimination as a result of chemotherapy.

[00327] System for Software Facilitation

Another aspect of the invention provides a system that is configured to implement the methods of the disclosure. The system can include a computer server ("server") that is programmed to implement the methods described herein. FIG. 42 depicts a system 4200 adapted to enable a user to detect, analyze, and process images of cells. The system 4200 includes a central computer server 4201 that is programmed to implement exemplary methods described herein. The server 4201 includes a central processing unit (CPU, also "processor") 4205 which can be a single core processor, a multi core processor, or plurality of processors for parallel processing. The server 4201 also includes memory 4210 (e.g. random access memory, read-only memory, flash memory); electronic storage unit 4215 (e.g. hard disk); communications interface 4220 (e.g. network adaptor) for communicating with one or more other systems; and peripheral devices 4225 which may include cache, other memory, data storage, and/or electronic display adaptors. The memory 4210, storage unit 4215, interface 4220, and peripheral devices 4225 are in communication with the processor 4205 through a communications bus (solid lines), such as a motherboard.

[00328] The storage unit 4215 can be a data storage unit for storing data. The server 4201 is operatively coupled to a computer network ("network") 4230 with the aid of the

communications interface 4220. The network 4230 can be the Internet, an intranet and/or an extranet, an intranet and/or extranet that is in communication with the Internet, a

telecommunication or data network. The network 4230 in some cases, with the aid of the server 4201, can implement a peer-to-peer network, which may enable devices coupled to the server 4201 to behave as a client or a server. The microscope and micromanipulator can be peripheral devices 4225 or remote computer systems 4240. The storage unit 4215 can store files, such as individual images, time lapse images, data about individual cells, cell colonies, or any aspect of data associated with the invention. The data storage unit 4215 may be coupled with data relating to locations of cells in a virtual grid.

[00329] The server can communicate with one or more remote computer systems through the network 4230. The one or more remote computer systems may be, for example, personal computers, laptops, tablets, telephones, Smart phones, or personal digital assistants. [00330] In some situations the system 4200 includes a single server 4201. In other situations, the system includes multiple servers in communication with one another through an intranet, extranet and/or the Internet.

[00331] The server 4201 can be adapted to store cell profile information, such as, for example, cell size, morphology, shape, migratory ability, proliferative capacity, kinetic properties, and/or other information of potential relevance. Such information can be stored on the storage unit 4215 or the server 4201 and such data can be transmitted through a network.

[00332] Methods as described herein can be implemented by way of machine (or computer processor) executable code (or software) stored on an electronic storage location of the server 4201, such as, for example, on the memory 4210, or electronic storage unit 4215. During use, the code can be executed by the processor 4205. In some cases, the code can be retrieved from the storage unit 4215 and stored on the memory 4210 for ready access by the processor 4205. In some situations, the electronic storage unit 4215 can be precluded, and machine- executable instructions are stored on memory 4210. Alternatively, the code can be executed on a second computer system 4240.

[00333] Examples

[00334] While some cases of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such cases are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the cases of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[00335] Example 1: Self-assembling peptide hydrogels

[00336] The diffusion rate of an agent or elution profile at an injection site in an in vivo drug stimulus response assay can be slowed by using a self-assembling peptide hydrogel material. Self-assembling peptide hydrogel materials allow for modulation of the hydrogel matrix pore size from 5 nanometers to 200 nanometers by changing the self-assembling peptide concentration from 0.1% (w/v) to 4% (w/v). Hydrogel matrix pore size can be tuned by modifying the c-terminal sequence with hydrophobic amino acid residues that have slight affinity for anionic bile salt micelles, nonionic Solutol micelles, or cyclodextrin drug delivery systems. [00337] Self-assembling peptide hydrogels can be pre-labeled with a lysine substituted self-assembling peptide with a VivoTag-680 reactive ester. The Vivo-Tag 680 labeled self- assembling peptide can be detected by fluorescence microscopy at an injection site. The self- assembling peptides can be labeled with hapten constructs, including but not limited to, biotinylated hapten, dinitrophenol hapten, or digoxigenin hapten to allow for visualization of injection site localization. A lipophilic modified self-assembling peptide construct can deliver Abraxane or cyclodextrin 292.

[00338] A self-assembling peptide can be labeled with a TSP1 epitope. SiHa cells can be grown on the self-assembling peptide hydrogel. The self-assembling peptide can be imaged. The image can show the induction of extensive migration along cracks within the hydrogel (FIG. 7). Additionally, the SiHa cells can be labeled with DAPI, and the f-actin network of the cells can be labeled with Alexa Fluor 488 WGA and Alexa Fluor 568 phalloidin (FIG. 8). A 0.4% puramatrix self-assembling peptide can be dispensed by non- contact methods on epoxy-silane ES slides into patterns of cell adhesive lines separated by non-adhesive cell migration zones. The SiHa cells can be imaged at a 48 hour timepoint (FIG. 9). At this timepoint, the SiHa cells can show a rapid downregulation of tight junction on the TSP1 designer self-assembling peptides versus the puramatrix control lines.

Additionally, the TSP1 desiger self-assembling peptides can be imaged with a confocal imaging method. Imaging can be down with WGA-AF488 fluorescence channel pseudo intensity colormap at lOOx SiHa image. The self-assembling peptides can be visible on the hydrogel matrix as a filamentous network under attached cells (FIG. 10A). Additionally, the cells can be imaged at 100X confocal immunofluorescence staining of the SiHa cells. The images can show long nano fibers bridging two cells (FIG. 10B). Additionally, the images can show a dense web of positive cell membrane sialic receptors and golgi body sialic residues with a fiuorescently labeled lectin, WGA-AF488 (FIG. 11). Cells can also be counterstained with DAPI to show cell nuclei.

[00339] Example 2: Injection site visualization and maintenance of tumor orientation

[00340] An injection site can be imaged prior to injection. A tumor can be imaged using methods such as, but not limited to CT or PET/CT or ultrasound (FIG. 12A). The injection site can be imaged by using stains such as, but not limited to ICG (FIG. 12A), H&E (FIG. 13B), DAPI (FIG. 13C). The injection site can be imaged using markers for apoptosis, such as, but not limited to CC3 (FIG. 15C). The injection site can be imaged using markers for endothelial cells such as, but not limited to CD31 (FIG. 12C). The injection site can be imaged using a UV dye, such as, but not limited to SPOT ink (FIG. 18A). The UV dye can be visible by, but not limited to, black light (FIG. 21A), human eye (FIG. 21B), or ICG channel of IVIS imaging system (FIG. 21C).

[00341] Based on the image of the tumor, a guide needle can be inserted into the tumor tissue. A second image can be taken of the position of the guide needle. The location of the guide needle can be in an area that is cellular and distal to fat, large vessels, or necrotic tissue. Upon placement of the guide needle (FIG. 23), an alignment post can be pulled down over the guide needle (FIG. 24A). A microinjection device comprising a plurality of needles can be placed over the alignment post (FIG. 24B). One or more combination of agents can be injected into the tumor tissue through the use of the microinjector device (FIG. 24C and FIG. 25). One or more agents can be, but is not limited to chemotherapeutic agents. After injection, the microinjection device can be removed with the guide needle and alignment post remaining in the tumor tissue (FIG. 25E). The alignment post can then be removed (FIG. 26 A) followed by threading a guide marker through the guide needle (FIG. 26B). The guide marker can be an anchor suture or hookwire (FIGS. 27 and 28). The guide marker can be inserted into the tumor tissue by using a pushrod (FIG. 26C). The guide needle can be removed from the tumor tissue by pulling the guide needle up and over the pushrod (FIG. 26D). The pushrod can then be removed leaving the guide marker in place (FIG. 26E).

[00342] After injection, the response of the tumor to the injection of the agent can be visualized using a tracking dye such as but not limited to IDS (FIG. 15B), markers for apoptosis, including but not limited to CC3 (FIG. 15C), stains, including but not limited to H&E (FIG. 15D).

[00343] Example 3: Detection of microinjected contrast agent and subsequent washout following protease cleavage

[00344] A gadolinium contrast agent was injected into xenograft tumors in mice using a microinjection system. Immediately after injection, the focal points of agent accumulation were detected and measured at Tl map (20 minutes, FIG. 41). Subsequent imaging showed a rapid washout of the agent demonstrating the ability of cleaved contrast agent to rapidly diffuse out of the tumor Tl map (40 minutes, FIG. 41).

[00345] Example 4: Use of a device of the present disclosure

The CIVO drug microinjection platform [00346] The CIVO platform consists of a device that simultaneously delivers microgram quantities of test drugs into defined positions within a living tumor, coupled with automated, quantitative image-based analysis of specific tumor responses (FIG. 43). Drugs are co-injected with a chemically inert, visible injection tracking dye (ITD) to denote injection position (FIG. 43, A to C). Injections are performed so that a constant microliter volume of drug is delivered per millimeter extruded via retraction of each needle through the tumor. This design leaves a uniform, column-like track of drug through the z axis of the tumor (FIG. 43D), allowing investigators to sample multiple tissue depths to assess consistency of tumor response to drug. Furthermore, accounting for tumor heterogeneity observed in solid tumors, the drug column provides the potential to interrogate how different tumor microenvironments affect drug efficacy; for instance, normoxia versus hypoxia where the drop in a GLUT1 signal, at distances greater than 250 μιη from the nearest endothelial cell, was due to tumor necrosis, a consequence of poor vascularization indicated by CD31 staining (FIG. 50).

[00347] Responses to drugs were assessed after resection of the tumor at a

predetermined time point (typically 24 to 72 hours). Multiple biomarkers, including those for mechanism-based drug effects, target or pathway engagement, and apoptotic response, were tracked by sampling 4^m-thick histological sections at 2-mm intervals along the injection column (FIG. 43E). High-resolution whole-slide scanning was used to capture images of every cell from each tissue section (FIG. 43F), and these images were then quantitatively processed by a custom image analysis platform called CIVO Analyzer, which comprises automated injection site detection, cross section registration, and biomarker- specific tissue and cellular segmentation to streamline analysis of induced tumor response (FIG. 43, G and H)

Spatially defined drug distribution enables multidrug analysis in tumors

[00348] CIVO platform performance and drug distribution were first assessed in xenografted human tumor models using a panel of radiolabeled compounds of varied physicochemical properties, including molecular weight, lipophilicity, and protein binding. Drug distribution was assessed by ¹⁴ C or ³ H count distribution within 2 mm of the injection epicenter and at an adjacent site microinjected with saline. Three different xenograft models [Ramos lymphoma, H2122 non-small cell lung cancer (NSCLC), and H292 NSCLC] and three time points (15 min, 4 hours, and 24 hours) were evaluated. On average, more than 96% of each drug remained within the 2-mm radius around the targeted injection site and was not detected at adjacent sites (Table 1).

Table 1. Drug biodistribution after CIVO microinjection of radiolabeled compounds.

Drugs were microinjected at one site and saline controls were injected at adjacent sites within the same tumor using the CIVO device. Core biopsies of all sites were measured for radioactivity by scintillation counting from a minimum of nine replicates per condition.

Vd (1/kg, Drug remaining in 2 unless Protein- mm radius vs. adjacent

MS otherwise bound Half- saline control*(%)

Isotope Drug Formula (g/mol) indicated) (%) life Ramos H2122 H292

C14 Chlorambucil C ₁₄ H ₁₉ C1 ₂ N0 ₂ 304.22 0.14-0.24 99 1.5 h 94 97

H3 Cordycepin C ₁₀ H ₁₃ N ₅ O ₃ 251.20 97 98 97

H3 Didanosine C ₁₀ H ₁₂ N ₄ O ₃ 236.50 0.77 < 5 99 97 96

H3 ,y- C ₁₀ H ₁₃ N ₅ O ₂ 235.20 99 98

Dideoxyadenosine

H3 Flucortisone C ₂₄ H ₃₀ F ₂ O6 452.50 1.3-1.7 h 98 97

C14 Mitoxantrone C ₂₂ H ₂ §N ₄ 0 ₄ 444.49 1000 1/m ² 78 5.8 97

days

H3 Paclitaxel C ₄₇ H ₅₁ N0 ₁₄ 853.91 89-98 5.8 h 95

C14 Mercaptopurine C ₅ H ₆ N ₄ OS 170.19 0.59-0.9 5-37 20- 97

47

* Represents combined data from 15 minute (n = 3-5), 4 hour (n = 3-5), and 24 hour (n = 3-5) time points.

[00349] Further characterization of drug distribution after CIVO microinjection was performed with the antimitotic agent vincristine. Vincristine distribution was directly tracked by microinjection of H-labeled drug into discrete positions of xenografted Ramos lymphoma tumors. Tumors were resected at 2, 8, 24, 48, and 72 hours after injection, and fixed cryosections cut perpendicular to each injection column were subjected to autoradiography. The resulting autoradiograms revealed spatially defined (with a maximum radial extent of about 1500 μιη) and graded regions of drug distribution with respect to the injection site origin (FIG. 44A). Radioactivity, plotted as a function of distance from the injection, confirmed a monotonic drop in drug content with increasing radial distance. Within 400 gm of the injection epicenter, a rapid drop in drug content over time was observed (FIG. 44 A). At radial distances between 500 and 1500 μιη, the activity remained fairly stable over time, suggesting a steady-state drug concentration profile with diffusion into the zone balancing drug elimination, at least through 72 hours after injection. These data indicate that none of the drugs tested distributed beyond 2 mm of the injection epicenter. [00350] The distribution pattern was compared with histological biomarkers of tumor response to drug in parallel Ramos tumors microinjected with the same amount of unlabeled vincristine. All injections of vincristine resulted in spatially constrained regions of tumor response, with a leading edge of phospho-histone H3 (pHH3)-positive cells arrested in mitosis (500 to 1500 μιη) surrounding an inner core of apoptotic cleaved caspase-3 (CC3)- positive cells (0 to 900 μιη) emanating from the site of drug delivery (FIG. 44B); these findings are consistent with the known mechanism of action of vincristine. In contrast, sites microinjected with vehicle were pHH3- and CC3-negative but exhibited variable evidence of modest tissue damage directly adjacent (≤200 μιη) to the region where the needle passed through the tissue. The same distribution pattern of biomarker response to vincristine was observed upon microinjection into a patient-derived xenograft model of diffuse large B cell lymphoma (FIG. 51A).

[00351] The graded drug distribution and corresponding response allowed each injection site to be evaluated as a range of drug concentrations, where the drug gradient is a function of distance from the original site of localized drug delivery. Our data demonstrate an increasing magnitude of tumor response upon increasing drug (vincristine) input concentration at each microinjection site, observed as a dose-dependent increase in CC3 ⁺ cells when plotted against radial distance (FIG. 44C). Dose-dependent increases were also observed for the local pHH3 ⁺ cell population. However, in contrast to CC3, pHH3 staining revealed a parabolic profile with cells nearest to injection sites (<500 μιη) no longer expressing the pHH3 antigen. Histological analysis suggests that pHH3 loss is due to extensive cell death in this core region, because loss of pHH3 directly correlates with increased CC3 and the appearance of morphologically damaged cells in a dose-dependent manner.

[00352] These experiments were repeated with mafosfamide (an active surrogate for the pro-drug cyclophosphamide), doxorubicin, and prednisolone (an active surrogate for the pro-drug prednisone), because these represent or simulate components of the CHOP

[cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin (vincristine), prednisone] regimen, a first-line lymphoma therapy. All positions were co-injected with an ITD, except for doxorubicin, because the autofiuorescence from doxorubicin interferes with detection of the dye (FIG. 52). Similar to responses observed with vincristine, CIVO microinjection of either mafosfamide or doxorubicin resulted in dose-dependent, spatially defined regions of cellular response in Ramos lymphoma xenograft tumors (FIG. 45 A). Tumor responses were specific to the established mechanism of action of each drug as shown in FIG. 45B and quantified in FIG. 45C. Of the four agents tested, only vincristine resulted in pHH3 ⁺ cells, corresponding with its antimitotic activity (FIG. 45, B and C). Consistent with their DNA- damaging activity, both doxorubicin and mafosfamide resulted in phosphohistone H2AX (γΗ2ΑΧ) expression in cells adjacent to injection sites (FIG. 45, B and C). Mafosfamide, doxorubicin, and vincristine all induced a measurable apoptotic response (CC3 ⁺ ) above vehicle control by 72 hours.

[00353] In contrast to the other agents tested, prednisolone exposure did not result in overt tumor responses at the time points and the concentrations of drug examined (FIG. 45B). Consistent with a role as a chemo-enhancer in some lymphomas, co-injection of prednisolone with vincristine resulted in a modest but significantly enhanced drug-induced tumor cell apoptotic response across a range of drug concentrations as observed by plotting response as a function of radial distance from the injection site (FIG. 53).

Localized tumor response predict response to systemically delivered drugs

[00354] To access the clinical predictive capabilities of the CIVO platform, we investigated whether localized tumor response correlated with responses to systemic delivery of drug in mice. Because most novel drugs are first tested in patients who have failed first- line therapies, we extended our analysis to include a new drug resistant variant of the Ramos lymphoma line, called Res-Ramos, which is refractory to 500 nM doxorubicin in vitro likely due in part to increased activity of the P-glycoprotein (Pgp/MDRl/ABCBl) drug efflux pump (Table 2). Nude mice bearing either parental Ramos or Res-Ramos tumors were CIVO- injected or treated systemically with one of the four CHOP agents. Short-term (24 to 72 hours) CIVO responses were compared to longer-term (up to 29 days) systemic responses to drug. Similar to previous experiments, CIVO microinjection of doxorubicin, vincristine, and mafosfamide all resulted in localized tumor responses in the parental Ramos tumors, with vincristine inducing the greatest apoptotic effect and prednisolone having no effect (FIG.

46A).

Table 2. Drug efflux pump expression in Ramos and Res-Ramos cells.

Accession Mean centered intensity (log ₂ ) Fold Change in Target ID number Ramos Res-Ramos Res-Ramos cells

ABCB1 (MDR1) DR000893 ^■ 2.569681 1.6603004 18.76511726 ABCB1 (MDR1) DR000893 ^■ 1.5897073 1.1753292 6.797651971 ABCB9 (MDR9) DR003131 ^. 2.4468904 0.14986765 4.914425417 ABCB9 (MDR9) DR003131 ^■ 2.1294227 0.18619162 3.845659633 ABCG1 DN996555 ^■ 1.0038419 -0.25202337 1.683914082 ABCG1 DN996555 -0.8154632 -0.17683513 1.556847972 ABCB4 (MDR4) NG 007118 -0.8673328 -0.3114056 1.470113151

[00355] Tumor cell response after CIVO microinjection mirrored tumor growth inhibition after systemic treatment of Ramos tumors (FIG. 46B). CIVO was also able to predict response in a tumor context-specific manner (both resistance and increased sensitivity). In our model of drug resistance, Res-Ramos tumors exhibited the expected reduced response to microinjection of doxorubicin, as well as to vincristine— also a Pgp substrate (FIG. 46, A and B). Failure to induce a localized response was mirrored by lack of tumor growth inhibition after systemic exposure to vincristine, doxorubicin, or prednisone (FIG. 46B).

[00356] In contrast to the other drugs, CIVO microinjection of mafosfamide induced significantly greater cell death responses in the Res-Ramos tumor model (FIG. 46A), which was confirmed in vivo after systemic cyclophosphamide administration (FIG. 46B). Res- Ramos mice treated with cyclophosphamide also demonstrated significant improvement in overall survival compared with Ramos mice treated with the same drug (FIG. 46B). At the end of the study, 70% of the Res-Ramos mice treated with cyclophosphamide were tumor- free (average tumor volume,≤60 mm ) compared with only 15% of the Ramos mice, in agreement with the prediction drawn from CIVO microinjections in FIG. 46 A.

[00357] Cell-based proliferation assays were also performed to assess the correlation between in vitro and in vivo responses to drug treatment. Drug sensitivity of the parental Ramos line in vitro approximated in vivo responses but did not register sensitivity to mafosfamide and falsely indicated sensitivity to prednisolone (FIG. 46C). Res-Ramos cells exhibited resistance to doxorubicin and vincristine in vitro (FIG. 46C), which was expected. However, in contrast, the increased sensitivity of Res-Ramos cells to mafosfamide, observed by both in vivo approaches (CIVO and systemic therapy), was not seen in the cell-based in vitro assay (FIG. 46C).

In vivo screening with CIVO indicates chemoresistant lymphoma sensitivity to mammalian target of rapamycin inhibitors

[00358] To expand on the CHOP results and to identify candidate agents to treat chemoresistant lymphoma, we performed a pilot CIVO-based screen in the Res-Ramos tumors using the 97-compound Developmental Therapeutics Program (DTP) version III set of approved oncology drugs from the National Cancer Institute (NCI). In vivo assessment of all 97 drugs was completed within 2 weeks from the initiation of the screen by microinjecting 8 compounds per tumor into replicate Res-Ramos xenografts and resecting and staining at 72 hours (FIG. 54A). Of the 202 injection sites (replicate DTP set plus vehicle controls), 195 resulted in clear detection of localized injection site dye (96.5% success rate).

[00359] Of the 97 drugs tested, 5 resulted in a measurable increase in apoptotic cells

(>15% CC3 ⁺ ) around the site of injection (Table 3). One of the five agents was the mammalian target of rapamycin (mTOR) complex 1 (mTORCl) inhibitor rapamycin, suggesting a dependence of Res-Ramos tumors on active mTORCl activity for survival (FIG. 54B). To confirm that rapamycin was exerting its expected mechanism of action, down-regulation of the mTOR pathway, we microinjected Res-Ramos tumors with rapamycin and resected them for examination of mTOR pathway status after 2 hours of localized (CIVO-mediated) drug exposure. Local exposure to rapamycin led to a marked decrease in phosphorylation of the mTORCl substrate eIF4E-binding protein 1 (4EBP1) (p4EBPl) (FIG. 54C). Pathway inhibition occurred before observable increase in apoptosis (CC3) or morphological changes indicative of cell death. This suggests that mTOR pathway inhibition precedes rapamycin-induced apoptosis. These results also suggest that use of CrVO is not limited to classic cytotoxic chemotherapy agents but can be used to evaluate inhibitors targeting specific pro-oncogenic pathways.

Table 3. The top five CIVO drug-screening hits were determined using CC3.

The total fraction of CC3-positive cells within 1200 mm from the injection site for each drug was quantified using CIVO Analyzer. The percentage of cells positive for CC3 are from the total number of cells within the same 1200 mm radius. A threshold of >15% total positive cells for CC3 was used to determine the top hits for CIVO-injected drugs in Res-Ramos tumors.

Drug name Mechanism of action CC3-positive cells (%)

Dactinomycin Noncovalent DNA binding 44.91

Bendamustine HC1 Alkylating agent 35.98

Plicamycin Noncovalent DNA binding 33.25

Rapamycin mTOR inhibitor 24.46

Vorinostat Histone deacetylase inhibitor 17.41

[00360] Two additional mTOR inhibitors— ridaforolimus (mTORCl inhibitor) and

CC-115 [inhibitor of mTORCl, mTORC2, and DNA-PK (DNA-protein kinase);

ClinicalTrials.gov: NCT01353625]— were microinjected into Ramos and Res-Ramos tumors, and p4EBPl and CC3 staining was analyzed. Both ridaforolimus and CC-115 inhibited the mTOR pathway in the Ramos and Res-Ramos tumors (FIG. 47A). Like rapamycin, ridaforolimus induced apoptosis central to the region of mTOR pathway inhibition, and this activity was similar in both parental and Res-Ramos models. Whereas ridaforolimus exhibited no cell line preference, CC-115 activity was significantly increased in the Res-Ramos background within 24 hours (FIG. 47, A and B). Similar to the case of mafosfamide (FIG. 46), this apparent cell line-dependent activity was not detected in a standard 72-hour in vitro cell-based viability assay (FIG. 47C).

[00361] In vivo, CC-115-treated parental Ramos xenografts were overtly smaller than vehicle controls but continued to grow through the course of the experiment. In contrast, systemic delivery of CC-115 resulted in complete regression of Res-Ramos xenograft tumors (FIG. 47, D and E), consistent with localized apoptosis observed upon CIVO microinjection (FIG. 47, A and B). Regardless of the mechanism, these results emphasize the importance of efficacy testing in vivo, because the context-specific effect of CC-115 was not detected in a cell-based in vitro assay.

Initial translation of CIVO into cancer patients

[00362] As a first step to establish feasibility for use of CIVO in vivo in humans, we initiated a trial in lymphoma patients with a prototype device for arrayed microinjection. The primary goals of this trial were to assess the physician and patient experience during the procedure, to establish the choreography of using CIVO technology in the clinical setting, and to learn what aspects of the technology and methodology need to be improved. Four patients with enlarged lymph nodes were injected with microdoses of vincristine (1.5 μg) using the CIVO prototype (FIG. 48A). Although clear cell death was observed in cells surrounding the vincristine injection (FIG. 48B), the ITD indocyanine green (ICG) commonly used in human studies was difficult to detect (displayed as a separate image panel from the CC3 stain in FIG. 48C) owing to the loss of ICG signal during tissue processing. Technology modifications, including an improved method of tracking injection sites, were implemented on the basis of what was learned from pilot testing in humans.

[00363] Early data regarding patient and physician experiences were encouraging. No grade 2, 3, or 4 adverse events were reported. All patients experienced transient grade 1 events, such as mild erythema and swelling, that resolved without intervention. An external review committee evaluated the interim results and raised no questions or concerns. On a pain scale of 0 to 10, with 0 being no pain and 10 being the worst possible pain, three patients reported 0.5, and one patient reported 3 (mild discomfort) (Table 4). Table 4. Patients' pain experience.

Pain was rated on a standard Mosby 0-10 scale, with 0 being no pain and 10 being the worst possible pain. Patient #4 did not respond to a request for follow-up after lymph node removal.

Patient ID #1 #2 #3 #4

Pain at microinjection 0.5 0 0.5 3

Pain after lymph node removal 2 2 0 N/A

[00364] Follow-up patient interviews revealed outstanding patient satisfaction, with one suggestion to improve the experience (Table 5). In response to feedback from the various stakeholders involved in the clinical procedure (oncologist, pharmacist, patient, surgeon, and pathologist), the prototype device was reengineered as follows: (i) inclusion of ultrasound-based guide needle placement for optimized targeting of CIVO microinjections to cancerous tissue, (ii) improved materials and associated methods for retaining tumor orientation and injection site identification after tumor injection, and (iii) increased needle density to allow more drug samples to be investigated per patient.

Table 5. Stakeholders' input on the early CIVO prototype.

CIVO device

user/stakeholder Input on early prototype

Oncologist Device was easy to insert through the skin and entire microinjecting drugs procedure was of short duration.

into tumor Device was easy to use after minimal training and the accompanied manual was a good reference.

Patients did not show signs of discomfort during injection.

Suggestion of improving the targeting of the ideal site of injection in the tumor using ultrasonography, rather than simply measuring tumor size and depth prior to injection.

Pharmacist loading The device was easy to load.

CIVO device Device loading procedure was simple enough to perform within a reasonable time period.

Suggestion of color coding the various needle positions to minimize drug swapping errors.

Suggestion of improving loading procedure to only require aseptic technique.

Surgeon removing It was sometimes difficult to identify the exact site injected tumor and orientation of the injection site. • Suggestion of improving internal and external

marking methodology of injection site.

Pathologist handling • It was sometimes difficult to retain orientation of the resected tumor injection sites without additional marks incorporated by

surgeon.

• Suggestion of improving on external marking

methodology to speed up the sample sectioning process.

Patient's comments at • Suggestion to improve patient experience by microinjection avoiding showing the multiple needles to the patient prior to injection

Post-pilot CIVO modifications result in performance improvements in canine patients with lymphoma

[00365] CIVO technology modifications made after pilot testing in humans were applied in canine patients with spontaneous lymphoma in a clinical setting. Anesthetized dogs were subjected to CIVO microinjection of vincristine along with a fiuorescent tracking dye (FIG. 49, A to D). Ultrasound-based image guidance, the use of a guide needle combined with modified needle positioning, and use of the improved ITD increased injection success in the clinic from 42% (n = 20 observed/48 total injection sites) before modifications, to >93% (n = 15 observed/16 total injection sites) in lymphoma tumors. The responses to vincristine in native tumors, as detected by both pHH3 and CC3 staining around easy-to- identify injection sites, were robust and reproducible in two canine patients, with a radial CC3 response similar in extent to that observed in xenograft tumors in mice (FIG. 49E). No toxicity was observed according to the Veterinary Cooperative Oncology Group standards. These clinical studies in both canine and human patients demonstrate that tumor response to drug can be tested in a toxicity-sparing, localized manner in vivo with CIVO technology. DISCUSSION

[00366] We engineered a platform technology called CIVO that enables safe, ethical, and efficient (multiplexed) testing of cancer drugs directly in human tumors. The CIVO technology intentionally bypasses bioavailability, biodistribution, metabolism, and excretion issues associated with systemic dosing, making it possible to focus to on whether a drug engages its target, how cancer cells respond to target engagement, and whether the ultimate fate of the exposed cells indicates potential for patient therapeutic response— all in the context of an actual tumor micro-environment.

[00367] Crossing the threshold into human studies required initial studies in the controlled environment of preclinical xenograft models of cancer. With an optimized device and protocol in place, we determined whether the short-term (24 to 72 hours) localized responses induced by CIVO predicted tumor response to systemic administration of the same drugs. The pair-matched set of Ramos and Res-Ramos (resistant) lymphoma tumors provided a simple model to test this correlation. Res-Ramos tumors were resistant to doxorubicin and had up-regulated Pgp drug efflux pumps; thus, the observed lack of responses to localized CIVO microinjection of doxorubicin and vincristine was expected and served as fundamental demonstrations that our technology could detect resistance to cytotoxic drugs. We unexpectedly observed sensitivity of the Res-Ramos tumors to microinjection of mafosfamide, which was subsequently confirmed in vivo with systemically administered cyclophosphamide; standard in vitro analysis in cell culture, however, failed to reveal this sensitivity, indicating the importance of localized in vivo testing of drugs for more accurate systemic therapeutic predictions.

[00368] CC-115 was identified as a new anticancer agent for Res-Ramos tumors after an in vivo CIVO screen of 97 compounds. CC-115 indeed was able to shrink tumors in vivo in animals with the resistant tumors, further demonstrating the use of CIVO in identifying drugs that are effective in tumors already resistant to most therapies. Furthermore, in vitro cell-based proliferation assays did not detect the sensitivity to CC-115.

[00369] In our study, CIVO was translated to the clinic for preliminary feasibility testing in four patients. None of the patients who received CIVO microinjections have exhibited adverse effects to date beyond mild grade 1 (transient erythema). From that pilot study, we further optimized the CIVO device and tested in canine patients. Similar to the responses observed in largely homogeneous lymphoma xenograft tumors in mice, we noted robust drug (vincristine)-specific cellular responses in heterogeneous, autochthonous lymphoma tumors. Although these results are preliminary, they support our expectation that CIVO will ultimately enable early drug discovery and treatment decisions in the clinic.

[00370] Ultimately, determination of whether a cancer drug can be commercialized depends on demonstrating activity in human patients. CIVO technology has the potential for testing experimental drugs directly in a patient with their own intact tumor

microenvironment, their own immune system, and unique oncogenomic profile without the toxicities associated with typical clinical exposures. In combination with traditional preclinical toxicology studies, drug developers may then focus resources on the agents that demonstrate superior efficacy in the context of the true human disease before advancement to conventional clinical trials. The quantities of drug required to induce localized tumor responses, both for cytotoxic chemotherapy agents and for targeted inhibitors represented here by the mTOR inhibitors, all fall under established FDA phase 0 guidelines for microdosing studies. CIVO technology will further complement genomic medicine.