PATIENT-DERIVED CTC-XENOGRAFT MODELS

FIELD OF THE INVENTION

The invention relates to oncology and medicine.

BACKGROUND

All publications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant and has the lowest survival of any human cancer. Extensive metastasis and therapeutic resistance are the two major contributors to its dismal prognosis. The mechanisms by which PDAC cells successfully spread and metastasize are largely unknown, and molecular events underlying its resistance to therapeutics remain undefined.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

Figures 1A-1C depicts, in accordance with various embodiments of the invention, an example of CTC induced R&R. FIG 1 A) organoid aggregate growth of CTC847 (200x), an ex vivo expanded CTC culture as 3-D spheroids from a prostate cancer patient blood sample. FIG IB) upper row, Representative H&E images of prostate CTC induced subcutaneous and intraosseous tumors and kideney metastasis (400x magnification). H&E assay of tumorigenicity and metastatic potential of CTC847 was determined by CTC-PDX tumor formation via s.c. (left) and intra-femoral (middle) inoculation. The bone tumor caused many secondary metastases in many organs including the kidney (right panel). Bottom row, Representative images of IHC staining of prostate cancer specific markers, PSA, AR, and PSMA in prostate CTC subcutaneous tumors (400x magnification). CTC-PDX tumors expressed prostate biomarkers, including prostate specific membrane antigen (PSMA), androgen receptor (AR) and prostate specific antigen (PSA). FIG 1C) Ex vivo cultured CTCs containing metastasis-initiating cell (MIC) properties (MIC-CTCs) transformed and reprogrammed non-tumorigenic prostate epithelial cells, DC-1, derived from primary prostate tumor to express MIC genes examined by qRT-PCR. DC-1 cells were co-cultured with PCa CTCs in 3-D suspension co-culture for 4 days before DC-1 cells were isolated from the co- culture by FACS analysis and antibiotic selection. DC-1 is a non-tumorigenic "dormant" epithelial cell line from a prostate tumor specimen. After co-culture with CTC847 in 3-D in mixture for 5 passages, DC-1 became tumorigenic in athymic mouse (not shown) and started to express elevated prostate cancer biomarkers indicative of EMT, sternness and neuroendocrine differentiation as detected by qRT-PCR.

Figure 2 depicts, in accordance with various embodiments of the invention, schematic diagram of the study for biomarkers of PDAC metastasis and therapeutic resistance.

Figure 3 depicts, in accordance with various embodiments of the invention, gene fusion nomination for the discovery of driver gene fusions in individual samples. We previously identified ETV1 and ERG gene fusions as driver mutations in certain cancers positive for ETS gene rearrangement (Palanisamy N., et al. Nat Med 2010; 16(7):793-798). We used a similar approach in ETS negative prostate cancer to identify SLC45A3-BRAF (top) and ESRPl-RAFl (bottom) and RAFl-ESRPl (not shown) as driver gene fusions in another subset of prostate cancers.

Figures 4A-4D depicts, in accordance with various embodiments of the invention, ex vivo CTC expansion as 3-D spheroids and the CTC-PDX model. Representative results are shown. FIG 4A) A matched pair of normal pancreatic tissue and a PaCa tumor specimens were cultured ex vivo for 14 days. Appearance of CTC-like cells in PaCa tumor culture was recorded. FIG 4B) A packed blood sample from the same PaCa patient was subjected to ammonium chloride hemolysis to isolate peripheral blood mononuclear cells (PBMCs), which were subjected to ex vivo culture as 3-D spheroids (1 ^χ 10 ⁶ /ml) for 8 weeks with a unique protocol developed in our laboratory. Compared to healthy donor samples (upper row), patient blood culture led to large aggregated growth, reminiscent of spheroid/organoid proliferation with immortality (> 15% samples). FIG 4C) Pancreatic mesenchymal stromal/stellate cells in the tumor microenvironment promote CTC growth and survival. Conditioned medium (CM) from a culture of the cancer-associated pancreatic mesenchymal cells was used (at 1 :4 dilution) to treat a CTC culture. Growth rate of the cells were determined by counting the cells with an automatic cell counter. FIG 4D) Establishment of CTC-PDX model. Expanded CTCs (2.5 ^χ 10 ⁶ /site) from two PaCa patients were used in subcutaneous (s.c.) inoculation to 6 - 8 week-old male NSG mice (n = 5). Tumor formation- induced animal death was documented and demonstrated by Kaplan-Meier survival curve. These CTCs also formed tumors and metastasis following intra-femoral (i.f.) or orthotopic pancreas (o.t.) inoculation.

Figures 5A-5D depicts, in accordance with various embodiments of the invention, characterization of the CTC-PDX model. Characteristics of the CTC-PDX-752 tumor formation and metastasis are shown. FIG 5A) The CTC-PDX model was established by inoculating ex vivo expanded CTCs as 3-D spheroids (2>< 10 ⁶ /site) to NSG mice via different routes to induce rapid PDX tumor formation. Both subcutaneous and intra-femoral (i.f.) inoculation resulted in multiple organ metastases and rapid animal death (< 80 days). Following i.f. inoculation, the CTCs formed bone tumors, which caused soft tissue metastasis in kidneys and spleen. FIG 5B) Demonstrated by microCT scan, the CTC bone tumor growth resulted in osteolytic lesion. FIG 5C) Examined with H&E stain, the CTC-PDX tumors bore similar histopatological features of PaCa (in situ PDAC patient tumor - 400 ^χ ). Intra-femoral tumor growth caused osteolytic lesion. FIG 5D) The s.c. CTC-PDX tumors displayed biomarker expression similar to clinical PaCa, as determined by immunohistopathologic stain (200x). Similarity between patient PDAC and CTC-PDX tumors validated with a PDAC panel of biomarkers at the Department of Pathology, VAGLA. CTC-PDX tumors show a typical CK7+/CK20-/panCK+/CA19-9+ stain pattern (200x).

Figure 6 depicts, in accordance with various embodiments of the invention, using CTC-PDX model to investigate PaCa progression and metastasis. CTCs were dually tagged with luciferase and green fluorescence protein (GFP) reporters and were inoculated o.t to mouse pancreas head (2 ^χ 10 ⁵ /site), to track CTC-PDX growth and metastasis by bioluminescence imaging. Subsequently, the CTC-PDX tumors were cultured ex vivo for detailed study of cancer-stromal interaction, and to identify participant resident stromal cells of the tumor microenvironment, with GFP as marker of cancer cells (not shown). Results of BLI tracking are shown at 1 day, 3 weeks and 9 weeks after orthotopic pancreas inoculation.

Figures 7A-7B depicts, in accordance with various embodiments of the invention, effects of anti-tumor agents in PaCa models. As a preliminary inquiry, we tested anti-PaCa activity of a near infrared ( R) IR-783-gemcitabine conjugate in human pancreatic MiaPaCa II and BX-PC3 tumor cell models. FIG 7A) In a PaCa prevention study, mice (n=5) were treated with intraperitoneal IR-783-gemcitabine immediately after MiaPaCa II inoculation, s.c. tumor volumes (left), NIR fluorescence imaging (middle), and mouse survival (right) were recorded. FIG 7B) In a PaCa inhibition study, IR-783-gemcitabine was given 10 weeks after tumor cell inoculation. IR-783-gemcitabine markedly inhibited MiaPaCa II tumor growth, and improved dramatically animal survival.

Figures 8A-8H depicts, in accordance with various embodiments of the invention, metastasis-initiating cell (MIC) signatures and functions in CTCs from human patients. FIG 8A) Morphology of CTCs. FIG 8B) CTC protein expression detected by the mQDL method. FIG 8C) In vitro expanded human CTCs consist of both MIC and non-MIC cells. FIG 8D) Percent MICs in CTCs. FIG 8E) Cultured CTCs as 3-D spheroids from prostate, pancreas, and kidney cancers express MIC markers. Cultured CTCs of prostate, pancreas, and kidney cancers express MIC markers with elevated EMT, neuroendocrine (NE), and stem cell markers detected by western blot analysis. FIG 8F) MIC marker expression of longitudinal PCa CTC samples from one of the CRPC patients before and after therapeutic interventions detected by western blot analysis. Ex vivo cultured CTCs as 3-D spheroids express EMT, stem and NE phenotype. FIG 8G) Ex vivo cultured CTCs containing MIC properties (MIC- CTCs) transformed and reprogrammed non-tumorigenic prostate epithelial cells, DC-1, derived from primary prostate tumor to express MIC proteins examined by western blot analysis. DC-1 cells were co-cultured with PCa CTCs in 3-D suspension co-culture for 4 days before DC-1 cells were isolated from the co-culture by FACS analysis and antibiotic selection. CTCs with MIC phenotype reprogram indolent DC-1 cells. FIG 8H) MIC-CTCs also up-regulates CK13 expression in DC-1 cells during the reprogramming process.

Figures 9A-9F depicts, in accordance with various embodiments of the invention, ex vivo cultured human CTCs from PC patients form tumors and metastases in mice. FIG 9A) Representative image of prostate CTC-induced intrafemoral tumors and kidney metastases. FIG 9B) Representative microCT scan of prostate CTC bone tumors with osteolytic lesions. FIG 9C) Kaplan-Meier survival curves of mice bearing prostate cancer CTCs. FIG 9D) Kaplan-Meier survival curves of mice bearing pancreatic, renal, and breast cancer CTCs. FIG 9E) Representative bioluminescence (BLI) images of mice bearing prostate orthotopic CTC- derived xenograph (CDX) tumors and metastases. PCa CTCs (847) were tagged with luciferase and implanted into the prostate of the NSG mice, which were monitored for tumor formation by BLI imaging once a week. FIG 9F) Representative bioluminescence images of mice bearing luciferase-tagged pancreatic CTC (752) metastases via intracardiac inoculation.

Figure 10 depicts, in accordance with various embodiments of the invention, that cultured CTCs induced in vitro osteoclastogenesis of mouse osteoclast precursors upon co- culture. Representative images of mature osteoclasts induced by cultured CTCs and stained with Tartrate-resistant acid phosphatase (TRAP). (200x magnification). Graph depicts the quantification of mature osteoclasts (nuclei > 3) induced by cultured CTCs of prostate and pancreas cancer.

Figures 11A-11B depicts, in accordance with various embodiments of the invention, STR DNA fingerprint authentication of a CTC line (CTC-752-S) established from a PDAC patient blood sample. FIG 11 A) Identical polymorphic alleles are detected between CD45+ PBMCs from the same patient and FIG 11B) cells of the newly established CTC-752-S line. An additional allele appearing in the D19S433 locus (lower right panel) of the CTC-782-S is suggestive of genomic instability, a common observation in malignant cells and not affecting the authentication.

Figures 12 depicts, in accordance with various embodiments of the invention, representative IHC staining of CK14 and CK13 in ex vivo expanded CTC organoids of pancreatic and prostate cancer (top panels, 400x magnification) and representative IHC staining of CK13 in pancreatic and prostate cancer-derived CDX tumors (lower panels, 200x magnification).

Figure 13 depicts, in accordance with various embodiments of the invention, a time course of CTC culture morphology.

Figure 14 depicts, in accordance with various embodiments of the invention, detection of the epithelial marker EpCAM on expanded CTCs by flow cytometry.

Figure 15 depicts, in accordance with various embodiments of the invention, CTC counts in primary prostate cancer patients and castration-resistant prostate cancer cases. Results obtained from culturing l x lO ⁷ peripheral blood mononucleated cells in defined medium for 21 days.

SUMMARY OF THE INVENTION

Various embodiments of the present invention provide for a method of establishing a personalized model for a cancer, comprising: isolating circulating tumor cells (CTCs) from a biological sample from a subject with the cancer; expanding the CTCs as 3-D spheroids ex vivo; inoculating the ex vivo expanded CTCs into a non-human animal, thereby generating a patient-derived xenograft non-human animal; and establishing the patient-derived xenograft non-human animal as the personalized model for cancer detection and therpy.

In various embodiments, the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In various other embodiments, pancreatic cancer can be pancreatic ductal adenocarcinoma. In yet other embodiments, the biological sample can be a liquid biopsy or tissue biopsy from the subject with cancer. In some other embodiments, a liquid biopsy can be a blood sample from the subject with cancer. In various other embodiments, the biological sample can be obtained from the subject with cancer before, during, and/or after therapeutic treatment. In some embodiments, CTCs may be isolated from the blood sample by separating the blood cells and plasma to obtain packed nucleated blood cells. In other embodiments, the packed blood cells may be lysed using ammonium chloride hemolysis to obtain a cell pellet comprising CTCs. In various embodiments, the cells from the cell pellet may be cultured in a defined medium to form spherical organoid aggregates. In various other embodiments, expanding CTCs ex vivo can result from culturing cells from the biological sample to form spherical organoid aggregates. In various embodiments, the expanded CTCs can be inoculated orthotopically, intrafemorally or intraosseosly, intracardiac, or subcutaneously. In some embodiments, the non-human animal can be a rodent, mouse, rat, rabbit or guinea pig.

In various embodiments, the method further comprises tagging the CTCs with a lentiviral reporter construct that expresses green fluorescent protein, red fluorescent protein, luciferase, or a combination thereof. In some embodiments, bioluminescence imaging can be used to monitor and track the tagged CTCs in the tumor.

In various embodiments, the method further comprises co-innoculating PaSCs with the expanded CTCs into a non-human animal. In various embodiments, MIC-CTCs are inoculated into a non-human animal.

Various embodiments of the present invention provide for a method of identifying one or more drugs to treat a cancer, comprising: providing a personalized model for a cancer, wherein the model is the personalized model established herein; administering one or more drugs to the model; and detecting a therapeutic response in the model and identifying the drug as being therapeutically effective to the cancer, or detecting no therapeutic response in the model and identifying the drug as being therapeutically ineffective to the cancer. In various embodiments, the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In various other embodiments, pancreatic cancer can be pancreatic ductal adenocarcinoma.

In some embodiments, the therapeutic response can be inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited cancer cell invasion, inhibited cancer cell mobility, inhibited cancer cell differentiation, promoted cancer cell death, inhibited cancer progression, inhibited cancer metastasis, or improved animal survival, or a combination thereof. In yet other embodiments, the method further comprises instructing the subject with cancer to receive the one or more drugs identified to treat the subject's cancer.

Various embodiments of the present invention provide for a method of identifying a subject that has cancer, as having resistance or not to a drug, comprising: providing a personalized model for a cancer; administering one or more drugs to the model; and detecting resistance in the model and identifying the subject as having resistance to the drug, or detecting no resistance in the model and identifying the subject as having no resistance to the drug.

In various embodiments, the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In some embodiments, pancreatic cancer can be pancreatic ductal adenocarcinoma.

In various embodiments, the personalized model for cancer can be the model established herein. In some embodiments, resistance to a drug can be detected when there is uninhibited cancer cell proliferation, uninhibited cancer cell growth, uninhibited cancer cell invasion, uninhibited cancer cell mobility, uninhibited cancer cell differentiation, diminished cancer cell death, uninhibited cancer progression, uninhibited cancer metastasis, a decline in animal survival, or a combination thereof. In other embodiments, no resistance to a drug can be detected when there is inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited cancer cell invasion, inhibited cancer cell mobility, inhibited cancer cell differentiation, inhibited cancer cell death, inhibited cancer progression, inhibited cancer metastasis, improved animal survival, or a combination thereof.

Various embodiments of the present invention provide for a method of identifying MIC-CTCs in a sample from a subject, comprising: obtaining a biological sample from a subject; assaying for metastasis-initiating cell - circulating tumor cells (MIC-CTCs), and identifying MIC-CTCs in a sample when mesenchymal, stem, neuroendocrine markers, or a combination thereof, are detected.

In various embodiments, the cancer is pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In various other embodiments, pancreatic cancer is pancreatic ductal adenocarcinoma.

In various embodiments, the mesenchymal, stem and neuroendocrine markers comprise RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDH1,SDF1, PR1, Lin28b, and/or SYP.

In yet other embodiments, the biological sample is a liquid biopsy or tissue biopsy from the subject.

In various embodiments, the mesenchymal, stem, neuroendocrine markers are assayed by mQDL.

In various other embodiments, identifying MIC-CTCs in the biological sample obtained from a subject, establishes the subject has cancer.

Various embodiments of the present invention provide for a method of establishing a model for a cancer, comprising: isolating circulating tumor cells (CTCs) from a subject with the cancer; expanding the CTCs as 3-D spheroids ex vivo, thereby establishing the ex vivo expanded CTCs as the model for the cancer.

Various embodiments of the present invention provide for a composition, comprising circulating tumor cells (CTCs) isolated from a subject with a cancer. In various embodiments, the cancer is pancreatic cancer or prostate cancer. In various embodiments, the CTCs are isolated from a biological sample from the subject. In some embodiments, the biological sample is a liquid biopsy of the cancer. In various other embodiments, the CTCs are expanded ex vivo. In yet other embodiments, the CTCs are inoculated into a non-human animal.

Various embodiments of the present invention provide for a non-human animal inoculated with circulating tumor cells (CTCs) isolated from a subject with a cancer. In various embodiments, the CTCs are inoculated subcutaneously, intrafemorally, orthotopically, or intraosseously, or a combination thereof. In various embodiments, the non- human animal is a rodent, mouse, rat, rabbit or guinea pig. DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, NY 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor NY, 2013); Kohler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 Jul, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U. S. Patent No. 5,585,089 (1996 Dec); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. Indeed, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The definitions and terminology used herein are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims.

As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

Unless stated otherwise, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

As used herein, the terms "treat," "treatment," "treating," or "amelioration" when used in reference to a disease, disorder or medical condition, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, reverse, alleviate, ameliorate, inhibit, lessen, slow down or stop the progression or severity of a symptom or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease, disorder or medical condition is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Also, "treatment" may mean to pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.

"Beneficial results" or "desired results" may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition, decreasing morbidity and mortality, and prolonging a patient's life or life expectancy. As non-limiting examples, "beneficial results" or "desired results" may be alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of pancreatic cancer, delay or slowing of pancreatic cancer, and amelioration or palliation of symptoms associated with pancreatic cancer.

"Diseases", "conditions" and "disease conditions," as used herein may include, but are in no way limited to any form of malignant neoplastic cell proliferative disorders or diseases. Examples of such disorders include but are not limited to cancer and tumor.

A "cancer" or "tumor" as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems, and/or all neoplastic cell growth and proliferation. A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are dormant tumors or micrometastasis. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. As used herein, the term "invasive" refers to the ability to infiltrate and destroy surrounding tissue. Melanoma is an invasive form of skin tumor. As used herein, the term "carcinoma" refers to a cancer arising from epithelial cells. Examples of cancer include, but are not limited to, nervous system tumor, brain tumor, nerve sheath tumor, breast cancer, colorectal cancer, colon cancer, rectal cancer, bowel cancer, carcinoma, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen- independent prostate cancer. Examples of brain tumor include, but are not limited to, benign brain tumor, malignant brain tumor, primary brain tumor, secondary brain tumor, metastatic brain tumor, glioma, glioblastoma, glioblastoma multiforme (GBM), medulloblastoma, ependymoma, astrocytoma, pilocytic astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma, mixed glioma such as oligoastrocytoma, low-grade glioma, high-grade glioma, supratentorial glioma, infratentorial glioma, pontine glioma, meningioma, pituitary adenoma, and nerve sheath tumor. Nervous system tumor or nervous system neoplasm refers to any tumor affecting the nervous system. A nervous system tumor can be a tumor in the central nervous system (CNS), in the peripheral nervous system (PNS), or in both CNS and PNS. Examples of nervous system tumor include but are not limited to brain tumor, nerve sheath tumor, and optic nerve glioma.

As used herein, the term "administering," refers to the placement of an agent, composition and/or drug into a subject by a method or route which results in at least partial localization of the agents or composition at a desired site. "Route of administration" may refer to any administration pathway known in the art, including but not limited to oral, topical, aerosol, nasal, via inhalation, anal, intra-anal, peri-anal, transmucosal, transdermal, parenteral, enteral, or local. "Parenteral" refers to a route of administration that is generally associated with injection, including intratumoral, intracranial, intraventricular, intrathecal, epidural, intradural, intraorbital, infusion, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravascular, intravenous, intraarterial, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the agent or composition may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the enteral route, the agent or composition can be in the form of capsules, gel capsules, tablets, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the topical route, the agent or composition can be in the form of aerosol, lotion, cream, gel, ointment, suspensions, solutions or emulsions. In an embodiment, agent or composition may be provided in a powder form and mixed with a liquid, such as water, to form a beverage. In accordance with the present invention, "administering" can be self-administering. For example, it is considered as "administering" that a subject consumes a composition as disclosed herein.

The term "biological sample" or "sample" or "liquid biopsy" as used herein denotes a sample taken or isolated from a biological organism, e.g., a tumor sample from a subject. Exemplary biological samples include, but are not limited to, cheek swab; mucus; whole blood, blood, serum; plasma; bone marrow aspirate, urine; saliva; semen; lymph; fecal extract; sputum; intestinal fluids or aspirate, and stomach fluids or aspirate, cerebral spinal fluid (CSF), other body fluid or biofluid; cell sample; tissue sample; tumor sample; tumor cells in blood circulation (circulating tumor cells (CTCs)) and/or tumor biopsy etc. The term also includes a mixture of the above-mentioned samples. The term "sample" also includes untreated or pretreated (or pre-processed) biological samples. The term "liquid biopsy" refers to any liquid sample obtained from a subject. In some embodiments, a sample can comprise one or more cells from the subject. In some embodiments, a sample can be a tumor cell sample, e.g. the sample can comprise cancerous cells, cells from a tumor, and/or a tumor biopsy. In various other embodiments, the sample can be a blood sample which comprises of cancerous cells.

As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. The terms, "patient", "individual" and "subject" are used interchangeably herein. In an embodiment, the subject is mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In addition, the methods described herein can be used to treat domesticated animals and/or pets.

"Mammal" as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, rabbits and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., pancreatic cancer) or one or more complications related to the condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for a condition or one or more complications related to the condition or a subject who does not exhibit risk factors. For example, a subject can be one who exhibits one or more symptoms for a condition or one or more complications related to the condition or a subject who does not exhibit symptoms. A "subject in need" of diagnosis or treatment for a particular condition can be a subject suspected of having that condition, diagnosed as having that condition, already treated or being treated for that condition, not treated for that condition, or at risk of developing that condition.

The term "statistically significant" or "significantly" refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p- value.

Described herein are methods of establishing a patient-derived xenograft, using CTC cells isolated from a subject to obtain a personalized model of cancer.

CTCs are the culprit cell pool for tumor spreading and metastasis. As a metastatic cancer cell type readily available from liquid biopsy, CTCs exist in a minute fraction among vast numbers of normal cells in a given clinical blood sample. Repeated investigation in a CTC preparation is nearly impossible due to the rarity of this cell type in the blood. The inventors have developed an ex vivo CTC expansion protocol. Expanding CTCs ex vivo is necessary for reproducible examination of their genomic makeups and behaviors in vitro in culture or in vivo as patient-derived xenografts (PDXs).

With conventional PDX modeling, pieces of patient tumor are implanted directly to athymic mice for tumor formation. Conventional PDX suffers from inherent drawbacks including extremely low tumor formation rates in mice and less tumor progression and metastasis. CTCs are in dynamic equilibrium with tumor cells at the primary and metastatic sites, thus reflecting the state of the in situ tumor in real time. The advantages of CTC-PDX over the conventional PDX include: 1) unlike PDX, CTC-PDX can be studied repeatedly in culture and in mice; 2) CTC-PDX tumor can metastasize in the mouse host, whereas PDXs rarely metastasize; and 3) multiple CTC-PDXs can be established with longitudinally acquired patient samples at return visits, to monitor tumor progression, metastasis and therapeutic resistance.

The present invention is based, at least in part, on these findings. The present invention addresses the need in the art for methods of obtaining reproducible examination of the genomic makeup and behavior of a tumor in a subject, in vitro in culture or in vivo as patient-derived xenografts (PDXs) established from live CTCs, to study cancer metastasis and aid in biomarker discovery for predicting survival and therapeutic resistance in PDAC patients.

Establishing a personalized model for cancer

Various embodiments of the present invention provide for a method of establishing a personalized model for a cancer, comprising: isolating circulating tumor cells (CTCs) from a biological sample from a subject with the cancer; expanding the CTCs as 3-D spheroids ex vivo; inoculating the ex vivo expanded CTCs into a non-human animal, thereby generating a patient-derived xenograft from CTCs in a non-human animal; and establishing the patient- derived xenograft non-human animal as the personalized model for the cancer.

In various embodiments, the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In various other embodiments, pancreatic cancer can be pancreatic ductal adenocarcinoma. In yet other embodiments, the biological sample can be a liquid biopsy or tissue biopsy from the subject with cancer. In some other embodiments, a liquid biopsy can be a blood sample from the subject with cancer. In various other embodiments, the biological sample can be obtained from the subject with cancer before, during, and/or after therapeutic treatment. In some embodiments, CTCs may be isolated from the blood sample by separating the blood cells and plasma to obtain packed blood cells. In other embodiments, the packed blood cells may be lysed using ammonium chloride hemolysis to obtain a cell pellet comprising CTCs. In various embodiments, the cells from the cell pellet may be cultured in a defined medium to form spherical organoid aggregates. In various other embodiments, expanding CTCs ex vivo can result from culturing cells from the biological sample to form spherical organoid aggregates. In various embodiments, the expanded CTCs can be inoculated orthotopically, intrafemorally, intracardiac, intraosseosly, or subcutaneously. In some embodiments, the non- human animal can be a rodent, mouse, rat, rabbit or guinea pig.

In various embodiments, the method further comprises tagging the CTCs with a lentiviral reporter construct that expresses green fluorescent protein, red fluorescent protein, luciferase, or a combination thereof. In some embodiments, bioluminescence imaging can be used to monitor and track the tagged CTCs in the tumor.

The construct of the invention encoding the luciferase or the fluorescent proteins may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801805. The DNA construct may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491. The DNA construct of the invention may be inserted into a vector which may be any vector which may conveniently be subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced. In various embodiments, the vector can be a viral vector. In various embodiments, the vector is a lentiviral vector.

Various embodiments of the invention comprise CTCs tagged with a fluorescent protein. Other suitable fluorescent proteins include, but are not limited to, Y66H, Y66F, EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, ECFP, CyPet, Y66W, mKeima- Red, TagCFP, AmCyanl, mTFPl, S65A, Midoriishi Cyan, Wild Type GFP, S65C, TurboGFP, TagGFP, S65L, Emerald, S65T (Invitrogen), copGFP (SABiosciences), EGFP (Clontech), Azami Green (MBL), ZsGreenl (Clontech), TagYFP (Evrogen), EYFP (Clontech), Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellowl (Clontech), Kusabira Orange (MBL), mOrange, mKO, TurboRFP (Evrogen), tdTomato, TagRFP (Evrogen), DsRed (Clontech), DsRed2 (Clontech), mStrawbeny, TurboFP602 (Evrogen), AsRed2 (Clontech), mRFPl, J-Red, mCheny, HcRedl (Clontech), Katusha, Kate (Evrogen), TurboFP635, (Evrogen), mPlum, and mRaspberry. Various other embodiments of the invention comprise CTCs tagged with luciferase. Luciferases are proteins which react with a suitable substrate to produce light as one of the reaction products. Luciferases catalyze the oxygen oxidation of an organic molecule, i.e., a luciferin (such as aldehydes, benzothiazoles, imidazolopyrazines, tetrapyrroles and flavins). Luciferases that use coelenterazine (an imidazoloyrazine derivative) as a substrate to produce luminescence include luciferases from the species Renilla, Gaussia, Metridia and Obelia. The amount of light produced by a bioluminescent reaction can be measured and used to determine the presence of or amount of luciferase in a sample. The term "luciferase" refers to a naturally occurring or mutant luciferase.

As used herein, bioluminescence refers to the emission of light from a cell tagged with a fluorescent protein or resulting from a reaction catalyzed by a luciferase. Bioluminescence can be measured using luminometer or imaging systems. In various embodiments, CTC bioluminescence can be assessed in vivo. In various other embodiments, CTC bioluminescence can be assessed in vitro.

In various embodiments, the method further comprises co-innoculating PaSCs with the expanded CTCs into a non-human animal. In various embodiments, MIC-CTCs are inoculated into a non-human animal. In various embodiments, MIC-CTCs are inoculated either alone or together with a dormant cell, DC- 1 , into a non-human animal.

The personalized model for cancer of the invention is useful for the screening of agents or treatment modalities, (e.g., anti-cancer drugs, antibodies, combination of drugs, or administration regimens), having efficacy in cancer therapy. For such screening, host rodents are inoculated with CTC cells and, following a time interval sufficient to allow development of a tumor, the host is then administered with the tested agent or treatment modality and the therapeutic effect of such agent or treatment can then be evaluated, for example by determining the animals' median survival time or by assessing tumorigenic cellular characteristics, such as, but not limited to tumor size, angiogenesis, cell proliferation, cellular migration, cellular differentiation, apoptosis and cancer metastasis.

Drug Identification to Treat Cancer

Various embodiments of the present invention provide for a method of identifying one or more drugs to treat a cancer, comprising: providing a personalized model for a cancer, wherein the model is the personalized model established herein; administering one or more drugs to the model; and detecting a therapeutic response in the model and identifying the drug as being therapeutically effective to the cancer, or detecting no therapeutic response in the model and identifying the drug as being therapeutically ineffective to the cancer. In various embodiments, the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In various other embodiments, pancreatic cancer can be pancreatic ductal adenocarcinoma. In some embodiments, the therapeutic response can be inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited angiogenesis in a tumor, inhibited cancer cell invasion, inhibited cancer cell mobility, inhibited cancer cell differentiation, promoted cancer cell death, inhibited cancer progression, inhibited cancer metastasis, or improved animal survival, or a combination thereof. In yet other embodiments, the method further comprises instructing the subject with cancer to receive the one or more drugs identified to treat the subject's cancer.

Identifying Resistance to a Drug in a Subject

Various embodiments of the present invention provide for a method of identifying a subject that has cancer, as having resistance or not to a drug, comprising: providing a personalized model for a cancer; administering one or more drugs to the model; and detecting resistance in the model and identifying the subject as having resistance to the drug, or detecting no resistance in the model and identifying the subject as having not having resistance to the drug.

In various embodiments, the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In some embodiments, pancreatic cancer can be pancreatic ductal adenocarcinoma.

In various embodiments, the personalized model for cancer can be the model established herein. In some embodiments, resistance to a drug can be detected when there is uninhibited cancer cell proliferation, uninhibited cancer cell growth, uninhibited cancer cell invasion, uninhibited cancer cell mobility, uninhibited cancer cell differentiation, diminished cancer cell death, uninhibited cancer progression, uninhibited cancer metastasis, a decline in animal survival, or a combination thereof. In other embodiments, no resistance to a drug can be detected when there is inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited cancer cell invasion, inhibited cancer cell mobility, inhibited cancer cell differentiation, inhibited cancer cell death, inhibited cancer progression, inhibited cancer metastasis, improved animal survival, or a combination thereof.

Identifying MIC-CTCs in a Subject

Various embodiments of the present invention provide for a method of identifying

MIC-CTCs in a sample from a subject, comprising: obtaining a biological sample from a subject; assaying for metastasis-initiating cell - circulating tumor cells (MIC-CTCs), identifying MIC-CTCs in a sample when mesenchymal, stem, neuroendocrine markers, or a combination thereof, are detected. In various embodiments, the mesenchymal, stem and neuroendocrine markers comprise RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP. In various embodiments, detecting the mesenchymal, stem and neuroendocrine markers comprises detecting 1 or combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and/or 24 of the markers disclosed herein. In various embodiments, the cells assayed for are cells with metastasis- initiating cell properties within the pool of circulating tumor cells.

In some embodiments, identifying MIC-CTCs in the biological sample obtained from a subject, and establishes that the subject has cancer. In some embodiments, the subject identified with MIC-CTCs in the biological sample may have a poor prognosis and can be treated and closely.

In various embodiments, the biological sample is obtained from a subject with cancer. In various embodiments, the cancer is pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In various other embodiments, pancreatic cancer is pancreatic ductal adenocarcinoma. In some embodiments, the biological sample, can be obtained before, during and/or after treatment.

In some embodiments, the biological sample is a liquid biopsy or tissue biopsy from the subject. In some other embodiments, the mesenchymal, stem, neuroendocrine markers are assayed by mQDL. In various embodiments, the mesenchymal, stem, neuroendocrine markers assayed for can be compared to a reference value.

The reference value can depend on the type of disease or condition that will be determined. Different types of diseases and conditions may have different reference values. The reference value can be established from biological samples from a healthy subject or the same subject with samples obtained from different time of disease progression. For example, if the biological sample is a blood sample, then the reference value can be obtained from the blood sample of a healthy subject or the same subject with samples obtained from different time of the disease progression. The reference value to be used to compare with the expression value of the subject will typically be from the same tissue, cell, and/or location in the cell. For example, if RANKL protein expression level in a cell is measured for the subject, it will be compared to RANKL protein expression level in a cell obtained from a healthy control sample(s) or the same subject with samples obtained from different time of disease progression. Further, the reference value used can typically be from control samples having known disease states and survival times.

Various embodiments of the invention provide for a method of treating prostate cancer in a subject, comprising obtaining the results of an analysis of a level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP in a subject; and administering a treatment when the level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP is increased compared to a healthy individual. In various embodiments, the subject with an increased RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP level is indicative of a MIC-CTC phenotype. As discussed in detail below, the analysis can be accomplished by multiplex quantum dot labeling (mQDL), qRT-PCR, western blotting, fluorescence in situ hybridization (FISH), immunohistochemistry and/or in situ hybridization.

Various embodiments of the invention provide for a method of treating prostate cancer in a subject, comprising: requesting the results of an analysis of a level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP in a subject; and administering a treatment to the subject when the level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP is increased compared to a healthy individual. In various embodiments, the subject with an increased RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDH1,SDF1, NPRl, NSE, Lin28b, and/or SYP level is indicative of a MIC-CTC phenotype. As discussed in detail below, the analysis can be accomplished by multiplex QD-based labeling (mQDL), qRT-PCR, western blotting, fluorescence in situ hybridization (FISH), immunohistochemistry and/or in situ hybridization.

Various embodiments of the invention provide for a method of treating prostate cancer in a subject, comprising administering a treatment to the subject which has been determined to have an increased level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDH1,SDF1, NPRl, NSE, Lin28b, and/or SYP compared to a healthy individual. In various embodiments, the subject with an increased RANKL, Vimentin, FOXM1, FOXA2, c- Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N- Cad, ALDH1,SDF1, NPRl, NSE, Lin28b, and/or SYP level is indicative of a MIC-CTC phenotype.

In some embodiments, the subject identified with increased RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDH1,SDF1, NPRl, NSE, Lin28b, and/or SYP in the biological sample may be treated. In other embodiments, the subject with a MIC-CTC phenotype is treated. As discussed in detail below, the analysis can be accomplished by multiplex quantum dot labeling (mQDL), qRT-PCR, western blotting, fluorescence in situ hybridization (FISH), immunohistochemistry and/or in situ hybridization.

In various embodiments, the subject is treated by administering a drug that interrupts MIC-non-MIC cell communication. In various embodiments, the subject is treated by administering a therapeutically effective amount of a drug that targets MIC-CTCs along with a pharmaceutically acceptable excipient.

The targeting of MIC-CTCs can be accomplished by modifying or modulating

RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP gene expression. In various embodiments, gene regulation is modified by inhibiting or inducing the MIC-CTCs associated genes and causing an over-expression or under- expression of the genes, mentioned above. In various embodiments, the modifications can occur in transcriptional initiation, RNA processing and/or during post-translation. In certain embodiments, the modifications can occur at the transcription and/or the translational level. The modifications at the transcriptional level can include, but are not limited to the administration of siRNA or shRNA to add or remove the gene. The modifications at the translational level can include, but are not limited to phosphorylation, methylation, acetylation and/or the use of the respective inhibitors and gene silencing and/or gene induction can occur through translational modifications.

In yet other embodiments, the genes can be modified by altering upstream and/or downstream effectors of the genes. The genes, the upstream effectors and/or the downstream effectors can be up-regulated and/or down-regulated. RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N- Cad, ALDH1,SDF1, PR1, NSE, Lin28b, and/or SYP targeting can occur through the activation and/or deactivation of receptors and/or ligands. The activation and/or deactivation of receptors and/or ligands can inhibit and/or induce gene binding. The activation and/or deactivation of receptors and/or ligands can modulate the growth and behaviors of cancer cells and can increase or reverse their MIC-CTCs characteristics which include, but are not limited to cell growth, invasion, migration and metastasis.

In some embodiments, the subject is treated by administering a cancer therapeutic. In some other embodiments, the cancer therapeutic includes but it not limited to heptamethine carbocyanine near-infrared ( R) dye-drug conjugate. In yet other embodiments, the subject is treated by administering the one or more drugs identified using the personalized model established herein.

"Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

Identifying a Subject with Cancer

Various embodiments of the present invention provide for a method of identifying a subject with cancer, comprising: obtaining a biological sample from a subject; assaying for metastasis-initiating cells (MIC)-CTCs, activated PaSCs, macrophages or a combination thereof; and identifying the subject with cancer when MIC-CTCs, activated PaSCs, macrophages or a combination thereof are detected. In various embodiments, the reciprocal activation of MIC-CTCs by activated PaSCs is assayed.

Various embodiments of the present invention provide for a method of identifying a subject with cancer, comprising: obtaining a biological sample from a subject; assaying for metastasis-initiating cells (MIC)-CTCs, activated PaSCs, macrophages or a combination thereof; and identifying the subject with metastatic cancer when MIC-CTCs, activated PaSCs, macrophages or a combination thereof are detected.

In various embodiments, the cancer is pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In various other embodiments, pancreatic cancer is pancreatic ductal adenocarcinoma. In various embodiments, MIC-CTCs are identified by assaying for mesenchymal, stem and neuroendocrine markers. In various embodiments, the mesenchymal, stem and neuroendocrine markers comprise RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and/or 24 of these markers are identified. As discussed in detail below, the assaying for metastasis-initiating cells (MIC)-CTCs, activated PaSCs, and/or macrophages can be accomplished by multiplex QD-based labeling (mQDL), qRT-PCR, western blotting, fluorescence in situ hybridization (FISH), immunohistochemistry and/or in situ hybridization. In various other embodiments, the subject identified with cancer can be treated, as discussed above.

Markers used for the detection of macrophages include, but are not limited to CD1 lb, F4/80, CD68, CSF1R, MAC2, CDl lc, LY6G, LY6C, IL-4Ra, and/or CD163. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of these markers are identified. Markers used for the detection of pancreatic stellate cells (PaSCs) include, but are not limited to a-SMA and/or collagen I.

Sample Preparation and Assaying the Biological Samples

Nucleic acid or protein samples derived from diseased and non-diseased cells of a subject that can be used in the methods of the invention can be prepared by means well known in the art. For example, surgical procedures or needle biopsy aspiration can be used to collect diseased samples from a subject. In some embodiments, it is important to enrich and/or purify the diseased tissue and/or cell samples from the non-diseased tissue and/or cell samples. In other embodiments, the diseased tissue and/or cell samples can then be microdissected to reduce the amount of non-diseased tissue contamination prior to extraction of genomic nucleic acid or gene expression RNAs for use in the methods of the invention. Such enrichment and/or purification can be accomplished according to methods well-known in the art, such as fine needle aspiration or biopsy, laser capture microdissection, fluorescence activated cell sorting, and immunological cell sorting.

In some embodiments, a biological sample can comprise one or more cells from the subject. In some embodiments, a sample can be a tumor cell sample, e.g. the sample can comprise cancerous cells, cells from a tumor, and/or a tumor biopsy. In various other embodiments, the sample can be a blood sample which comprises of cancerous cells. In various other embodiments, the biological sample comprises MIC-CTCs, activated PaSCs, macrophages.

One of ordinary skill in the art will readily appreciate methods and systems that can be used to detect the expression level of the biomarkers described herein.

The biological sample can be assayed by various methods. These methods include but are not limited to multiplex QD-based labeling (mQDL), diaminobenzidine (DAB) immunohistochemical methods, fluorescent immunohistochemical methods, ELISA methods, Western blotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR). These methods and systems also include but are not limited to enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry, fluorescence in situ hybridization (FISH), radioimmuno assays, and affinity purification. Examples of ELISAs include but are not limited to indirect ELISA, sandwich ELISA, competitive ELISA, multiple and portable ELISA.

In various embodiments, assaying the biological sample comprises using multispectral quantitative imaging analysis. In certain embodiments, assaying the biological sample comprises using multiplex quantum dot labeling. This method is quantitative in comparison to the conventional method for assaying the samples to determine expression levels in tissues, which uses the intensity of IHC staining scored based on a combined intensity and percentage positive scoring cells as previously reported by De Marzo et al. (De Marzo AM, Knudsen B, Chan-Tack K, Epstein JL E-cadherin expression as a marker of tumor aggressiveness in routinely processed radical prostatectomy specimens. Urology 53(4):707-713, 1999). Recently, however, many other methods have achieved success by using semi -quantitative analyses of gene expression by in situ hybridization, and by the use of apatamer or nanoparticle amplification system. Accordingly, those methods can also be used to detect the expression levels of the biomarkers described herein.

The analysis of gene expression levels may involve amplification of an individual's nucleic acid by the polymerase chain reaction. Use of the polymerase chain reaction for the amplification of nucleic acids is well known in the art (see, for example, Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994)).

As used herein, the term "nucleic acid" means a polynucleotide such as a single or double-stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA. The term nucleic acid encompasses nucleic acid molecules of both natural and synthetic origin as well as molecules of linear, circular or branched configuration representing either the sense or antisense strand, or both, of a native nucleic acid molecule.

Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis is described in Ginzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR may also be used in the methods of the invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green.

Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.

A DNA sample suitable for hybridization can be obtained, e.g., by polymerase chain reaction (PCR) amplification of genomic DNA, fragments of genomic DNA, fragments of genomic DNA ligated to adaptor sequences or cloned sequences. Computer programs that are well known in the art can be used in the design of primers with the desired specificity and optimal amplification properties, such as Oligo version 5.0 (National Biosciences). PCR methods are well known in the art, and are described, for example, in Innis et al., eds., 1990, PCR Protocols: A Guide to Methods And Applications, Academic Press Inc., San Diego, Calif. It will be apparent to one skilled in the art that controlled robotic systems are useful for isolating and amplifying nucleic acids and can be used.

Hybridization

The nucleic acid samples derived from a subject used in the methods of the invention can be hybridized to arrays comprising probes (e.g., oligonucleotide probes) in order to identify RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP and in instances wherein a housekeeping gene expression is also to be assessed, comprising probes in order to identify housekeeping genes. A variety of different housekeeping genes can be used based on the experimental technique use and the sample being analyzed. One of ordinary skill in the art would be readily able to identify the appropriate housekeeping gene. In particular embodiments, the probes used in the methods of the invention comprise an array of probes that can be tiled on a DNA chip (e.g., SNP oligonucleotide probes). Hybridization and wash conditions used in the methods of the invention are chosen so that the nucleic acid samples to be analyzed by the invention specifically bind or specifically hybridize to the complementary oligonucleotide sequences of the array, preferably to a specific array site, wherein its complementary DNA is located. In some embodiments, the complementary DNA can be completely matched or mismatched to some degree as used, for example, in Affymetrix oligonucleotide arrays. The single-stranded synthetic oligodeoxyribonucleic acid DNA probes of an array may need to be denatured prior to contact with the nucleic acid samples from a subject, e.g., to remove hairpins or dimers which form due to self-complementary sequences.

Optimal hybridization conditions will depend on the length of the probes and type of nucleic acid samples from a subject. General parameters for specific {i.e., stringent) hybridization conditions for nucleic acids are described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 ^th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012); Ausubel et al., eds., 1989, Current Protocols in Molecules Biology, Vol. 1, Green Publishing Associates, Inc., John Wiley & Sons, Inc., New York, at pp. 2.10.1- 2.10.16. Exemplary useful hybridization conditions are provided in, e.g., Tijessen, 1993, Hybridization with Nucleic Acid Probes, Elsevier Science Publishers B. V. and Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Calif.

Oligonucleotide Nucleic Acid Arrays

In some embodiments of the methods of the present invention, DNA arrays can be used to determine the expression levels of genes, by measuring the level of hybridization of the nucleic acid sequence to oligonucleotide probes that comprise complementary sequences. Various formats of DNA arrays that employ oligonucleotide "probes," (i.e., nucleic acid molecules having defined sequences) are well known to those of skill in the art. Typically, a set of nucleic acid probes, each of which has a defined sequence, is immobilized on a solid support in such a manner that each different probe is immobilized to a predetermined region. In certain embodiments, the set of probes forms an array of positionally-addressable binding (e.g., hybridization) sites on a support. Each of such binding sites comprises a plurality of oligonucleotide molecules of a probe bound to the predetermined region on the support. More specifically, each probe of the array is preferably located at a known, predetermined position on the solid support such that the identity (i.e., the sequence) of each probe can be determined from its position on the array (i.e., on the support or surface). Microarrays can be made in a number of ways, of which several are described herein. However produced, microarrays share certain characteristics, they are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other.

In some embodiments, the microarrays are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions. The microarrays are preferably small, e.g., between about 1 cm ² and 25 cm ² , preferably about 1 to 3 cm ² . However, both larger and smaller arrays are also contemplated and may be preferable, e.g., for simultaneously evaluating a very large number of different probes. Oligonucleotide probes can be synthesized directly on a support to form the array. The probes can be attached to a solid support or surface, which may be made, e.g., from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, gel, or other porous or nonporous material. The set of immobilized probes or the array of immobilized probes is contacted with a sample containing labeled nucleic acid species so that nucleic acids having sequences complementary to an immobilized probe hybridize or bind to the probe. After separation of, e.g., by washing off, any unbound material, the bound, labeled sequences are detected and measured. The measurement is typically conducted with computer assistance. DNA array technologies have made it possible to determine the expression level of RA KL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N- Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP and housekeeping genes, as mentioned above.

In certain embodiments, high-density oligonucleotide arrays are used in the methods of the invention. These arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface can be synthesized in situ on the surface by, for example, photolithographic techniques (see, e.g., Fodor et al., 1991, Science 251 :767- 773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91 :5022-5026; Lockhart et al., 1996, Nature Biotechnology 14: 1675; U.S. Pat. Nos. 5,578,832; 5,556,752; 5,510,270; 5,445,934; 5,744,305; and 6,040, 138). Methods for generating arrays using inkjet technology for in situ oligonucleotide synthesis are also known in the art (see, e.g., Blanchard, International Patent Publication WO 98/41531, published Sep. 24, 1998; Blanchard et al., 1996, Biosensors And Bioelectronics 11 :687-690; Blanchard, 1998, in Synthetic DNA Arrays in Genetic Engineering, Vol. 20, J. K. Setlow, Ed., Plenum Press, New York at pages 111-123). Another method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al. (1995, Science 270:467-470). Other methods for making microarrays, e.g., by masking (Maskos and Southern, 1992, Nucl. Acids. Res. 20: 1679-1684), may also be used. When these methods are used, oligonucleotides {e.g., 15 to 60-mers) of known sequence are synthesized directly on a surface such as a derivatized glass slide. The array produced can be redundant, with several oligonucleotide molecules corresponding to each informative locus of interest {e.g., SNPs, RFLPs, STRs, etc.).

One exemplary means for generating the oligonucleotide probes of the DNA array is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (Froehler et al., 1986, Nucleic Acid Res. 14:5399-5407; McBride et al., 1983, Tetrahedron Lett. 24:246-248). Synthetic sequences are typically between about 15 and about 600 bases in length, more typically between about 20 and about 100 bases, most preferably between about 40 and about 70 bases in length. In some embodiments, synthetic nucleic acids include non-natural bases, such as, but by no means limited to, inosine. As noted above, nucleic acid analogues may be used as binding sites for hybridization. An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., 1993, Nature 363 :566-568; U.S. Pat. No. 5,539,083). In alternative embodiments, the hybridization sites {i.e., the probes) are made from plasmid or phage clones of regions of genomic DNA corresponding to SNPs or the complement thereof. The size of the oligonucleotide probes used in the methods of the invention can be at least 10, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. It is well known in the art that although hybridization is selective for complementary sequences, other sequences which are not perfectly complementary may also hybridize to a given probe at some level. Thus, multiple oligonucleotide probes with slight variations can be used, to optimize hybridization of samples. To further optimize hybridization, hybridization stringency condition, e.g., the hybridization temperature and the salt concentrations, may be altered by methods that are well known in the art.

In various embodiments, the high-density oligonucleotide arrays used in the methods of the invention comprise oligonucleotides corresponding to RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPR1, NSE, Lin28b, and/or SYP and housekeeping genes, as mentioned above. The oligonucleotide probes may comprise DNA or DNA "mimics" {e.g., derivatives and analogues) corresponding to a portion of each informative locus of interest {e.g., SNPs, RFLPs, STRs, etc.) in a subject's genome. The oligonucleotide probes can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone. Exemplary DNA mimics include, e.g., phosphorothioates. For each SNP locus, a plurality of different oligonucleotides may be used that are complementary to the sequences of sample nucleic acids. For example, for a single informative locus of interest {e.g., SNPs, RFLPs, STRs, etc.) about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more different oligonucleotides can be used. Each of the oligonucleotides for a particular informative locus of interest may have a slight variation in perfect matches, mismatches, and flanking sequence around the SNP. In certain embodiments, the probes are generated such that the probes for a particular informative locus of interest comprise overlapping and/or successive overlapping sequences which span or are tiled across a genomic region containing the target site, where all the probes contain the target site. By way of example, overlapping probe sequences can be tiled at steps of a predetermined base interval, e. g. at steps of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases intervals. In certain embodiments, the assays can be performed using arrays suitable for use with molecular inversion probe protocols such as described by Wang et al. (2007) Genome Biol. 8, R246. For oligonucleotide probes targeted at nucleic acid species of closely resembled {i.e., homologous) sequences, "cross-hybridization" among similar probes can significantly contaminate and confuse the results of hybridization measurements. Cross-hybridization is a particularly significant concern in the detection of S Ps since the sequence to be detected {i.e., the particular SNP) must be distinguished from other sequences that differ by only a single nucleotide. Cross-hybridization can be minimized by regulating either the hybridization stringency condition and/or during post- hybridization washings. Highly stringent conditions allow detection of allelic variants of a nucleotide sequence, e.g., about 1 mismatch per 10-30 nucleotides. There is no single hybridization or washing condition which is optimal for all different nucleic acid sequences, these conditions can be identical to those suggested by the manufacturer or can be adjusted by one of skill in the art. In some embodiments, the probes used in the methods of the invention are immobilized {i.e., tiled) on a glass slide called a chip. For example, a DNA microarray can comprises a chip on which oligonucleotides (purified single-stranded DNA sequences in solution) have been robotically printed in an (approximately) rectangular array with each spot on the array corresponds to a single DNA sample which encodes an oligonucleotide. In summary the process comprises, flooding the DNA microarray chip with a labeled sample under conditions suitable for hybridization to occur between the slide sequences and the labeled sample, then the array is washed and dried, and the array is scanned with a laser microscope to detect hybridization. In certain embodiments there are at least 250, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000,34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000 or more or any range in between, of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPR1, NSE, Lin28b, and/or SYP or the housekeeping genes for which probes appear on the array (with match/mismatch probes for a single locus of interest or probes tiled across a single locus of interest counting as one locus of interest). The maximum number of RA KL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP or the housekeeping genes being probed per array is determined by the size of the genome and genetic diversity of the subject's species. DNA chips are well known in the art and can be purchased in pre-5 fabricated form with sequences specific to particular species. In other embodiments, SNPs and/or DNA copy number can be detected and quantitated using sequencing methods, such as "next-generation sequencing methods" as described further above.

Labeling

In some embodiments, the protein, polypeptide, nucleic acid, fragments thereof, or fragments thereof ligated to adaptor regions used in the methods of the invention are detectably labeled. For example, the detectable label can be a fluorescent label, e.g., by incorporation of nucleotide analogues. Other labels suitable for use in the present invention include, but are not limited to, biotin, iminobiotin, antigens, cofactors, dinitrophenol, lipoic acid, olefinic compounds, detectable polypeptides, electron rich molecules, enzymes capable of generating a detectable signal by action upon a substrate, and radioactive isotopes.

Radioactive isotopes include that can be used in conjunction with the methods of the invention, but are not limited to, 32P and 14C. Fluorescent molecules suitable for the present invention include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, texas red, 5 'carboxy -fluorescein ("FAM"), 2', 7'-dimethoxy-4', 5'-dichloro-6- carboxy-fluorescein ("JOE"), N, N, N', N'-tetramethyl-6-carboxy-rhodamine ("TAMRA"), 6-carboxy-X-rhodamine ("ROX"), HEX, TET, IRD40, and IRD41.

Fluorescent molecules which are suitable for use according to the invention further include: cyamine dyes, including but not limited to Cy2, Cy3, Cy3.5, CY5, Cy5.5, Cy7 and FLUORX; BODIPY dyes including but not limited to BODIPY-FL, BODIPY-TR, BODIPY- TMR, BODIPY-630/650, and BODIPY-650/670; and ALEXA dyes, including but not limited to ALEXA-488, ALEXA-532, ALEXA-546, ALEXA-568, and ALEXA-594; as well as other fluorescent dyes which will be known to those who are skilled in the art. Electron rich indicator molecules suitable for the present invention include, but are not limited to, ferritin, hemocyanin and colloidal gold.

Two-color fluorescence labeling and detection schemes may also be used (Shena et al., 1995, Science 270:467-470). Use of two or more labels can be useful in detecting variations due to minor differences in experimental conditions (e.g., hybridization conditions). In some embodiments of the invention, at least 5, 10, 20, or 100 dyes of different colors can be used for labeling. Such labeling would also permit analysis of multiple samples simultaneously which is encompassed by the invention.

The labeled nucleic acid samples, fragments thereof, or fragments thereof ligated to adaptor regions that can be used in the methods of the invention are contacted to a plurality of oligonucleotide probes under conditions that allow sample nucleic acids having sequences complementary to the probes to hybridize thereto. Depending on the type of label used, the hybridization signals can be detected using methods well known to those of skill in the art including, but not limited to, X-Ray film, phosphor imager, or CCD camera. When fluorescently labeled probes are used, the fluorescence emissions at each site of a transcript array can be, preferably, detected by scanning confocal laser microscopy. In one embodiment, a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used. Alternatively, a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al. (1996) Genome Res. 6, 639-645). In a preferred embodiment, the arrays are scanned with a laser fluorescence scanner with a computer controlled X-Y stage and a microscope objective. Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser, and the emitted light is split by wavelength and detected with two photomultiplier tubes. Such fluorescence laser scanning devices are described, e.g., in Schena et al. (1996) Genome Res. 6, 639-645. Alternatively, a fiber-optic bundle can be used such as that described by Ferguson et al. (1996) Nat. Biotech. 14, 1681-1684. The resulting signals can then be analyzed to determine the expression of RA KL, Vimentin, FOXMl, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP and the reference genes, using computer software.

In other embodiments, where genomic DNA of a subject is fragmented using restriction endonucleases and amplified prior to analysis, the amplification can comprise cloning regions of genomic DNA of the subject. In such methods, amplification of the DNA regions is achieved through the cloning process. For example, expression vectors can be engineered to express large quantities of particular fragments of genomic DNA of the subject (Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 ^th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012)).

In yet other embodiments, where the DNA of a subject is fragmented using restriction endonucleases and amplified prior to analysis, the amplification comprises expressing a nucleic acid encoding a gene, or a gene and flanking genomic regions of nucleic acids, from the subject. RNA (pre-messenger RNA) that comprises the entire transcript including introns is then isolated and used in the methods of the invention to analyze and provide a genetic signature of a cancer. In certain embodiments, no amplification is required. In such embodiments, the genomic DNA, or pre-RNA, of a subject may be fragmented using restriction endonucleases or other methods. The resulting fragments may be hybridized to SNP probes. Typically, greater quantities of DNA are needed to be isolated in comparison to the quantity of DNA or pre-mRNA needed where fragments are amplified. For example, where the nucleic acid of a subject is not amplified, a DNA sample of a subject for use in hybridization may be about 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ng of DNA or greater. Alternatively, in other embodiments, methods are used that require very small amounts of nucleic acids for analysis, such as less than 400 ng, 300 ng, 200 ng, 100 ng, 90 ng, 85 ng, 80 ng, 75 ng, 70 ng, 65 ng, 60 ng, 55 ng, 50 ng, or less, such as is used for molecular inversion probe (MTP) assays. These techniques are particularly useful for analyzing clinical samples, such as paraffin embedded formalin-fixed material or small core needle biopsies, characterized as being readily available but generally having reduced DNA quality (e.g., small, fragmented DNA) and/or not providing large amounts of nucleic acids.

Once the expression levels have been determined, the resulting data can be analyzed using various algorithms, based on well-known methods used by those skilled in the art.

CTC Composition

As discussed herein, CTCs exist in a minute fraction among vast numbers of normal cells in a given clinical blood sample. Repeated investigation in a CTC preparation is nearly impossible due to the rarity of this cell type in the blood. Expanding CTCs ex vivo is thus necessary for reproducible examination of their genomic makeups and behaviors in vitro in culture or in vivo as patient-derived xenografts (PDXs).

In various embodiments, the present invention provides a composition, comprising circulating tumor cells (CTCs), grown as 3-dimensional (3-D) spheroids, isolated from a subject with a cancer. In various embodiments, the CTCs are cultured and/or expanded ex vivo. In various embodiments, the CTCs are inoculated into a non-human animal. In certain embodiments, the composition is used as a model of the cancer.

In various embodiments, the CTCs and a buffer solution are inoculated in the non- human animal. In other embodiments, the buffer solution can include, but is not limited to, PBS, TBS, TAPS, bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, and/or MES. In some embodiments, the buffer solution is PBS. In some embodiments, the CTCs, a buffer solution and a gelatinous protein mixture are inoculated in the non-human animal. In various embodiments, the gelatinous protein mixture is Matrigel and/or Geltrex. In various embodiments, the gelatinous protein mixture is Matrigel. In various embodiments, a clinically useful number of CTCs are obtained through the CTC expansion protocol. In various embodiments, the amount of cells obtained can range between lxl0 ² to lOxlO ² , lxlO ³ to 10xl0 ³ , lxl0 ⁴ to lOxlO ⁴ , lxlO ⁵ to lOxlO ⁵ , lxlO ⁶ to lOxlO ⁶ , lxlO ⁷ to lOxlO ⁷ , lxlO ⁸ to lOxlO ⁸ cells or a combination thereof.

In various embodiments, the present invention also provides a method of establishing a model for a cancer, comprising isolating circulating tumor cells (CTCs) from a subject with the cancer; culturing and/or expanding the CTCs as 3-D spheroids ex vivo, thereby establishing the ex vivo cultured and/or expanded CTCs as the model for the cancer.

In various embodiments, the present invention provides a non-human animal inoculated with circulating tumor cells (CTCs) isolated from a subject with a cancer. In various embodiments, the CTCs are inoculated subcutaneously, intrafemorally, orthotopically, or intraosseously, or a combination thereof. In various embodiments, the CTCs are cultured and/or expanded ex vivo. In various embodiments, the non-human animal is a rodent, mouse, rat, rabbit or guinea pig. In certain embodiments, the non-human animal is used as a model of the cancer.

In various embodiments, the present invention also provides a method of establishing a model for a cancer, comprising: isolating circulating tumor cells (CTCs) from a subject with the cancer; culturing and/or expanding the CTCs as 3-D spheroids ex vivo; and inoculating the ex vivo cultured and/or expanded CTCs into a non-human animal, thereby establishing the non -human animal as the model for the cancer.

In various embodiments, the cancer is pancreatic cancer or prostate cancer.

In various embodiments, the CTCs are isolated from a biological sample from the subject. In various embodiments, the biological sample is a liquid biopsy of the cancer. In various embodiments, the biological sample is cheek swab; mucus; whole blood, blood, serum; plasma; urine; saliva; semen; lymph; fecal extract; sputum; other body fluid or biofluid; cell sample; tissue sample; tumor sample; tumor biopsy, or their combinations.

In various embodiments, the present invention provides a method of identifying a drug as being therapeutically effective or ineffective to a cancer, comprising: providing a model for the cancer; administering the drug to the model; and detecting a therapeutic response in the model and identifying the drug as being therapeutically effective to the cancer, or detecting no therapeutic response in the model and identifying the drug as being therapeutically ineffective to the cancer. In some embodiments, the model is a composition, comprising circulating tumor cells (CTCs) isolated from a subject with the cancer. In various other embodiments, the composition is cultured in RPMI1640, 10% FBS and antibiotics. In some embodiments, the antibiotics include but are not limited to, actinomycin D, ampicillin, carbenicillin, cefotaxcime, fosmidomycin, gentamicin, kanamycin, neomycin, penicillin streptocysin, polymyxin C and/or streptomycin. In some embodiments, the antibiotic are penicillin streptocysin, penicillin and/or streptocysin.

In various embodiments, the composition comprises CTCs which can range between lxlO ² to lxlO ⁹ cells/ml. In some embodiments, the composition comprises lxlO ⁶ cells/ml.

In various other embodiments, the CTCs are tagged with a fluorescent protein or a luciferase, as discussed above.

In other embodiments, the model is a non-human animal inoculated with circulating tumor cells (CTCs) isolated from a subject with the cancer. In various embodiments, the non-human model is a rodent, mice, rat, rabbit or guinea pig. In various embodiments, the method is used for screening drugs.

In various embodiments, the therapeutic response is inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited cancer cell invasion, inhibited cancer cell mobility, promoted cancer cell differentiation, promoted cancer cell death, inhibited cancer progression, inhibited cancer invasion, inhibited cancer metastasis, or improved animal survival, or a combination thereof.

In various embodiments, the present invention provides a method, comprising: providing a model for a cancer, wherein the model is a composition comprising circulating tumor cells (CTCs) isolated from a subject with the cancer; administering a drug to the model; and detecting a therapeutic response in the model and instructing the subject to receive the drug to treat the cancer, or detecting no therapeutic response in the model and instructing the subject not to receive the drug to minimize exposure to side effects associated with the drug. In various embodiments, this method is used for selecting appropriate drugs for individual cancer patients. In various embodiments, this method is used for personalizing treatments for individual cancer patients.

In various embodiments, the present invention provides a method, comprising: providing a model for a cancer, wherein the model is a non-human animal inoculated with circulating tumor cells (CTCs) isolated from a subject with the cancer; administering a drug to the model; and detecting a therapeutic response in the model and instructing the subject to receive the drug to treat the cancer, or detecting no therapeutic response in the model and instructing the subject not to receive the drug to minimize exposure to side effects associated with the drug. In various embodiments, this method is used for selecting appropriate drugs for individual cancer patients. In various embodiments, this method is used for personalizing treatments for individual cancer patients.

In various embodiments, the present invention provides a method of identifying a subject as having resistance or not to a drug, wherein the subject has a cancer, comprising: providing a model for the cancer; administering the drug to the model; and detecting resistance in the model and identifying the subject as having resistance to the drug, or detecting no resistance in the model and identifying the drug as having no resistance to the drug. In some embodiments, the model is a composition, comprising circulating tumor cells (CTCs) isolated from the subject. In other embodiments, the model is a non-human animal inoculated with circulating tumor cells (CTCs) isolated from the subject.

In various embodiments, the subject is a human. In various embodiments, the subject is a mammalian subject including but not limited to human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse and rat. Dosages and Administration

Typical dosages of an effective amount of the drug can be in the ranges recommended by the manufacturer where known therapeutic molecules or compounds are used, and also as indicated to the skilled artisan by the in vitro responses in cells or in vivo responses in animal models. Such dosages typically can be reduced by up to about an order of magnitude in concentration or amount without losing relevant biological activity. The actual dosage can depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of relevant cultured cells or histocultured tissue sample, or the responses observed in the appropriate animal models. In various embodiments, the drug may be administered once a day (SID/QD), twice a day (BID), three times a day (TDD), four times a day (QID), or more, so as to administer an effective amount of the drug to the subject, where the effective amount is any one or more of the doses described herein.

In various embodiments, the drug is administered at about 0.001-0.01, 0.01-0.1, 0.1- 0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600- 700, 700-800, 800-900, or 900-1000 mg/kg, or a combination thereof. In various embodiments, the drug is administered at about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10- 20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800- 900, or 900-1000 mg/m ² , or a combination thereof. In various embodiments, the drug is administered once, twice, three or more times. In some embodiments, the drug is administered 1-3 times per day, 1-7 times per week, 1-9 times per month, or 1-12 times per year. Still in some embodiments, the drug is administered for about 1-10 days, 10-20 days, 20-30 days, 30-40 days, 40-50 days, 50-60 days, 60-70 days, 70-80 days, 80-90 days, 90-100 days, 1-6 months, 6-12 months, or 1-5 years. Here, "mg/kg" refers to mg per kg body weight of the subject, and "mg/m ² " refers to mg per m ² body surface area of the subject. In certain embodiments, the drug is administered to a human.

In various embodiments, the effective amount of the drug is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 μg/kg/day, or a combination thereof. In various embodiments, the effective amount of the drug is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 μg/m ² /day, or a combination thereof. In various embodiments, the effective amount of the drug is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200- 300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg/kg/day, or a combination thereof. In various embodiments, the effective amount of the drug is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg/m ² /day, or a combination thereof. Here, '^g/kg/day" or "mg/kg/day" refers to μg or mg per kg body weight of the subject per day, and '^g/m ² /day" or "mg/m ² /day" refers to μg or mg per m ² body surface area of the subject per day.

In accordance with the invention, the drug may be administered using the appropriate modes of administration, for instance, the modes of administration recommended by the manufacturer for each of the drug. In accordance with the invention, various routes may be utilized to administer the drug of the claimed methods, including but not limited to intratumoral, intravascular, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, aerosol, nasal, via inhalation, oral, transmucosal, transdermal, parenteral, implantable pump or reservoir, continuous infusion, enteral application, topical application, local application, capsules and/or injections. In various embodiments, the retinoid agonist is administered intracranially, intraventricularly, intrathecally, epidurally, intradurally, topically, intravascularly, intravenously, intraarterially, intratumorally, intramuscularly, subcutaneously, intraperitoneally, intranasally, or orally.

In accordance with the present invention, examples of the drug include, but are not limited to, Temozolomide, Actinomycin, Alitretinoin, All-trans retinoic acid, Azacitidine, Azathioprine, Bevacizumab, Bexatotene, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cetuximab, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Fluorouracil, Gefitinib, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Ipilimumab, Irinotecan, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizumab, Ofatumumab, Oxaliplatin, Paclitaxel, Panitumab, Pemetrexed, Rituximab, Tafluposide, Teniposide, Tioguanine, Topotecan, Tretinoin, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vorinostat, Romidepsin, 5 -fluorouracil (5- FU), 6-mercaptopurine (6-MP), Cladribine, Clofarabine, Floxuridine, Fludarabine, Pentostatin, Mitomycin, ixabepilone, Estramustine, prednisone, methylprednisolone, dexamethasone, thiostrepton, JQ1 (OTX015/MK-8628), Tideglusib or a combination thereof.

Various embodiments of the present invention provide for the inoculation of isolated CTC cells into a non-human animal, which have been expanded as 3-D spheroids ex vivo. In various embodiments, the amount of cells inoculated can range between lxlO ⁶ to 2xl0 ⁶ cells, 2xl0 ⁶ to 3xl0 ⁶ cells, 3xl0 ⁶ to 4xl0 ⁶ cells, 4xl0 ⁶ to 5xl0 ⁶ cells, 5xl0 ⁶ to 6xl0 ⁶ cells, 6xl0 ⁶ to 7xl0 ⁶ cells, 7xl0 ⁶ to 8xl0 ⁶ cells, 8xl0 ⁶ to 9xl0 ⁶ cells, or 9xl0 ⁶ to lOxlO ⁶ cells or a combination thereof. In various embodiments, the amount of cells inoculated is 2xl0 ⁶ CTC cells per 50μ1 phosphate buffer saline (PBS) mixed with equal volume of Matrigel. In some embodiments, the inoculated animal is maintained for a period of months for tumor growth and metastasis. In various embodiments, the animal is maintained for 1-2 months, 2-3 months, 3-4 months, 4-5 months, 5-6 months, 6-8 months, or 8-12 months. In some embodiments, the animal is maintained for 4 months.

In various embodiments, the CTC cells can be inoculated orthotopically, intrafemorally, intracardiac, intraosseosly, or subcutaneously. In various embodiments, the animal can be inoculated multiple times with CTC cells. In some embodiments, the animal receives 2, 3, 4 or 5 inoculations of CTC cells. In various embodiments, the inoculation can be administered simultaneously, consecutively, or subsequently in a series of administrations. In various embodiments, the CTCs inoculated can be from samples obtained before, during, and/or after therapeutic treatment.

Many variations and alternative elements have been disclosed in embodiments of the present invention. Still further variations and alternate elements will be apparent to one of skill in the art. Among these variations, without limitation, are the selection of constituent modules for the inventive methods, compositions, kits, and systems, and the various conditions, diseases, and disorders that may be diagnosed, prognosed or treated therewith. Various embodiments of the invention can specifically include or exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

EXAMPLES

The invention will be further explained by the following Examples, which are intended to be purely exemplary of the invention, and should not be considered as limiting the invention in any way. The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1

Pancreatic cancer and military service

PDAC cases can be categorized into 3 clinical phases: 1) primary localized resectable tumor; 2) locally advanced unresectable disease; and 3) metastatic disease. All cases of PDAC are eventually metastatic, which accounts for the poor prognosis. Accumulating evidence indicates that military service per se may be a risk factor for PDAC, since military personnel and veterans have a higher incidence of PDAC, and veteran patients are more frequently seen with unresectable diseases. There is a 40% increased risk overall and a 90% increased risk in men between ages 48-52, indicating a association of PDAC oncogenesis with military service. Two major risk factors of the disease are alcohol consumption and smoking, which leads to higher PDAC incidence among veterans.

Over ten phase II/III clinical trials worldwide have addressed PDAC during the past decade without significant success. At Cedars-Sinai Medical Center (CSMC) we treat approximately 250 cases and at the VA Greater Los Angeles Health Care System (VAGLA) we treat approximately 60 cases per year.

These treatments are aimed at blocking DNA replication and preventing DNA repair, enhancing a beneficial immune response, and improving dietary nutritional effects, all conventional approaches in oncology. These trials may lead to some benefit, but a breakthrough in PDAC management depends on a better understanding of the specific features of this cancer that make it so recalcitrant. We bridge knowledge gaps between basic research and clinical treatment through: 1) the development of clinically relevant experimental models to simulate PDAC metastasis and therapeutic resistance in a personalized manner; 2) the role of host mesenchymal cells in promoting PDAC growth, invasion and metastasis; and 3) the mechanism of acquisition of therapeutic resistance by PDAC cells. We obtain patient samples and key clinical information from clinical trials through institutional IRB approved BioBank protocols. We focus on the role of pancreatic stellate cells (PaSCs) in modulating cancer cell behavior, the effectiveness of treatments against therapy resistance, and the identification of novel biomarkers for diagnosis, staging and treatment. The role of the tumor microenvironment in PDAC metastasis and acquisition of therapeutic resistance

A salient feature of PDAC histopathology is a marked desmoplastic reaction by actively proliferating PaSCs and macrophages. Activated PaSCs and macrophages participate in modulating the malignant behavior of the tumor, and their contribution to the development of therapeutic resistance is currently unknown. We employ in vitro co-culture and in vivo co- inoculation to study the interaction of CTCs with PaSCs in the tumor microenvironment to determine their mechanistic roles and interplay to identify new opportunities for PDAC therapy.

CTCs and PDAC progression

CTCs are the culprit cell pool for tumor spreading and metastasis. As a metastatic cancer cell type readily available from liquid biopsy, CTCs exist in a minute fraction among vast numbers of normal cells in a given clinical blood sample. Though CTCs are being tested widely for diagnosis and prognosis, repeated investigation in a CTC preparation is nearly impossible due to the rarity of this cell type in the blood. Expanding CTCs as 3-D spheroids ex vivo is thus necessary for reproducible examination of their genomic makeups and behaviors in vitro in culture or in vivo as patient-derived xenografts (PDXs).

CTC-PDX versus PDX

With conventional PDX modeling, pieces of patient tumor are implanted directly to athymic mice for tumor formation. Conventional PDX suffers from inherent drawbacks including extremely low tumor formation rates in mice and less tumor progression and metastasis. CTCs are in dynamic equilibrium with tumor cells at the primary and metastatic sites, thus reflecting the state of the in situ tumor in real time. The advantages of CTC-PDX over the conventional PDX include: 1) unlike PDX, CTC- PDX can be studied repeatedly in culture and in mice; 2) CTC-PDX tumor can metastasize in the mouse host; and 3) multiple CTC-PDXs can be established with longitudinally acquired patient samples at return visits, to monitor metastasis and therapeutic resistance.

Tumor recruitment and reprogramming (R&R) of bystander cells in cancer progression

We found that cancer cells could transform normal host cells during xenograft tumor formation. Using 3-dimensional (3-D) cancer- stromal co-culture (Rhee HW et al., In Vitro Cell Dev Biol Anim 2001;37(3): 127-140 and Sung SY et al., Cancer research 2008;68(23):9996-10003) and chimeric xenograft tumor formation (Chu GC et al., Endocr Relat Cancer 2014;21(2):311-326), we identified a subcluster of metastasis-initiating cells (MICs) in the cancer cell population. Importantly, we found that MICs function as "drivers" to recruit and reprogram dormant cells as an integral part of the tumor (Chu GC et al., Endocr Relat Cancer 2014;21(2):311-326). Bystander cell R&R may be one source of cancer progression and metastasis. Identification and characterization of MICs within CTCs has important implications for the study of cancer metastasis, and could aid novel biomarker discovery for predicting survival and therapeutic resistance in PDAC patients.

We investigate PDAC progression and metastasis and treatment using clinical specimens available from prospective trials and retrospective specimens (Table 1). A CTC- PDX model was established by using clinical patient blood samples with consent from the BioBank (Wang RX, American Pancreatic Association 2015 Annual Meeting, Loews Coronado Bay, San Diego, November 4-7 2015). We elucidate the mechanism of PDAC progression and metastasis by studying the role of PaSCs in this CTC-PDX model. We target CTC-PDX models with new anti-tumor agents of tumor-homing gemcitabine conjugates, and examine the expression of predictive biomarkers of metastasis and therapeutic resistance.

Table 1. CSMC PaCa Clinical Trials

Trial Code name Stage Treatment PaCa indication Protocol

Locally advanced/borderline resectable

1 Phase I Veliparid (ABT- Locally IRB25528

888) with advanced,

gemcitabine and unresectable

intensity- modulated

radiation therapy

2 NLG-505 Phase III Folfirinox with Borderline IRB32122

hyperacute- resectable or

pancreas locally advanced

(algenpantucel- unresectable

1)

immunotherapy

Adjuvant

3 RTOG0848 Phase II-R Erlotinib (Ph II- Adjuvant IRB38099

Phase III R) and treatment for

chemoradiation patients with

(Ph III) resected

pancreas head

Metastatic

4 GS-US-370-1296 Phase II Gemcitabine Previously IRB36872

Randomized with nab- untreated

double blind, paclitaxel and metastatic

preceded by a momelotinib, dose-finding

lead-in phase

5 HALO-109-202 Phase II PEGPH20 with Metastatic (stage IRB31934

Randomized, nab-paclitaxel IV), previously

multicenter and gemcitabine, untreated

versus nab- paclitaxel and

gemcitabine

6 IMMU-107-04 Phase III 90Y- Metastatic (stage IRB37324

(PANCRIT-1) cliatuzumab IV), with at least

International tetraxetan with 2 prior

multicenter, low-dose treatments

randomized gemcitabine,

double blinded versus low-dose

gemcitabine

7 PanCax A longitudinal, Enteral feeding Advanced PaCa IRB38239

single

institutional

study

The clinical relevance of CTCs in cancer metastasis is being established in many human cancer types. An ex vivo 3-D spheroid CTC expansion protocol has been developed to: 1) elucidate the mechanism of CTC shedding, seeding, growth and survival at secondary sites; 2) assess the clinical value of CTCs as a marker for PDAC diagnosis and treatment evaluation; and 3) treat CTCs as a target for personalized PDAC therapy.

Expansion of CTCs from clinical PDAC patients

We reported our success in culturing CTCs from PDAC patients and characterization of CTC-PDX for the first time (Wang RX, American Pancreatic Association 2015 Annual Meeting, Loews Coronado Bay, San Diego, November 4-7 2015). Packed blood cells of PDAC patients, after the plasma fraction was harvested for clinical diagnostic use, were obtained from CSMC and VAGLA with IRB approvals and patient consents. Our study has investigated 62 PDAC pre-surgery blood samples. Of those, 13 yielded unique organoid aggregated growth with unlimited proliferation capabilities up to 60 passages, and more in the process of expansion (Figure 4B). Our success rate of ex vivo expansion remains 20% to 30%), most likely because all the PDAC patients are under cytotoxic therapy, which probably affected the viability of all the blood cells including CTCs in the sample. We also confirmed in 59 healthy donor samples that normal PBMCs do not form organoid aggregate growth with our ex vivo expansion protocol. Establishment of CTC-PDX models

We established CTC-PDX models by inoculating ex vivo expanded CTCs for xenograft tumor formation in athymic mice via subcutaneous, intraosseous or orthotopic routes (Figure 5A). The CTCs formed rapid tumor growth at inoculation sites, and also caused widespread metastases in soft tissues including lymph node, kidney, liver and lung (Figure 5A). CTC growth in long bones resulted in osteoclastic (osteolytic) lesions (Figure 5B). Orthotopic inoculation into mouse pancreas caused local invasion and distant metastasis (Figure 5C). CTC-PDX tumors exhibited histopathological features of the original adenocarcinoma in situ and stained consistently with PDAC markers (Figure 5D). As far as we know, this is the first CTC-PDX model that can grow and metastasize in mice. This model is of immense value for PDAC metastasis and therapeutic resistance studies. To facilitate the use of CTC-PDXs in translational research, we established methodologies to tag CTCs with a lentiviral reporter construct that bicistronically expresses luciferase (Luc) and green or red fluorescence protein (GFP/RFP). Luc activity facilitated tracking of tumor formation and metastasis of CTC-PDX tumors by bioluminescence imaging (BLI) (Figure 6).

Chemotherapeutic sensitivity of CTCs to conventional and therapeutics

Metastasis takes place once cancer cells acquire the capability of epithelial- mesenchymal transition (EMT), while therapeutic resistance can be acquired and has been suggested as a trait of cancer stem cells. We have developed a targeted therapeutic agent against cancer metastasis and examined mechanisms of chemotherapy resistance. The targeted therapeutic agent is a heptamethine carbocyanine near-infrared (NIR) dye-drug conjugate targeting tumor cells specifically with the tumor-homing NIR heptamethine carbocyanine dye. We demonstrated that a specific delivery of cytotoxic payload, gemcitabine, was dependent on increased organic anion transporting peptides (OATPs) carriers, which are further induced by hypoxia via hypoxia inducible factor (HIF-la) upstream of OATP promoter for transcription. The NIR dye-gemcitabine conjugate for metastatic PDAC treatment can be used in combination with other therapeutics (see below). Establishment of matched pairs of mesenchymal stromal cells from clinical PDAC specimens We have established 6 matched pairs of normal and cancer-associated stromal cultures from surgical PDAC specimens, following our published protocol (Sun X et al., In Vitro Cell Dev Biol Anim 2010;46(6):538-546). A general observation from ex vivo culture was that cancer-associated stromal cells grew faster than their normal counterparts. Interestingly, we also found that cancer-associated stromal culture could boost CTC growth (Wang RX, American Pancreatic Association 2015 Annual Meeting, Loews Coronado Bay, San Diego, November 4-7 2015), suggesting that some of the established stromal cells produced soluble paracrine factors.

Molecular profiling

Molecular profiling analyses expedite biomarker discovery and are used to characterize CTCs as a new cancer cell type. Palanisamy et al. has completed many molecular profiling projects, and comprehensively characterized prostate cancer PDXs in collaboration with Dr. Nora Navonne from MDACC (Palanisamy et al, American Association for Cancer Research Annual Meeting 2013, Washington, DC, April 6-10, 2013). We identify cancer gene fusions using Next Generation Sequencing (NGS), and detecting genomic abnormalities by fluorescence in situ hybridization (FISH) (see e.g., US Patents 7,964,345 and 7,585,964). This techniques is used for analyzing chromosomal translocations. FISH procedures have been standardized for formalin-fixed paraffin embedded (FFPE) tissues and FISH probes have been developed for the 27 ETS family genes and PTEN under two and four color schemes for simultaneous detection of ERG and PTEN status in the same nucleus. For FISH detection of candidate genes we can generate probes using bacterial artificial chromosome (BAC) clones on FFPE tissues from CTC-PDX tumor and biopsy tissues.

Without wishing to be bound by any particular theory, based on the results and our previous experience, we believe that ex vivo cultured CTCs and pancreatic mesenchymal cells generate chimeric tumors in vivo that represent the biology and metastatic behavior of the PDAC patient. Behaviors can be associated with molecular biomarkers and subsequently validated in the clinical setting for choosing specific treatments. CTC-PDX models can respond to therapeutics with significant improvement in survival. Example 2

We have successfully established CTC-PDX models of using banked specimens from PDAC patients who were recruited by CSMC to participate in clinical trials. We also confirmed the expression of PDAC biomarkers in CTC-PDX using retrospective PDAC specimens collected from VAGLA as reference standards. Tumor-stroma interactions and biomarkers associated with PDAC progression and acquisition of therapeutic resistance are further addressed.

1. Determine the biology and metastasis of PDAC in CTC-PDX models.

2. Assess the role of pancreatic stroma in modulating PDAC metastasis and therapeutic resistance.

3. Determine PDAC biomarkers in cultured CTCs and in chimeric CTC-PDX models. First, we establish new CTC-PDX models using clinical trial patient blood samples from 4 subtypes of PDAC disease: 1) primary localized surgically resectable disease; 2) recurrent tumors after surgery; 3) advanced tumors responsive to therapy; and 4) advanced tumors with therapeutic resistance. CTC-PDX from longitudinal samples of selected patients is studied to correlate CTC-PDX tumor behavior with therapeutic responses of the patients. These models are studied for the evolution of PDAC through interaction with bystander cells in the tumor microenvironment via R&R mechanisms, and treated with our newly developed anti-tumor agents to overcome therapeutic resistance.

Second, we define the functional roles of cancer-associated PaSCs in promoting or impeding PDAC progression. PaSC and CTC factors that mediate PDAC metastasis and therapeutic resistance are identified, including extracellular matrices and soluble factors secreted immune cells. The effects of therapeutics and their transport carriers are evaluated in PaSCs and CTCs in an immune-deficient PDAC mouse model.

Third, state-of-art technologies are used to profile CTC-PDX and patient tumor specimens. Information on genomic abnormalities and expressional aberrations is critical to understanding the mechanism of PDAC metastasis and therapeutic resistance. Special emphasis is placed on discovering new PDAC cell, tissue, and EVs-associated biomarkers based on biological and molecular read-outs from the model systems, with the intent of developing clinically relevant surrogate biomarkers of PDAC metastasis and therapeutic resistance. A focus of our approach analyzes and integrates results from each of these strategic approaches. We identify important associations and design new statistically powered prospective studies to further investigate the findings, including focused preclinical studies using the agents we have developed. This overall approach can lead to personalized applications of treatments to dramatically improve the outcome.

Determine the biology and metastasis of PDAC in CTC-PDX models

We develop 3 ex vivo CTC cultures for each of the 4 PDAC subtypes from clinical trials and a total of 12 CTC-PDX tumor models. All results from the cultures and animal models are correlated with key clinical information (i.e., time of disease-free survival after surgery, and responsiveness to chemotherapy) from the same PDAC patients.

Our recent success establishing ex vivo cultures of CTCs and CTC-PDX models allows us assess if ex vivo CTC expansion per se can be used to detect disease subtype, predict patient course, and to study the molecular and cellular mechanisms driving PDAC metastasis; assess if there are any CTC-specific biomarkers that can predict PDAC progression, invasion, metastasis and therapeutic resistance; assess if the concept of MICs in R&R can be used to develop innovative biomarkers associated with tumor cells or their secreted EVs by molecular profiling to predict PDAC progression and therapeutic resistance; and assess if the CTC cultures or CTC-PDX model can be used as a tool to screen for optimal clinical treatments of PDAC in a personalized manner.

We focus on a comparative study of established CTCs and CTC-PDX models among the 4 subtypes of PDAC, to identify potential mechanisms driving PDAC metastasis. CTCs and CTC-PDX models are also employed for assessing the therapeutic response to the agents. Establish CTC-PDX models

Blood and tumor biopsy samples from PDAC patients are obtained from CSMC and VAGLA, which is the referral center for veteran PDAC patients. Patient grouping is based on clinical diagnosis and subtyping at the time of blood sampling. For patients undergoing surgery, matched pairs of normal and cancerous specimens along with a blood sample from the same patient are collected. Tissue specimens are kept at -80°C. IRB approved written informed consent is used to collect packed blood cells and fresh tissue specimens. We conduct 3-D spheroid CTC expansion ex vivo. Fresh samples of packed blood cells, usually 2-3 ml in volume, are transported on ice. Red blood cells (RBCs) are removed by hemolysis and whole PBMCs are cultured at l x l0 ⁶ /ml in a specified medium for 4 weeks to obtain outgrowth of CTCs in organoid aggregates with unlimited proliferation potential (Figure 4B). At passage 10, CTCs are expanded to 2>< 10 ⁸ cells for animal inoculation, karyotyping, preparation of genomic DNA, total RNA, and whole cell lysates for molecular analyses.

To establish the CTC-PDX models, 2>< 10 ⁶ CTCs in single cell suspension in 50 μΐ of phosphate buffered saline (PBS), mixed with equal volume of Matrigel, are inoculated subcutaneously (s.c.) to the flank of male NOD SCID Gamma (NSG) mice (The Jackson Laboratory, Inc.) under anesthesia. For each patient-derived sample, 5 mice aged of 4 - 6 weeks are used, with each animal receiving 2 s.c. inoculations. After recovery from anesthesia, the animals are maintained for 4 months with tumor growth and metastasis monitored by our previous established molecular imaging protocols (Yang et al, J Urol 2013; 189(2):702-710 and Yang et al., Clin Cancer Res. 2010; 16(10):2833-2844).

With a similar protocol, expanded CTCs are also assessed for metastatic potential in CTC-PDX models following intra-femoral (i.f), orthotopic (o.t.) and intra-cardiac (i.e.) inoculation (Figures 4B, 5A-5D and 6). In these models where tumors form internally, growth and metastasis are monitored by signs of general wellbeing of the host, and confirmed by non-invasive R tumor imaging method developed in our laboratory. At the end of the study, animals are euthanized and complete necropsy performed to determine the extent of tumor metastasis, which is confirmed by histopathological analyses.

Analyze the growth and behavior of longitudinally established CTCs

It is documented that virulence of the CTCs reflects the malignant potential of the disease. The behavior of ex vivo expanded CTCs, therefore, may serve as a measure of tumor status in situ in the patient. The modeling is complemented by additionally analyzing longitudinally cultured CTCs from blood samples of 4 returning patients from the on- going clinical trial. They are sampled every 3 months for at least 3 consecutive time points. Behavior of the CTCs is correlated to status of patient disease progression, including the extent of metastasis and responsiveness to therapy. These CTCs are invaluable for elucidating the relationship of genomics, epigenetics, and gene expression with the mechanisms of cancer metastasis and therapeutic resistance.

Genomic characterization and expression profiling

For a mechanistic understanding of PDAC metastasis and therapeutic resistance, 3

CTC representative cultures for each of the 4 disease groups and a total of 12 CTC- PDX models are analyzed. Together with matching pairs of normal and cancerous tumor specimens, this analysis processes at least 36 data sets. To reduce the workload and conduct the analysis, we adopt a stepwise strategy for genomic characterization. The major part of the initial work sis performed and superposed with molecular identification of actionable biomarkers.

Determine the mechanism by which R&R drives PDAC metastasis

Utilizing a prostate cancer xenograft model, we found that cancer cells could transform host cells and a subpopulation of cancer cells, MICs, could recruit and reprogram "dormant" bystander cells in the tumor microenvironment. RNASeq analysis of the reprogrammed cells revealed that transcription factors c-Myc and FOXMl were obligatory for R&R and for activated expression of EMT, stem and neuroendocrine markers (Figures 1 and 8).

We investigate the PDAC metastasis by tracking CTCs in R&R of bystanders, co- culturing CTCs with normal pancreatic epithelial cells (to gain tumorigenic and metastatic phenotype) and PaSCs (to become reactive stroma driving PDAC growth). After R&R during co-culture, bystander cells are re-isolated and characterized for acquired malignant behavior.

Normal pancreatic epithelial or PaSCs are dually tagged with RFP and Luc and co- cultured with CTCs under 3-D conditions for 4 days. Tagged cells are then be isolated from the co- culture by fluorescence-activated cell sorting (FACS) and characterized in vitro and in vivo as we reported (Chu GC et al., Endocr Relat Cancer 2014;21(2):311-326). Acquired tumorigenicity in re-programmed pancreatic epithelial cells is assessed by inoculation through o.t. (N=10) and i.e. (N=10) routes in 6-week-old NSG mice. To assay for activation of stromal cells, PaSCs from the co-culture are tested in co-inoculation for chimeric CTC- PDX tumor formation. Compared to CTC-PDX without PaSCs, accelerated tumor formation and metastasis can reflect the activation status of the PaSCs. Once CTCs are shown to recruit and re- program bystander pancreatic epithelial or stromal cells, three modes of cancer-stromal interaction are examined. First, we determine whether R&R occurs through intercellular communication, using gene expressional and behavioral changes in bystander cells as read- out. Conditioned media (CM) from CTC culture are used to treat bystander epithelial or PaSCs under 3-D conditions as we reported (Chu GC et al., Endocr Relat Cancer 2014;21(2):311-326, Wang RX, American Pancreatic Association 2015 Annual Meeting, Loews Coronado Bay, San Diego, November 4-7 2015). Changes in growth rate and behavior are monitored. After 5 passages, any acquired tumorigenicity in the bystander cells is examined by xenograft tumor formation.

Second, to distinguish whether R&R is due to CTC secretion of soluble factors or via EVs transmission, CM from CTC culture is subjected to EV enrichment by ultracentrifugation, as we have reported (Morello M. et al., Cell cycle 2013; 12(22):3526- 3536). Both enriched EVs in ΙΟΟ ^χ concentration and EV-depleted CM is used to treat bystander epithelial cells and PaSC monolayers, with the same protocol described above, to observe changes in growth rate and behavior. After treatment with CM for 5 passages, any acquisition of tumorigenicity in treated cells is examined by xenograft tumor formation.

Subsequently, the growth factors mediating R&R are identified with biochemical methods and as we previously reported (Chu GC et al., Endocr Relat Cancer 2014;21(2):311- 326). To elucidate the mechanism of exosome-mediated R&R, enriched EVs from CTC culture medium are processed by miRNeasy kit (Qiagen) for miRNA isolation, which are used in RNASeq analysis to determine the identity of prevalent non-coding RNA species, which are studied further to evaluate their role in R&R, following our reported method (Josson S. et al., Clinical cancer research: an official journal of the American Association for Cancer Research 2014;20(17):4636-4646, Josson S. et al., Oncogene 2015;34(21):2690-2699 and Josson S. et al., Prostate 2008;68(15): 1599-1606). Briefly, the non-coding RNA is overexpressed via lentiviral infection to bystander PaSCs. Altered behavior of the overexpressing cells is assayed in vitro in cell culture and in vivo in chimeric CTC-PDX xenograft tumor formation.

Third, we examine whether CTCs recruit and re-program bystander cells through epigenetic mechanisms. Besides paracrine communication, we have found that cancer cells can fuse to bystander cells to cause epigenetic reprogramming. CTCs are tagged with RFP (with neor selection) and subjected to co-culture under 3-D conditions with PaSCs for spontaneous CTC-PaSC fusion by our reported protocol (Wang R. et al., PloS one 2012;7(8):e42653). At the end of co-culture after removing CTCs in suspension, the remaining stromal monolayer is treated with G418 (200 μg/ml) for 3 days to remove the PaSCs that are not involved in cell fusion. Survival CTC-PaSC hybrids are subjected to functional analyses in in vitro cell culture and by chimeric CTC-PDX tumor formation. The hybrids are also examined for signs of epigenetic modification (e.g., histone modifications, genomic methylation and imprinting).

Evaluate targeting potential of newly developed anti-tumor agents

CTC-PDX models are subjected to treatment with our newly developed anti -tumor

NIR dye-gemcitabine conjugates. We assessed the clinical usefulness of the experimental agent in mice. CTC inoculated mice are maintained till tumor size reaches 300 mm ³ in volume, and then treated with the test agent disclose herein, to assess whether the treatment can eradicate previously formed PDAC tumors in mice. NIR dye and gemcitabine are used separately as a control treatment for NIR dye- gemcitabine conjugates.

Once the efficacy of the new anti-tumor agents on s.c. CTC-PDX tumors has been determined, the same treatment schemes are tested in animals bearing i.e. and o.t CTC-PDX tumors. These experiments also provide clues to which types of PDAC, based on biomarker expression and the ratios of MICs/bystander cells in the clinical specimens, might be optimal targets for the newly developed agents in clinical trials.

We established a method for expanding CTCs from patient blood samples, and obtained 13 CTC cultures so far from primary resectable and post- surgery recurrent diseases. We can successfully establish CTC cultures from therapeutic sensitive metastatic and therapeutic resistant metastatic subtypes of PDAC in the first year, based on an estimated 260 blood samples from both CSMC and VAGLA, of which about 100 samples are from patients with advanced disease. Blood samples for longitudinal study are acquired from returning patients undergoing clinical trials at CSMC and from veteran patients at VAGLA. We believe that normal pancreatic cells can be provoked by CTCs to become tumorigenic and metastatic through R&R. EVs prepared from CTC- MICs have the ability to mediate R&R. To enhance the effect of NIR dye-gemcitabine conjugate on suppression or eradication of preformed tumors in mice, we additionally test if JQl (or OTX015/MK-8628), bromo-domain inhibitor drugs targeting transcription factor c-Myc, or thiostrepton, a thiazole antibiotic natural inhibitor of FOXM1 transcription, could synergistically yield potentially interesting results in blocking R&R and inhibiting tumor growth and distant metastasis. NIR dye-gemcitabine conjugate is highly effective agents promoting the death of PDAC cells in culture and as tumors in mice.

Assess the role of pancreatic stroma in modulating PDAC metastasis and therapeutic resistance.

We focus on the definition of cancer-associated PaSCs for their role in promoting PDAC progression. Several features of the pancreatic mesenchymal compartment may be clinically relevant to PDAC metastasis and therapeutic resistance. 1) The tumor-promoting role by PaSCs of the tumor microenvironment is well known. PDAC is the most desmoplastic of all malignancies, and PaSCs are the key participants in desmoplasia. 2) The loss of transporters for uptake of therapeutic agents by PDAC cells. Responsiveness to gemcitabine has been associated with the presence of human equilibrative nucleoside transporter 1 (hENTl), while unresponsiveness to therapy is related to a decreased hENTl in desmoplasia. 3) Cancer cell acquisition of therapeutic resistance and metastasis during therapy. We address these issues and provides new therapeutic agents designed by our laboratory expressly to address the key resistance issues.

There has been general agreement that PaSCs promote PDAC metastasis and therapeutic resistance, with strong stroma reaction associated with worse outcome. Secretion of large amounts of extracellular matrix proteins by PaSCs is responsible for the fibrotic characteristics of PDAC and for enhanced cancer cell survival. We identified activated PaSCs and extracellular matrix deposition in the earliest stages of tumor development in both human PDAC and mouse models of PDAC. More recent data suggest an anti-tumor effect of PaSCs as well. Studies show that deletion of PaSCs in transgenic mice led to invasive and undifferentiated tumors, accompanied by enhanced EMT and cancer sternness. Importantly, the authors observed an association between fewer activated PaSCs and worse patient survival. As described below, our approach distinguishes these seemly opposite effects by comparing the effects of normal PaSCs to those derived from advanced PDAC.

For these studies we use ex vivo expanded CTCs. Normal PaSCs as a byproduct of islet cell isolation are obtained. Both normal and cancer-associated PaSCs are available. Examine the effect ofPaSCs on CTC behavior

CTCs in suspension aggregate growth are co-cultured with PaSC monolayer for 72 hours. After separating CTCs and PaSCs from co-culture, activation status of the PaSCs is assayed by a-smooth muscle actin (a-SMA) expression. Proliferation of the treated CTCs is measured and apoptosis is detected. EMT and invasion are assessed by measuring its markers, E- cadherin, N-cadherin and vimentin, by western blot and Matrigel invasion assays. Cancer sternness is assessed by detecting stem cell markers of CD133, sox2 and Nanog and by colony formation assays.

To determine the mediators between CTCs and PaSCs, we conduct proteomic analysis with CMs from the cultures as described herein, through identification of proteins in both soluble and EV fractions using our reported procedure (Nomura T. et al., J Urol 2007; 178(l):292-300, Nomura T. et al., Clinical cancer research: an official journal of the American Association for Cancer Research 2006; 12(24):7294-7305 and Huang WC. et al., Cancer research 2005;65(6):2303-2313). Potential mediators are then tested for mechanistic roles through detailed experimentation. Although the focus of these experiments is on the interplay between CTCs and PaSCs, we pay attention to identify mediators that modulate both CTC-PaSC interaction and immune function. Examples include interleukin-4 (TL-4), IL- 6, IL-10, IL-13 and colony stimulating factor- 1 which we and others have found to recruit and convert monocytes into tumor promoting macrophages.

Identify cell targets of anti-tumor agents

Using the same experimental setting, we compare the efficacy of our anti-tumor agents versus standard PDAC chemotherapeutics, as well as the potential synergism between our new agents and standard chemotherapeutics. These comparative studies use both CTC- PaSC co-cultures and mono-cultures to pinpoint cells where the anti-tumor agents are acting.

Based on the results, we determine the role of OATPs in target cells following our published study of OATP function. Expression of OATPs in target CTCs or PaSCs is nullified by lentivirus- mediated siRNA knockdown. Target cells become insensitive to NIR dye-gemcitabine concomitantly with reduced drug uptake, which can be detected by NIR imaging under both normoxia and hypoxia conditions.

In another study, target CTCs or PaSCs are examined for loss of hENTl protein by western blot to identify cells lacking hENTl expression. These cells are infected with a lentiviral construct for hENTl overexpression to make the cells re-gain sensitivity toward treatment. Potential synergism between the two new agents is assessed by controlling doses of the agents in CTC-PaSC co-culture. Validate the effects of newly developed anti-tumor agents

Efficacy of our newly developed anti-tumor agents is evaluated in an orthotopic chimeric CTC-PDX tumor model established by co-inoculating ex vivo expanded CTCs with pair-matched PaSCs into the pancreas of NSG mice. 20 female NSG mice at 6 weeks of age are operated on one occasion and inoculated to pancreas head with a CTC and PaSC mix (2.5x l0 ⁵ /25 μΐ, each) as we have shown (Figure 6). Post inoculation transversal pancreatic ligation is applied, to avoid interfering with metastasis.

In CTC-PDX models, extensive necropsy is done following euthanasia. Pancreatic tumor size is measured and metastatic lesions in abdominal and thoracic cavities identified. Tissues are histopathologically assessed and immunostained for aSMA and PCNA and CK- 19 to identify activated PaSCs and proliferating CTC cancer cells. Comparative analysis is used to detect changes in cancer stem cell marker expression with our established protocol.

Despite a recent report suggesting that the stromal compartment has an inhibitory effect on PDAC tumor growth in genetically engineered mice, without wishing to be bound by any particular theory, we believe co-culture with cancer-associated PaSCs to stimulate CTC-PDX tumor growth, EMT, and metastasis, while PaSCs from normal tissues may have less effect or even an inhibiting effect. This study thus resolves the controversy over the role of PaSCs. Proteomic analysis of CMs from matched pairs of PaSCs additionally provides us with potential new targets of CTC-PaSC interaction.

Without wishing to be bound by any particular theory, we believe that our anti-tumor agent inhibits PDAC growth and metastasis in CTC-PDX mouse models. CTCs from the 4 subtypes of PDAC disease and pair-matched PaSCs from normal and cancerous specimens are tested. The number of combinations are decided based on the in vitro data. We treat all the mice in one inoculation with the same preparations of CTCs and PaSCs, in order to avoid any confounding variability in the cell source. Determine PDAC biomarkers in ex vivo expanded CTCs and CTC-PDX models

Genomic abnormalities and expressional aberrations are identified and validated as PDAC biomarkers. Actionable and "driver" biomarkers are selected as tools for PDAC diagnosis and therapeutic evaluation.

CTCs in PDAC patients are readily available "liquid biopsy" materials. Due to the lack of collaborative clinical and basic research, however, comprehensive characterization of this specified tumor cell type has not been attempted. Given the rarity of CTCs in patient blood samples, we pioneered an approach for successful expansion of CTCs and generation of CTC-PDX. This approach overcomes several technical limitations in obtaining high quality DNA and RNA from CTCs, which are urgently needed for reproducible molecular studies to avoid artifacts inherent to conventional genomic and expressional assay methods.

We focus on comprehensive molecular characterization and validation of CTC and CTC-PDX tumors established, in comparison with surgery specimens or biopsies from primary or metastatic sites and normal control tissues. Comparative analysis of CTC and CTC-PDX is carried out with state-of- the-art technologies including array comparative genomic hybridization (aCGH), spectral karyotyping (SKY), multiplex quantum dot labeling (mQDL), FISH, RNA in situ hybridization (RNA-ISH), and RNASeq by NGS. Analyses are targeted to validate newly found genomic abnormalities, coding gene and non-coding RNA expressional aberrations, and common known PDAC biomarkers (see below). Abnormality is determined by comparative analysis of normal tissue, primary and metastatic PDAC specimens, cultured CTCs, CTC-PDX tumors and chimeric CTC-PDX tumors of the same patient. The innovative approaches and unique capabilities of this study are the first to generate an integrated molecular signature for PDAC tumor heterogeneity, metastatic potential, drug sensitivity and treatment follow-up.

Characterizations are systematically conducted at various levels as shown in the schematic diagram (Figure 2). Result from these studies also provide mechanistic elucidation of PDAC metastasis and therapeutic resistance as described herein.

Genomic characterizations

Two studies are conducted at the genomic level. The first analyze candidate mutations known to be associated with PDAC progression and metastasis. The second study focuses on identifying novel genomic abnormalities and evaluating their application as PDAC biomarkers.

Though detailed genomic characterization of CTC in PDAC has not been reported, recent PDAC genomic studies revealed a spectrum of genetic abnormalities, including SMAD4, CDKN2A, ARJDIA and ROB02, KDM6A and PREX2. SND1-BRAF gene fusion has been identified in PDAC, together with frequent somatic mutations in P53, KRAS or BRAF genes. High frequency amplifications encompassing oncogenes such as c-Myc, ERBB2, MET, FGFRl, CDK6, PIK3R3 and PIK3CA are known. CTCs and CTC-PDX tumors, together with primary and metastatic biopsies from the same patient, are examined for known mutations by established PCR protocols and Sanger sequencing. The results are corroborated with similar mutations during CTC-PDX tumor progression and metastasis in mice.

A stepwise strategy is used to identify novel genomic abnormalities associated with metastasis and therapeutic resistance in PDAC patients and CTC-PDX mouse models. Each ex vivo expanded CTC sample is analyzed successively with: 1) SKY assay; 2) aCGH comparison; and 3) NGS analysis. Whole genome sequencing with NGS technology is conducted by NantHealth Systems (Los Angeles, CA).

Karyotyping of live CTCs is conducted first to visualize gross structural and numerical abnormalities. Chromosome complements with high level of aneuploidy and structural faults is indicative of cancer. SKY analysis is used to determine the clonal nature of CTCs. Given the heterogeneous nature of PDAC, without wishing to be bound by any particular theory, we believe that subpopulations may carry variant structural and numerical changes.

To detect deletions, insertions and amplifications, we use genomic DNA from the respective samples for aCGH analysis to assess genomic fidelity from CTC to CTC-PDX, and in comparison to primary and metastatic tumor tissues. The aCGH assay is an essential tool to assess the genomic changes. Based on the results of SKY and aCGH analyses, representative CTC cultures are subjected to mutation screening in CTC, CTD-PDX and biopsy materials by whole genome sequencing using NGS for comprehensive molecular profiling of abnormalities at the nucleotide level. NGS data are transferred for biostatistics and bioinformatics analysis. Given the unbiased nature of NGS analysis, we can identify new druggable genomic mutations on a personalized basis. Expression profiling

We employ high-throughput transcriptome sequencing (RNASeq) as it provide s a direct assessment of most transcripts in CTCs and matched normal samples in an unbiased manner. High quality RNA is prepared for the inclusion of miRNA in the initial sample, and is confirmed by quality analysis before RNASeq analysis. The sequencing data are analyzed for both the identity and transcriptional level of: 1) each coding gene; 2) non -coding RNA genes; 3) miRNA transcripts; and 4) previously known and novel gene-fusion events.

To select transcripts for further study, stringent statistical analysis is used to examine the RNASeq data to identify transcripts that are differentially expressed between samples. Differential expression of top outliner candidate transcripts is validated with alternative methods of RT -PCR, western blot, mQDL, or RNA-ISH assays. Subsequently, extensive literature search and bioinformatics are used to identify transcripts that may play causal role in PDAC metast asis and therapeutic resistance.

Based on our experience in the RNASeq analysis of multiple solid cancer types, many transcriptomes carry multiple gene fusions, posing a bioinformatics challenge for identifying the driving gene fusion. Similarly, our RNASeq analysis revealed that PDAC transcriptomes carry multiple gene fusions as well. Pioneering studies from our group have firmly established that despite the presence of multiple gene fusions, critical gene fusions can be identified with a ranking strategy based on sequence read counts because the driver gene fusions may be expressed at a higher level.

Identify gene fusion biomarkers for therapeutic targeting

The function of many coding gene is known and protein products from "driver" gene fusion can often be exploited as therapeutic targets. Our research on druggable gene fusions with RAF kinase (Figure 3) provides an example to explore expression profiling and NGS data for identifying new "druggable" gene fusions and other actionable molecular markers.

Given the unprecedented prevalence of gene rearrangements in PDAC, without wishing to be bound by any particular theory, we believe additional "druggable targets" to occur in our study population. Based on the sequencing data, we validate gene fusions by RT-PCR at the RNA level followed by FISH to confirm gene rearrangement. Recurrent fusions are evaluated for oncogenic activity using expression constructs in normal pancreatic epithelial cells.

Without wishing to be bound by any particular theory, we can identify a set of new actionable fusion proteins specifically for PDAC. Our studies enhance our understanding of the molecular changes useful for selection of treatment and follow-up.

PDAC biomarker identification and validation

Biomarkers in ex vivo expanded CTCs are recognized by stringent statistical analysis and subjected to further characterization in three studies. First, we confirm the association of biomarkers with CTCs and CTC-PDX models. Second, the biomarkers are validated for correlation with clinical PDAC metastasis and therapeutic resistance. Finally, we retrospectively test the application of selected biomarkers in PDAC diagnosis, treatment evaluation and disease prognosis. As biomarkers could be in the form of genomic abnormalities or transcriptional aberrations, corresponding methods are used for validation, which we have previously reported (Wang R. et al., Biochemical and biophysical research communications 2009;389(3):455-460, Wang R. et al., Clinical cancer research: an official journal of the American Association for Cancer Research 2007; 13(20):6040-6048 and Wang R. et al., Cancer research 2004;64(5): 1589-1594). We develop biomarker detection protocols for cost-effective and non-invasive detection of clinical PDAC in a highly sensitive and specific fashion.

Genomic PCR is tested extensively as a convenient and preferred tool for detecting biomarkers of genomic abnormalities. After confirmation on ex vivo expanded CTCs, optimal PCR settings are established and tested for detection of CTCs directly from blood samples, first from the blood of mice bearing CTC-PDX tumors, then from clinical blood samples of PDAC patients at CSMC and VAGLA. Clinical PDAC specimens from the BioBank at CSMC are examined retrospectively with our established methods (Wang R. et al., Biochemical and biophysical research communications 2009;389(3):455-460, Wang R. et al., Clinical cancer research: an official journal of the American Association for Cancer Research 2007; 13(20):6040-6048 and Wang R. et al., Cancer research 2004;64(5): 1589-1594). Besides the more than 200 archived PDAC specimens at CSMC, there are 40 PDAC archived tissues at VAGLA (Table 2), an invaluable source of veteran PDAC materials for a disease highly relevant to military service. Table 2. VAGLA Human Pancreas Specimen Archives

Disease Staging

Cases Age Tis Tl T2 T3 T4 Lymph Distal Unknown nodes metastasis

Male 38 34-84 1 3 7 5 2 7 4 9 Female 2 42-54 2

Note: The table summarizes retrospective tissues that are presently available at the VAGLA site. There is another more than 200 archived PDAC tissues at CSMC BioBank available for the proposed study (not shown).

The specimens are also used in FISH assays as a supplementary to confirm the PCR detection. Sensitivity and specificity of the detection are critically analyzed by stringent statistical examination.

In a similar study setting, candidate coding-gene expressional aberrations are validated. Though RT-PCR may be used in the initial phase, differential expression of coding genes is confirmed by western blot, especially for biomarkers of gene fusion. Immune-based techniques such as enzyme-linked immunosorbent assay (ELISA), FACS, mQDL and IHC is used depending on subcellular localization of the protein. Patient plasma samples from CSMC BioBank are tested retrospectively for detection of protein biomarkers.

Biomarkers of non-coding RNA and miRNA are confirmed with special real-time PCR as we reported previously (Josson S. et al., Oncogene 2015;34(21):2690-2699, Josson S. et al., Prostate 2008;68(15): 1599-1606 and Gururajan M. et al., Int Immunol 2010;22(7):583- 592). Their validation as candidate markers for clinical PDAC detection is tested first with CTC-PDX tumor specimens and then with PDAC specimens by FISH. Plasma samples from CSMC BioBank are used for retrospective validation with our established PCR method (Morello M. et al., Cell cycle 2013; 12(22):3526-3536).

Compared to the observed high heterogeneity in clinical PDAC tumors, we determined that ex vivo expanded CTCs seemed to be composed of a morphologically homogenous population, and single-cell progenies can be cloned by limiting dilution. This unique feature provides favorable conditions for genomic and expression profiling, ensuring comparative analyses of abnormalities and aberrations on the same cell lineage.

We can achieve reliable data acquisition from RNASeq expression profiling with high quality RNA preparation. We have conducted stringent statistical analysis of RNASeq data, and confirmed and validated biomarkers of differential transcription, both for coding and non-coding genes and miRNA transcripts. Both CTCs and CTC-PDX tumor specimens are high quality, while more than 300 retrospective clinical PDAC specimens are available at the CSMC BioBank with our approved access to disease information.

We have designed a stepwise strategy to maximize the chances of discovering actionable molecular markers and to identify genomic and gene signatures indicative of treatment response, taking advantage of the unique CTC features. First, SKY analysis is applied to all CTC cultures to detect gross chromosomal changes. Whether a consensus can be made and whether gross abnormalities can be exploited as biomarkers are determined with clinical specimens. Second, representative CTC cultures are subjected to aCGH to identify smaller genomic abnormalities. These abnormalities are evaluated as biomarkers with clinical specimens. Finally, selected CTCs are subjected to NGS analysis to obtain a comprehensive view of genomic abnormalities at the nucleotide level. Next generation paired-end transcriptome sequencing is a well-established platform, particularly the Illumina sequencing platform. This is the most economical thoroughfare to a full understanding of the genomic base for PDAC metastasis and therapeutic resistance.

Statistical analysis and power considerations: We have translated scientific questions into meaningful statistical hypotheses with the most efficient design. While the statistical analysis attempts to encompass all the complexity of the experimental design, the power considerations are crafted in a very conservative and simple fashion, so at the time of data analysis we can have a higher power of detecting differences than the initial results. Our strategy to maximize precision while minimizing animal use is based on the results of CTC- PDX tumor formation. A Student's two-sided t-test is used to determine the tumorigenicity of each CTC culture (N=5) and two-way analysis of variance is used in testing therapeutic evaluation of the newly developed anti- tumor agents (N=10) to ensure that the underlying assumptions (i.e., normality, homoscedasticity) of the ANOVA model are met. If these assumptions are not met, we conduct data transformation or rank transformation to meet the assumptions. Our study design and animal group sizes for the survival and treatment studies were proven effective (Figure ID). For identifying biomarkers from NGS and RNASeq data, biostatistics and bioinformatics tools are used for mutation detection and quantification. List of Abbreviations, Acronyms, and Symbols

3-D 3 -dimensional

aCGH array comparative genomic hybridization

ANOVA analysis of variance

ARID 1 A AT -rich interactive domain-containing protein 1 A

a-SMA a-smooth muscle actin

β-2Μ β-2 microglobulin

BAC bacterial artificial chromosome

BDNF brain-derived neurotrophic factor

BLI bioluminescence imaging

BRAF v-raf murine sarcoma viral oncogenes homolog Bl

CCL5 chemokine (C-C motif) ligand 5

CD31 cluster of differentiation 31

CD133 cluster of differentiation 133

CDK6 cyclin-dependent kinase 6

CDKN2A cyclin-dependent kinase inhibitor 2A

CK-19 cytokeratin 19

c-Myc a proto-oncoprotein

Cre a PI bacteriophage tyrosine recombinase enzyme

CSMC Cedars-Sinai Medical Center

CTC circulating tumor cell

CTC-MIC metastasis-initiating cells in a circulating tumor cell population

CTC-PDX circulating tumor cell as patient-derived xenograft

CXCL5 chemokine (C-X-C motif) ligand 5

CXCL16 chemokine (C-X-C Motif) Ligand 16

DC-1 a non-tumorigenic human prostate cancer cell line established in our own laboratory

DNA deoxyribonucleic acid

ELISA enzyme-linked immunosorbent assay

EMT epithelial to mesenchymal transition

ERBB2 a member of the epidermal growth factor (EGF) receptor family

ERG ETS -related gene ESRP1 epithelial splicing regulatory protein 1

ETS E26 transformation-specific family transcription factors

ETV1 ETS translocation variant 1

EV exosome vesicle

FACS fluorescence-activated cell sorting

FFPE formalin-fixed, paraffin-embedded

FGFR1 a member of the fibroblast growth factor receptor family

FISH fluorescence in situ hybridization

FOXM1 forkhead box protein Ml

GFP green fluorescence protein

GSK-3p glycogen synthase kinase-3p

HDAC histone deacetylases

hENTl human equilibrative nucleoside transporter 1

HIF-la hypoxia inducible factor

i.e. intra-cardiac

IHC immunohistochemical

IL-4 interleukin 4

IL-6 interleukin 6

IL-10 interleukin 10

IL-13 interleukin 13

i.p. intra-peritoneal

IRB institutional review board

JQ1 an inhibitor of the BET family of bromodomain proteins

KC mouse tumor model with transgenic KRAS mutation

KDM6A lysine (K)-specific demethylase 6A

kg kilogram

Ki67 a cellular marker protein for proliferation

KRAS V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog

LSL a loxP-STOP-loxP cassette

Luc luciferase

MDACC M.D Anderson Cancer Center

MET a tyrosine kinase proto-oncoprotein mg milligram

MIC metastasis-initiating cell

μΐ microliter

miRNA microRNA

MK-8628 a BET-Bromodomain Inhibitor

mm millimeter

mQDL multiplex quantum dot labeling

ncRNA non-coding RNA

NGS next generation sequencing

NIR near infrared

nM nanomolar

NOD non-obese diabetic

NSG NOD scid gamma

OATP organic anion transporting peptide

o.t. orthotopic

OTX015 a BET Bromodomain Inhibitor

P53 tumor suppressor of tumor protein p53

PanIN pancreatic intraepithelial neoplasia

PaCa pancreatic cancer

PaSC pancreatic stellate cell

PBMC peripheral blood mononuclear cell

PBS phosphate buffered saline

PCNA proliferating cell nuclear antigen

PDAC pancreatic ductal adenocarcinoma

PDX patient-derived xenograft

Pdxl pancreatic and duodenal homeobox 1

PIK3CA phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform

PIK3R3 phosphatidylinositol 3-kinase regulatory subunit gamma

PREX2 phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2

PTEN phosphatase and tensin homolog protein

qRT-PCR quantitative reverse transcription polymerase chain reaction

RAF rapidly accelerated fibrosarcoma protein RAF 1 a proto-oncoprotein of the RAF family

R&R recruitment and re-programming

RANKL receptor activator of nuclear factor kappa-B ligand

RBC red blood cells

RFP red fluorescence protein

RNA ribonucleic acid

RNA-ISH RNA in situ hybridization

RNASeq RNA sequencing

ROB02 recombinant protein of human roundabout, axon guidance receptor, homolog 2

RT-PCR reverse transcription polymerase chain reaction

SAHA a hi stone deacetylase inhibitor

s.c. subsutaneous

sCM stromal cell conditioned medium

SKY spectral karyotyping

SLC45A3 solute carrier family 45 member 3

SMAD4 SMAD Family Member 4 protein

S D1 staphylococcal nuclease domain-containing protein 1

Sox2 a member of the Sox family of transcription factors

TP53 tumor protein P53

TU EL terminal deoxynucleotidyl transferase dUTP nick end labeling

TWIST a basic helix-loop-helix transcription factors

VA Veterans Affairs

VAGLA VA Greater Los Angeles Health Care System

VEGF vascular endothelial growth factor

Example 3

Patient-derived CTC-xenograft models for the study of pancreatic cancer biology, metastasis and therapy

Pancreatic cancer (PaCa) often spread rapidly to form metastatic tumors. We expanded respectively circulating tumor cells (CTCs) and stromal cells from liquid and tissue biopsies as source materials to examine the pathobiology and therapeutic responses of CTCs from PaCa patients. Peripheral blood mononuclear and pancreatic stromal cells were isolated respectively from the whole blood and PaCa tumor biopsies from the same patient. Ex vivo expansion of CTCs and stromal cells was performed by standardized outgrowth protocols and characterized. The tumorigenicity of CTCs and their responses to pancreatic stromal fibroblasts were assessed. Therapeutic responses of CTCs and CTC-derived patient xenografts (CTC-PDXs) were evaluated.

We have successfully expanded 9 candidate CTC cultures from 42 patients. Undergoing surgery for their cancers. CTCs grew as spherical organoids with high proliferative activity, which was not observed in healthy donor blood cultures. CTC-PDX tumors expressed PaCa markers and exhibited local invasive and metastatic behaviors when inoculated orthotopically or intraosseously but not subcutaneously. CTC growth was enhanced by stromal conditioned media. We found the growth of PaCa tumors in mouse models was inhibited, and mouse survival was improved by treatment with the anti-tumor agent dye-gemcitabine conjugate.

For the first time in this feasibility study, we succeeded in establishing CTC-PDX models using PaCa liquid biopsy. CTC-PDX tumors share malignant features with in situ tumors of the patients, and can be used as a reproducible model for the study of PaCa metastasis to facilitate personalized oncological research on PaCa diagnosis, treatment and prognosis.

Taken together, the inventors established a protocol for ex vivo CTC expansion from clinical PaCa patients, and characterized tumorigenic and metastatic CTC-PDX mouse models. CTCs and CTC-PDX models can be used to study the mechanism of PaCa progression, may be used as tools for testing therapeutic agents, and can facilitate application in personalized oncology.

Example 4

Circulating tumor cells (CTCs) from patient's blood represent a promising noninvasive liquid biopsy compartment useful for prognosis and diagnosis of prostate cancer (PC) patients. Since CTCs reflect the pool of tumor cells originated from the primary as well as metastatic sites, molecular and phenotypic characterizations of CTCs allow us understand the biology and metastatic process. The challenges of CTC research are a small number of PC cells in blood, the difficulties of repeating molecular and phenotypic assays, and validating these results with patients' clinical outcome. We established here an in vitro CTC expansion protocol, evaluated gene expression and behavioral profiles of CTCs, and correlated these results with the clinical status of PC patients. The cultured CTCs were used for the development of CTC-Derived patient Xenografts (CDXs) and for the study of CTCs with MIC phenotype that recruit and reprogram non-tumorigenic PC cells to participate in tumorigenesis.

An in vitro methodology of CTCs expansion was established using post-RBC lysed peripheral blood mononuclear cells (PBMCs) as the source materials. The ability of in vitro CTC growth, in vivo tumor formation and distant metastases are determined and compared between PC patients and within PC patients with PBMCs harvested at various time points of hormonal/ chemotherapy. CTCs are genetically tagged with GFP, RFP or Luc for ease of detection of local tumor growth and distant dissemination in mice and for subsequent tumor cell derivation for molecular analyses using microarray, qRT-PCR, western blots and in vitro behavioral assays including migration, invasion and anchorage-independent 3-D growth.

We have established 10 CTC-PDX models in mice from PC patients with two patients longitudinally before, during and after therapeutic intervention. Some but not all models expressed PC-associated biomarkers, androgen receptor, PSA and PSMA. Within the same patient, CTC-PDXs models established post therapy had increased tumor growth, invasiveness, metastasis and earlier death than prior therapy in mice. We identified metastasis-initiating cells (MICs) in CTCs, overexpressing genes associated with mesenchymal-, stem-, and neuroendocrine ( E)-cells. CTCs with MIC phenotype, remarkably recruit and re-program non-tumorigenic PC cells from primary tissues to mimic the MIC phenotype and gain tumorigenic and bone colonizing capability. Table 3. Incidence of tumor formation and metastasis of CTCs from prostate (PCa), pancreas (Pan), kidney (RCC), and breast (BCa) cancers in mice. In vitro cultured CTC lines of prostate, pancreas, kidney, and breast cancers form subcutaneous and bone tumors and induce metastases to soft tissues in mice.

Total Metastases

0/10 4/10 0/5 0/5 0/5

PCa (861) 5/6 (83%) 4/4 (100%)

(0%) (40%) (0%) (0%) (0%)

0/10 2/10 1/5 1/5 0/5

PCa (870) 5/6 (83%) 3/4 (75%)

(0%) (20%) (20%) (10%) (0%)

0/10 0/10 0/5 0/5 0/5

PCa (887) 6/6 (100%)

(0%) (0%) (0%) (0%) (0%)

0/10 3/10 0/5 0/5 1/5

PCa (847) 6/6 (100%) 4/4 (100%)

(0%) (30%) (0%) (0%) (20%)

0/10 2/10 1/5 1/5 0/5

PCa (71 IAD) 5/6 (83%) 3/4 (75%)

(0%) (20%) (20%) (10%) (0%)

0/10 1/10 0/5 3/5 3/5

PCa (711BD) 6/6 (100%) 4/4 (100%)

(0%) (10%) (0%) (60%) (60%)

0/10 0/10 0/5 0/5 0/5

PCa (711 CD) 6/6 (100%) 2/4 (50%)

(0%) (0%) (0%) (0%) (0%)

0/10 3/10 3/5 3/5 0/5

PCa (838) 6/6 (100%) 3/4 (75%)

(0%) (30%) (60%) (60%) (0%)

0/6 0/6 0/6 0/3 0/3

PCa (889) 6/6 (100%) 2/4 (50%)

(0%) (0%) (0%) (0%) (0%)

0/6 2/3 2/3 2/3 0/3

Pea (903) 6/6 (100%) 0/4 (0%)

(0%) (33%) (66%) (66%) (0%)

1/8 2/8 0/8 1/4 0/5

PCa (913) 4/6 (66.6%) 2/4 (50%)

(10%) (40%) (0%) (25%) (0%)

1/8 2/8 0/8 1/4 0/5

PCa (999) 6/6 (100%) 2/4 (50%)

(10%) (40%) (0%) (25%) (0%)

0/10 3/10 0/5 0/5 0/5

Pan (752) 6/6 (100%) 4/4 (100%)

(0%) (30%) (0%) (0%) (0%)

Pan (CS-P- 1/10 4/10 0/5 1/5 0/5

6/6 (100%) 4/4 (100%)

26Ca) (10%) (40%) (0%) (20%) (0%)

0/6 6/6 0/5 5/5 0/5

Pan (1122) 6/6 (100%) 2/4 (50%)

(0%) (100%) (0%) (100%) (0%)

Pan (1287- 6/6 (100%) 2/4 (50%) 0/10 0/10 0/10 0/5 0/10 S2) (0%) (0%) (0%) (0%) (0%)

0/10 4/10 0/10 3/5 0/10

PDAC-5-S2 6/6 (100%) 4/4 (100%)

(0%) (40%) (0%) (60%) (0%)

0/10 0/10 0/5 0/5 0/5

RCC (866) 6/6 (100%) 4/4 (100%)

(0%) (0%) (0%) (0%) (0%)

0/10 0/10 1/5 1/5 1/5

RCC (923) 6/6 (100%) 3/4 (75%)

(0%) (0%) (20%) (20%) (20%)

RCC (865) 0/6 (0%) 0/4 (0%)

Bca (1188- 0/10 2/10 0/10 2/5 3/5

6/6 (100%) 0/4 (0%)

Sl) (0%) (20%) (0%) (40%) (60%)

Methods are established expanding CTCs in culture and CTC-PDX models in mice. CTCs with MIC phenotype are shown to promote tumorigenic and metastatic potential in primary non-tumorigenic PC cells. MICs can be developed as a novel diagnostic, prognostic and therapeutic targets. PBMCs collected from 3 prostate cancer patients were used to establish 10 CDXs in mice. CDXs were found to express variable levels of prostate epithelial and cancer-associated biomarkers, including EpCAM, AR, PSA and PSMA. Metastasis- initiating cells (MIC) are identified in CTCs, by the mQDL method, demonstrating the overexpression of genes associated with mesenchymal-, stem-, and neuroendocrine cells. CDXs consist of MIC and non-MIC cells in which cellular interactions occur between tumorigenic/metastatic (MIC) and non-tumorigenic (non-MIC) populations of cells in the tumors. We observe that MIC can recruit and reprogram non-MIC or indolent prostate cancer cells to participate in tumorigenic and metastatic processes. Therapeutic interruption of MIC -non-MIC communication reduced tumor burden and the aggressiveness of prostate cancer cells in mice.

EXAMPLE 5

Circulating tumor cells (CTCs) represent an important component of liquid biopsy, which have the potential of predicting cancer metastasis and therapeutic responsiveness. The number, genomic alteration, and phenotypic feature of CTCs could offer unique insights of cancer plasticity and virulence. Such studies are limited by the recovery of adequate number of live CTCs for thorough evaluation since the number of CTCs in blood samples is often small. This deficiency can be overcome by expanding CTCs in ex vivo culture prior to biochemical and molecular characterization.

Peripheral blood samples, in the form of packed blood cells after removal of plasma, were scavenged from clinical laboratory. Mononuclear cells were isolated following ammonium chloride hemolysis and cultured in defined medium, which was formulated in our laboratory. Surface epithelial marker stains were used to detect CTC expansion, and cytotoxicity assays were conducted to evaluate the sensitivities of cultured CTCs to chemotherapeutic or differentiation agents.

Blood samples of a cohort of 204 patients with primary prostate cancer were subjected to ex vivo culture, together with 62 samples obtained from repeated visits by 8 patients with castration-recurrent prostate cancer (CRPC). A rigid culture procedure led to the finding that, under the defined culture conditions, a CTC-like population was formed from the blood of 38% of primary cases, versus an almost all (97%) of the samples from CRPC patients. Immunologic staining identified substantial numbers of the population expressing epithelial markers, suggesting the expansion of CTCs of the epithelial cell lineage. Interestingly, except for an antibiotics and a histone deacetylase inhibitor, clinically used therapeutic agents consistently yielded negative cytotoxic effects against the expansion of CTCs in culture.

A CTC-like population can be cultured consistently from patient's blood samples, while the presence of epithelial marker presence supports the cancer origin of the expanded CTCs. Further optimization of the ex vivo culture protocol could provide unique opportunities for reliable characterizations of the genomics, gene expression, and behavior of the CTCs. Without being bound to any particular theory, results of this study could lead to improved patient care in predicting disease progression and selecting effective therapy, at an individual basis for cancer targeting.

The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention.