FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under award number

U01CA128416 awarded by the National Cancer Institute of the National Institutes of Health and support under award number R56AI118464 awarded by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. The government has certain rights in the invention. OVERVIEW

Various diseases have cells with antigens that can be targeted for treatment purposes and/or other purposes. Epitopes of the antigens associated with diseases, such as cancer, can be used as biomarkers for targeting diagnosis or treatment. In cancer, circulating tumor cells (CTCs) and cancer stem cells (CSCs) have been identified as a prognostic factor in the development of cancer and progression to metastases. CSCs, in particular, are a progenitor of metastases and relapse in cancer patient. CTCs are rare cancer cells in blood circulation that are shed from the primary tumor and play key roles in disseminating metastatic tumor cells to remote sites. Detection of CTCs has been explored as a non-invasive "liquid biopsy" for tumor diagnosis and prognosis. CSCs, which are sometimes referred to as circulating cancer stem cells (cCSCs), belong to a subpopulation of undifferentiated tumor cells with embryonic characteristics. With epithelial-to- mesenchymal transition traits, CSCs are capable of escaping the primary tumor and entering the bloodstream as a subset of CTCs with high metastatic potential. Developing targeted immunotherapy to eradicate metastatic tumor cells in vivo is beneficial for precision tumor medicine.

Identifying CTC-specific and CSC-specific cell-surface biomarkers is substantially challenging. First, CTCs and CSCs originate from self-epithelial cells. Biomarkers currently in use for detection and isolation of these cells, such as the cell surface marker epithelial cell adhesion molecule (EpCAM), are commonly expressed by normal epithelial cells. The lack of specific immunological targets to detect CTCs and CSCs is a road block to development of highly specific immunotherapy against tumor metastasis. Second, CTCs and CSCs are extremely rare. The number of CTCs detectable in blood is approximately 1 CTC per 10 ⁶ — 10 ⁷ of peripheral mononuclear blood cells (PBMCs). The number of detectable CSCs is even smaller than CTCs. Conventional molecular and cellular techniques may not detect them and the molecular targets they express.

The above issues as well as others have presented challenges to identifying and isolating human antibodies for a variety of applications. SUMMARY

The present disclosure is directed to overcoming the above-mentioned challenges and others related to detecting CSCs in blood circulation (cCSCs) or tissue-associated CSCs (tCSCs) as discussed above and in other implementations. The present disclosure is exemplified in a number of implementations and applications, some of which are summarized below as examples.

Various aspects of the present disclosure are directed to methods for detecting CSCs in a biological sample of a subject via a glycan biomarker. The detection can be directly from a blood sample or tissue sample, such as from a human patient. A physical interaction between the biological sample and an antibody can be caused by exposing the biological sample to the antibody. The exposure of the biological sample to the antibody, when the biomarker is present within the sample, results in specific binding of the antigen by the antibody. In specific embodiments, the physical interaction is caused by forming an immuno-assay, such as an enzyme-linked immunosorbent assay (ELISA) or an immuno- sandwich, a microarray and/or nanoarray. The antibody or the biological sample is immobilized on a substrate and subsequently exposed to the other of the biological sample or the antibody. The biological sample, which is obtained from a subject suspected of or known to have cancer, is suspected of containing CSCs. CSCs are present in blood or in tissue at low frequencies. A presence of CSCs in the biological sample can be determined by detecting binding between the antibody and the glycan biomarker. In specific embodiments, the antibody is labeled with a detection agent. For example, the (targeting) antibody can be labeled with a fluorescent, enzymatic, or radioactive label. In some embodiments, the detection agent is applied to the antibody prior to exposing the biological sample to the antibody. In other embodiments, the detection agent is applied to a substrate after exposing the biological sample to the antibody, and may bind to the antigen-bound antibody via the FC segment of the antibody.

The glycan biomarker is an epitope of a blood group precursor antigen. The blood group precursor can include Tij II 20% fraction 2nd 10% (Tij II), OG 10% 2X (OG), and a combination thereof. More specifically, the glycan biomarker is an O-core cryptic epitope of a blood group precursor antigen. The glycan biomarker includes one or more of polylactosamine chains and/or oligosaccharide chains with branches of Πβ

(Gaipi,4GlcNAcpi,6), ip(Gaipi,3GlcNAcpi,6), and/or Πβ/Ιβ (Gal βΐ, 4/3GlcNAc β1,6) - moieties. For example, the glycan biomarker can include at least one chain and/or a plurality of chains selected from the group consisting of polylactosamine chains, oligosaccharide chains, and combinations thereof, and with the at least one chain and/or the plurality of chains having and/or including branches selected from the group consisting of Πβ (Gaipi,4GlcNAcpi,6), ip(Gaipi,3GlcNAcpi,6), Πβ/Ιβ (Gal βΐ, 4/3GlcNAc β1,6) - moieties, and combinations thereof.

In a number of specific embodiments, the antibody can include an anti -tumor glycan monoclonal antibody, such as CI, HAE3, or Gl . For example, the glycan biomarker can be associated with cancer, and more specifically, epithelial cancer, although embodiments are not so limited. Determining the presence of the CSCs in the biological sample can include the use of optical circuitry to detect the binding via the detection agent. In response to detecting the binding (e.g., via the detection agent bound to the antibody), the subject is analyzed for particular immunotypes of cancer and/or the presence of metastatic cancer.

In a number of related and more specific embodiments, the biological sample can be further analyzed. The biological sample can include a cell population. Responsive to the exposure of the biological sample to the antibody, the cell population can be analyzed. For example, the cell population can be identified and characterized based on the detected binding, including immunotyping the CSCs and/or the CTCs, detecting a presence of metastatic cancer in the subject responsive to the detected presence of the CSCs within the cell population and/or characterizing at least a portion of the cell population as CSCs based on the binding. The cell population can be characterized using morphological and immunological analysis via a fiber-optic array scanning technology (FAST) scan to distinguish CSCs from benign cells in the cell population. Additionally, the cell population can be classified as CTCs, CSCs, and benign by the morphological and/or immunological analysis.

Detecting the CSCs in the biological sample can be used for detecting cancerous cells, diagnostic and/or treatment purposes. For example, a metastatic cancer can be detected in the subject responsive to immunotyping the CSCs and based on the detected presence of the CSCs and/or monitoring the presence or absence of CSCs during treatment or therapy of the subject for epithelial cancer. The detected CSCs can be used for detecting cancerous cells associated with a variety of cancers such as indicating a presence of breast cancer, ovarian cancer, lymphoma, myeloma, lung cancer, rhabdomyosarcoma, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, pancreatic cancer, urinary bladder cancer, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, cervical cancer, endometrial cancer, adrenal cortical cancer, and prostate cancer and/or for detecting specific immunotypes of CSCs and/or CTCs.

In more specific embodiments, analyzing the cell population can include detecting a presence or a level of the CSCs within the cell population of the biological sample responsive to the identified binding. The cell population is then analyzed based on the detected presence or the level of the CSCs and/or immunotyping the CSCs for monitoring of a status of epithelial cancer. Analyzing the cell population can include identifying a cell population indicative of epithelial cancer and/or immunotyping of the detected CSCs in response to the detected presence of the CSCs. In more specific embodiments, immunotyping the CSCs (and/or CTCs) can be based on the detected binding of the antibody, such as the percentage of cells that bind to the antibody (e.g., percentage of staining) and intensity of the signal associated with binding (e.g., intensity of the staining).

In other specific and related embodiments, analyzing the cell population includes comparing the detected level of the CSCs within the cell population to a previously determined level of CSCs of a different biological sample of the subject. For example, an efficacy of a drug candidate compound for treatment of cancer or other treatment procedure in a subject can be determined. The drug candidate compound can be administered to the subject (or other treatment can be performed) suspected of having cancer. Biological samples are obtained from blood or tissue of the subject before and after treatment with the drug candidate compound (or other treatment). As described above, the biological samples are exposed to the antibody and the cell population of the biological sample is analyzed by detecting the presence or absence of the glycan biomarker and identifying levels of the CSCs in the biological samples before treatment with the drug candidate compound compared to after treatment with the drug candidate compound (or before other types treatment and after the other types of treatment). The presence of a decreased number of the CSCs after treatment compared to a number of the CSCs before treatment can indicate a relative efficacy of the drug candidate compound in treating the cancer in the subject.

More specific example methods can include exposing immobilized cells from a biological sample (e.g., a blood sample or tissue sample) to an antibody. The biological sample can be immobilized or fixed on a substrate and exposed to an antibody that is labeled via the detection agent. If CSCs are present, the antibody binds to the glycan biomarker, which can be identified by scanning the immobilized cells. From the scan, cells (among the immobilized) that are bound to the antibody are identified as cancerous. The bound cells can be further classified as CTCs or CSCs, in some specific embodiments, using a FAST scan. And, the CTCs and/or CSCs can be immunotyped responsive to the detected binding, as described above.

Other specific embodiments are directed to an apparatus which includes the optical circuitry (e.g., fiber optic scanner), a substrate, and processing circuitry. An example of optical circuitry can include a fiber optic bundle array, a laser, and imaging circuitry (e.g., camera). In specific aspects, the optical circuitry is used to scan the biological sample, as immobilized and exposed to an antibody to identify antibodies bound to the glycan biomarker. The optical circuitry and processing circuitry can further assess the cell population. For example, the assessment can include immunotyping the CSCs and/or CTCs within the cell population and/or classifying the cell population of the biological sample as benign cells, CSCs, and CTCs. In some specific embodiments, the processing circuitry can determine a level of CSCs (and/or CTCs) present in the cell population, which can be used to diagnose the subject, monitor progress of the subject, and/or evaluate efficacy of treatment and/or a drug candidate. The apparatus can include various additional , such as processing circuitry for controlling the various instruments, memory circuit for storing data sets, and various computer-readable instructions for controlling the optical circuitry and computer-executable instructions (e.g., software) for analyzing data obtained therefrom.

In various embodiments, gly comic tools are used to uncover the glycan biomarkers of CTCs and CSCs. Surprisingly, the glycan biomarker in experimental embodiments is identified as being a cell-surface glycan biomarker for CSCs and CTCs. This is particularly surprising given the morphological, functional, and size differences between CSCs and CTCs, as well as the very low concentration levels of CSCs present in biological samples. Example gly comics tools, in specific embodiments, are introduced to probe cell- surface glycan biomarkers of breast CTCs (bCTCs). Specifically, carbohydrate microarrays are applied to screen anti-tumor antibodies to identify those that are specific for tumor glycan biomarkers. A FAST scan is applied to verify whether the identified targets are CTC-specific cell-surface biomarkers and/or CSC-specific cell-surface biomarkers. The above overview is not intended to describe each illustrated embodiment or every implementation of the present disclosure. The figures and detailed description that follow more particularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Various example embodiments may be more completely understood in

consideration of the following detailed description in connection with the accompanying drawings, in which:

FIG. 1 illustrates an example apparatus in accordance with various embodiments; FIG. 2 illustrates an example process for detecting the presence of CSCs and specific immunotypes of CSCs in a biological sample, in accordance with various embodiments;

FIG. 3 illustrates an example process for assessing a cell population of a biological sample and/or assessing efficacy of a treatment using a glycan biomarker, in accordance with various embodiments;

FIGs. 4A-4C illustrate example substrates for detecting the presence of the glycan biomarker, in accordance with various embodiments;

FIG. 5A illustrates an example of a gly can-array, in accordance with various embodiments;

FIG. 5B illustrates an example of a resulting glycan biomarker, in accordance with various embodiments;

FIG. 5C illustrates an example of optical circuitry, in accordance with various embodiments;

FIG. 5D illustrates an example of a resulting immunotype of CTCs and CSCS from a scan of blood samples using optical circuitry, in accordance with various embodiments;

FIG. 6 illustrates an example of a blood group substance with a conserved O-glycan core and the epitopes recognized by antibody CI, HAE3, and Gl, in accordance with various embodiments;

FIGs. 7A-7B illustrates example experimental results of a carbohydrate microarray used to identify glycan biomarkers using antibody HAE3, in accordance with various embodiments;

FIGs. 8A-8B illustrate example experimental results of expression of surface tumor biomarkers, in accordance with various embodiments; and FIGs. 9A-9B illustrate example experimental results of characterizing blood samples from five Stage IV breast cancer patients, in accordance with various

embodiments;

FIGs. 10A-10D illustrate example experimental results of use of carbohydrate microarrays for detection of glycan biomarkers using antibodies Gl and HAE3, in accordance with various embodiments; and

FIG. 11 illustrates example experimental results of staining cell lines using antibody Gl and CSC biomarkers, in accordance with various embodiments.

While various embodiments discussed herein are amenable to modifications and alternative forms, aspects thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the disclosure to the particular embodiments described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure including aspects defined in the claims. In addition, the term "example" as used throughout this application is only by way of illustration, and not limitation.

DETAILED DESCRIPTION

Aspects of the present disclosure are believed to be applicable to a variety of different types of methods, systems and arrangements used for detecting cancer stem cells (CSCs) in a biological sample of a subject. A presence of CSCs is determined, more specifically, by detecting the presence of a surface-CSC glycan biomarker. The glycan biomarker is an epitope of a blood group precursor specifically recognizes (e.g., binds to) an anti-tumor (glycan) monoclonal antibody. In certain implementations, aspects of the present disclosure have been shown to be beneficial when used in the context of exposing the biological sample, such as a human blood sample or tissue sample (e.g., biopsy specimen), to an antibody and detecting binding between the antibody and the glycan biomarker using a detection agent. In other implementations, the cell population of the biological sample is analyzed by immunotyping the CSCs, classifying cells as benign, CSCs, and circulating tumor cells (CTCs) based on the identified antibodies bound to the glycan biomarker. For example, the cells can be classified as CSCs in blood circulation (cCSCs), tissue-associated CSCs (tCSCs), and CTCs in blood circulation (cCTC). The analysis can be used to identify cancerous cells within the biological sample, monitor progress of cancer in the subject, and/or determine an efficacy of treatment for the subject over time. While the present disclosure is not necessarily limited to such applications, various aspects of the disclosure may be appreciated through a discussion of various examples using this context.

Accordingly, in the following description various specific details are set forth to describe specific examples presented herein. It should be apparent to one skilled in the art, however, that one or more other examples and/or variations of these examples may be practiced without all the specific details given below. In other instances, well known features have not been described in detail so as not to obscure the description of the examples herein. For ease of illustration, the same reference numerals may be used in different diagrams to refer to the same elements or additional instances of the same element.

Various embodiments in accordance with the present disclosure include systems, apparatuses and methods for identifying CSCs present in a biological sample using an antibody that is specific for, e.g., binds to, a glycan biomarker. Tumor biomarkers can be overexpressed by cCSCs and/or tCSCs, and can be used for targeted immunotherapy against tumor metastasis. In specific experimental embodiments, suprisingly, the tumor biomarkers are surf ace-gly can biomarkers of CSCs, as well as CTCs, and are used to detect CSCs and/or immunotype the CSCs. CSCs, as previously described, have a variety of functional, morphological, and size differences, including different functional groups, as compared to CTCs, such as co-expression of CSC markers, such as CD44+CD24- phenotype, etc. The glycan biomarker, more specifically, is an epitope of a blood group precursor antigen. For example, the glycan biomarker is an O-core cryptic epitope of a blood group precursor antigen, the blood group precursor antigen including Tij II 20% fraction 2nd 10% (Tij II) and/or OG 10% 2X (OG). The glycan biomarker can bind to an anti-tumor antibody, such as CI, HAE3, and/or Gl . Presence of the glycan biomarker can indicate the presence of cancerous cells in the subject, such as metastatic cancer in the subject and/or early stage cancer. As used herein, a glycan biomarker is interchangeably referred to as "CSC/CTC glycan biomarker" and "cell-surface CSC/CTC biomarker".

As may be appreciated by one of ordinary skill, experimental antibodies are proteins that can be used by the immune system to detect, neutralize, and/or kill various target cells which may be harmful to the host organism, such as tumor cells and pathogens. The antibody can recognize and bind to a unique molecule of the target cell, called an antigen, via a binding region of the antibody. An antibody bound to the antigen can directly or indirectly (e.g., by triggering other parts of the immune system), detect, neutralize, and/or kill the target cell. For example, the antibody HAE3, CI, and/or Gl binds to the glycan biomarker, which is an epitope of an antigen associated with CSCs and CTCs, and the binding is used to detect for the presence of CSCs and/or immunotype the CSCs and/or CTCs. For more general and specific information on the antibodies HAE3, CI, and Gl, which are sometimes herein referred to as anti -human carcinoma antigen (HCA) antibodies, reference is made to US Patent No. 5,693,763, entitled "Antibodies to human carcinoma antigen", filed June 6, 1995; Rongshan Li, et. al, "Frequent Expression of Human Carcinoma- Associated Antigen, a Mucin-Type Glycoprotein, in Cells of Prostatic Carcinoma", Archives of Pathology & Laboratory Medicine: December 2004, Vol. 128, No. 12, pp. 1412-1417; and Jorge L. Yao, et. al, "Overexpression of Human Carcinoma-Associated Antigen in Urothelial Carcinoma of the Bladder", Archives of

Pathology & Laboratory Medicine: July 2004, Vol. 128, No. 7, pp. 785-787, each of which are fully incorporated herein for their general and specific teachings related to the antibodies HAE3, CI, and Gl. As provided by the above-noted references, antibody HAE3 is deposited at the American Tissue Type Culture Collection (ATCC), Rockville, MD under accession no. HB-9467. HAE3, from which CI is prepared (e.g., is subclone of the parent murine hybridoma, HAE3, as described further below) and Gl are available from Egenix, Inc., located in Rochester, New York, now Bantam Pharmaceutical, LLC. Gl is additionally available from Creative BioMolecules, Inc., located in Hopkinton, Massachusetts, now Curis, Inc., located in Cambridge, Massachusetts. HAE3 is additionally available from Maine Biotechnology Services, located in Portland, Maine.

In specific experimental embodiments, the glycan biomarkers of CSCs are identified using a carbohydrate microarray. The carbohydrate microarray includes a panel of carbohydrate antigens that are scanned against anti-tumor monoclonal antibodies (mAbs) to identify potential glycan biomarker therefrom. Flow cytometry and fiber-optic array scanning technology (FAST) is then applied to determine if the identified antigens are tumor-specific cell-surface biomarkers, e.g., are CSC glycan biomarkers. The antibodies identified can also be validated for performance in CSC and/or CTC-detection and immunotyping analysis using cancer patient's blood samples.

The glycan biomarkers can be used for identification of cancerous cells and/or treatment of the organism, such as for cancer treatment. In specific embodiments, CSCs in a biological sample of a patient are identified by exposing the biological sample to the antibody, e.g., the anti-tumor mAb, and determining the presence of CSCs in the biological sample by detecting binding between the antibody and the glycan biomarker. Exposing the biological sample to the antibody can cause a physical interaction between the antibody and glycan biomarkers present in the biological sample. Antibodies bound to the glycan biomarker are detected using a detection agent, which binds to the antibody or another epitope of the antigen associated with the glycan biomarker.

In specific embodiments, the physical interaction includes immobilizing the biological sample to a substrate, such as a glass substrate, a bead, or a nano or micro-array, and exposing the immobilized biological sample to the antibody. In other embodiments, the antibody is immobilized to a substrate and exposed to the biological sample. The substrate can be scanned to identify binding between the antibody and the glycan biomarker using optical circuitry by applying a detection agent that is conjugated to or specific for the anti-glycan antibody (e.g., the antibody bound to the glycan biomarker). The detection agent can be applied to the antibody prior to exposing the biological sample to the antibody or can be applied after exposing the biological sample to the antibody. The detection agent is man-made. In response to identifying binding, the presence of CSCs within a cell population of the biological sample is detected and the CSCs (as well as the CTCs) can be immunotyped.

The detection agent can include a label and/or a second antibody. For example, the antibody itself may be labeled with a detection agent, such as with a fluorescence, enzymatic, or radioactive label and/or a second antibody (e.g., an anti-antibody that binds to the antibody). The second antibody, which is labeled, can be exposed to the substrate after allowing for binding between the biological sample and the antibody to occur and washing away unbound antibody substance. In other embodiments, the antibody can be immobilized to the substrate and then the immobilized antibody is exposed to the biological sample. A second antibody, that binds to a different epitope of the antigen and is labeled, is subsequently exposed to substrate.

The detected CSCs within the biological sample and/or the cell population of the biological sample can be further analyzed in various more specific embodiments. The analysis can include characterizing at least a portion of the cell population as CSCs responsive to the detected binding and using morphological and immunological analysis via a FAST scan to distinguish CSCs from benign cells and/or to immunotype the CSCs in the cell population. For example, the cells in the cell population can be classified as benign cells, CSCs, and CTCs based on the identified bound antibodies, differences in

morphology, and the absence of an antibody bound to a glycan biomarker at portions of the substrate. The CSCs can further be immunotyped to classify a type of cancer present in the biological sample, such as a cell line of breast cancer, and which can be used to refine treatment of the subject. Example treatment or therapy can include administration of a specific antibody or other drug candidate that targets the immunotype of CSCs and/or CTCs.

Alternatively and/or in addition, in various embodiments, the detection of CSCs can be used to identify metastatic cancer, assess efficacy of treatment of the user, and/or assess efficacy of a drug candidate compound. In some embodiments, the detection of the presence of CSCs can be used to detect a presence of metastatic cancer in the subject. Additionally, the cell population can be characterized to determine the level (e.g., frequency) of CSCs presence and/or the immunotype of CSCs. The level and/or immunotype of CSCs can provide an indication of the stage of cancer and can be monitored over time to assess the development of cancer in the subject and the efficacy of treatment. In some specific embodiments, the treatment can be adjusted in response to the assessment. For example, the efficacy of a drug candidate compound can be assessed by comparing CSC levels and immunotype in a biological sample of subject prior to treatment with the drug candidate compound to CSC levels and immunotype in another biological sample of the subject after treatment. Further, the glycan biomarkers can be used for targeted immunotherapy against tumor metastasis based on the immunotype of CSCs and/or CTCs.

Various other embodiments of the present disclosure are directed toward an apparatus used to perform the various methodologies described herein. The apparatus can include (high speed) optical circuitry and processing circuitry. An example optical circuitry is a fiber optic scanner, which includes a fiber optic bundle array, a laser, and imaging circuitry (e.g., camera), such as FAST as further described herein. The FAST system scans a biological sample with the laser and collects a high resolution image of the sample using the fiber optic array. As previously described, in specific embodiments, the biological sample is plated on a substrate (e.g., glass slide) and can be attached to a stage. The substrate is treated with a fluorescently, enzymatically, or radioactively labeled antibody (e.g., an antibody with a detection agent applied) and is scanned using the optical circuitry. The optical circuitry can scan the entire biological sample and generate a digital image of the locations of an antibody bound to a glycan biomarker (via label). The optical circuitry and/or processing circuitry can identify cells (e.g., locations on the substrate) that do not result in binding of the antibody, and tissue and/or cellular compartment locations of the bound antibody. The processing circuitry can, responsive to the identification, identify CSCs, including cCSCs and tCSCs and, optionally, other CTCs. The classification between CSCs and CTCs can be made based on size, shape, and/or co-expression of CSC markers, such as CD44+CD24- phenotype, etc. Further, the CSCs and/or CTCs can be immunotyped based on the detected binding of the antibody. In other embodiments, the antibody can be plated on the substrate and treated with the biological sample (and subsequently exposed to another antibody that is labeled prior to the scan). Although this disclosure describes scanning with the FAST system, embodiments are not so limited and one skilled in the art will recognize that other types of scanning can also serve the same purpose including those based on multispectral and/or hyperspectral imaging.

The apparatus can additionally include various circuitry such as processing circuitry for controlling the various instruments, memory circuitry for storing data sets, and various computer-readable instructions for controlling the optical circuitry and processing circuitry for analyzing data obtained therefrom. Optionally, in various specific-embodiments, the apparatus can include a microengraving platform. The microengraving platform includes a multiple-well array, an immuno-assay, and/or an immuno-sandwich.

Turning now to the figures, FIG. 1 illustrates an example apparatus in accordance with various embodiments. The apparatus 102 can scan a biological sample to detect the presence of CSCs using an antibody.

The apparatus 102 includes optical circuitry 106 combined with processing circuitry 108. The optical circuitry 106 can include a platform termed FAST, as further illustrated and described in connection with FIG. 5C. The optical circuitry 106 can be used to directly identify antibodies bound to glycan biomarkers from a biological sample immobilized on a substrate 104 (such as the blood sample 103 from a human 101 illustrated by FIG. 1). An example scanning technology, the FAST system is based on the concept of "Xeroxing" a blood sample with a scanning laser and collecting a high resolution capture image of the sample using a densely packed fiber optic array bundle. The FAST system can allow for rapid scanning of cells at speeds of between 1 million and 25 million cells per minute. For example, the optical circuitry 106 can scan the substrate 104 containing or otherwise associated with a biological sample of a subject. The subject is suspected of having (or known to have) cancer. The biological sample can include a cell population that is exposed to an antibody, either a labeled antibody exposed to the biological sample or labeled via a secondary antibody (e.g., a labeled anti-antibody) via formation of an immuno-sandwich. In other related embodiments, the antibody can be immobilized to the substrate 104 and exposed to the biological sample. After washing away unbound cells from the biological sample, the substrate 104 can be exposed to a secondary antibody that is labeled and that binds to another epitope of an antigen associated with the glycan biomarker (e.g., another epitope of the blood group precursor antigen).

In specific embodiments, the exposure of the biological sample to the antibody, when the biomarker is present within the biological sample, causes (specific) binding between the antibody and the glycan biomarker. The antibodies bound to the glycan biomarker are labeled, in various embodiments, by applying a detection agent. The detection agent is man-made and can include a fluorescent, enzymatic and/or radioactive label that is configured to bind to the antibody and/or a second antibody configured to bind to either the antibody or another epitope associated with the glycan biomarker. The second antibody is labeled with a fluorescent, enzymatic and/or radioactive label. The detection agent can bind to antibody or the glycan biomarker via an epitope of the antibody or an epitope associated with the glycan biomarker. In some embodiments, the detection agent is applied to the antibody prior to exposing the biological sample to the antibody. In other embodiments, the detection agent is applied to the substrate 104 after exposing the biological sample to the antibody, and may bind to either the antibody or the epitope associated with the glycan biomarker (e.g., to the antigen associated with the glycan biomarker and via another epitope than the glycan biomarker).

The optical circuitry 106 scans for antibodies bound to the glycan biomarker that are bound to the substrate 104 responsive to the exposure to the antibody (or biological sample) and provides an image indicative of locations of the bound antibodies to the processing circuitry 108. For example, using the optical circuitry 106, glycan biomarkers bound to the antibody are identified via the detection agent and used to identify respective CSCs. The optical circuitry 106 and processing circuitry 108 can, responsive to the identified glycan biomarker bound to an antibody, provide an indication of the detected presence of CSCs. The presence of CSCs can indicate a presence of metastatic cancer in the subject and/or early stage cancer.

In some embodiments, the optical circuitry 106 and processing circuitry 108 can further assess a cell population of the biological sample (e.g., the blood sample 103 or a tissue sample, such as a biopsy specimen). The assessment can include identification and characterization of the cell population, such as using morphological and immunological analysis to characterize at least a portion of the cell population as CSCs, CTCs or benign. More specifically, identified glycan biomarkers bound to the antibodies can be used to distinguish CSCs and CTCs from benign cells. CSCs and CTCs can be distinguished from one another by identifying size, immunotype and morphology differences between the cells bound to the antibody via an optical scan of the substrate 104 by the optical circuitry 106. In more specific embodiments, once the cancerous (CSC or CTC tumor) cells are identified, further investigation can examine the characteristics of single cells by immunohistochemistry and other analyses such as fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and single nucleotide polymorphism (SNP) analysis.

In some specific embodiments, the detection of CSCs is used to monitor treatment of the subject, as further illustrated by FIGs. 3-4. Additionally and/or alternatively, the detection of CSCs can be used assess the efficacy of a drug candidate compound, as illustrated by FIG. 4 and further discussed herein.

In various specific embodiments, exposing the biological sample to the antibody can include forming an immuno-assay. For example, a glass substrate is coated with a biological sample suspected of containing the glycan biomarker (e.g., the epitope of the antigen) and used to form an immuno-sandwich by exposing the immobilized biological sample to the antibody (and optionally, a labeled anti-antibody). The immuno-sandwich is used to detect antibodies bound the glycan biomarkers. The glass substrate can be treated with a detection agent that includes a labeled anti-antibody, and the optical circuitry 106 scans the glass substrate to identify a signal (e.g., fluorescence) indicative of the labeled anti-antibody. If a CSC is present, the antibody binds to the glycan biomarker of the CSC on the glass substrate and the anti-antibody binds to the antibody. For example, the detection agent can bind to the FC segment of the antibody. Subsequently detected label (associated with the labeled anti-human detection antibody) indicates presence of the glycan biomarker. The anti-antibody can include various organism-specific antibodies, such as an anti-human antibodies, anti-horse antibodies, anti-dog antibodies, anti-cat antibodies, anti-fish antibodies, anti-cattle antibodies, anti-bird antibodies, among other organisms that have white blood cells which produce antibodies. As may be appreciated by one of ordinary skill in the art, the detection antibody used can be specific to the organism, such as an anti -horse detection antibody or an anti-dog detection antibody. Similarly, embodiments are not limited to first immobilizing the biological sample and can include immobilizing the antibody, as further illustrated by FIGs. 4A-4C.

FIG. 2 illustrates an example process for detecting the presence of CSCs and specific immunotypes of CSCs in a biological sample, in accordance with various embodiments. More specifically, FIG. 2 illustrates an example method for detecting the presence of CSCs from a biological sample of a subject.

As illustrated by FIG. 2, a blood sample 213 is obtained from a human 211.

Although the embodiment illustrates the blood sample 213 being obtained directly from a human 211, embodiments are not so limited and the blood sample may be previously obtained and/or may be from other organisms and used to identify antibodies used to treat the particular organism (e.g., other vertebrates, such as horses, dogs, cats, cattle, fish, birds).

The blood cells are immobilized, such as on a substrate, and attached to an apparatus 210. The apparatus 210 can include the apparatus 102, as previously illustrated and described by FIG. 1. In specific embodiments, the cell population for a blood sample is either fixed to one or more glass slide plates, or immobilized in a soft matrix such as agar or matrigel to maintain cell viability.

At 212, the immobilized blood cells are exposed to an antibody. For example, the immobilized blood cells can be exposed to an anti -tumor mAb, such as CI, HAE, and/or Gl. The exposure, in specific examples, includes treating the substrate with a

(fluorescently) labeled antibody. Blood cells that exhibit the glycan biomarker can bind to the labeled antibody. Further, as previously described, a detection agent can be applied, which can occur prior to or after immobilizing the blood cells. The detection agent can bind to the antibody bound to the glycan biomarker via an epitope of the antibody or another epitope associated with the glycan biomarker, as previously discussed.

At 214, the binding between the glycan biomarker of CSCs and/or CTCs and the antibody can be identified by scanning the substrate. The scan, in specific embodiments, can be by the optical circuitry, such as a FAST scan of the substrate, which identifies and locates the glycan biomarker-bound antibodies via the detection agent.

At 216, responsive to the detected binding, the presence of CSCs is determined. The presence of CSCs can be used to identify cancerous cells, diagnose the user with cancer, and/or further identify the stage of cancer. In specific embodiments, the cell population, at 222, is further analyzed and/or profiled. For example, the CSCs and/or CTCs can be immunotyped, in specific embodiments and as further described herein.

Alternatively and/or in addition, the assessment can include classifying the cell population and determining levels (e.g., percentage of total cell population) of CSCs, CTCs, and/or benign cells within the cell population. The levels can be compared to thresholds and/or previously determined cell levels to assess the user progress, stage of cancer, efficacy of treatment, and/or to adjust a treatment. The presence of a decreased number of CSCs compared to a number of CSCs during a previous assessment can indicate successful treatment and/or regressing cancer in the subject. Similarly, the presence of an increased number of CSCs compared to the number of CSCs during the previous assessment can indicate treatment is not effective and/or progressing cancer in the subject.

FIG. 3 illustrates an example process for assessing a cell population of a biological sample and/or assessing efficacy of a treatment using a glycan biomarker, in accordance with various embodiments. As previously described, an apparatus, such as the apparatus 102 illustrated by FIG. 1, can be used to detect for the presence of CSCs in a biological sample of a user. The biological sample, in specific embodiments, is a blood sample of a subject suspected of having, or known to have, cancer. The blood sample comprises a cell population.

A blood sample is obtained from an organism, such as a human as illustrated, although embodiments are not so limited. At 330, the blood sample is immobilized on a substrate. In some specific embodiments, the cell population is deposited into a nanowell array. The nanowell array includes a plurality of wells arranged in an array, as further illustrated herein. Each well of the nanowell array can have an individual blood cell deposited therein. Further, the wells can include a cell culture media that allows for the cells deposited in the wells to remain viable.

At 332, a physical interaction between the blood sample and an antibody is caused by exposing the blood sample to the antibody. In some embodiments, the exposure includes exposing the substrate (e.g., glass substrate or nanowell array) to a solution containing the antibody that is labeled. The antibody is labeled with a detection agent, such as a fluorescent, enzymatic, or radioactive label and/or second antibody. The detection agent can be used for identifying the CSCs and for subsequently phenotyping the blood cells. In other specific embodiments, the substrate is further exposed, after washing away unbound antibodies or cells, to a detection agent, such as a secondary antibody (e.g., an anti-antibody or a second antibody that binds to another epitope of the antigen associated with the glycan biomarker) that is labeled. Although embodiments are not so limited, and can include exposing the substrate, which has immobilized antibodies thereon, to the biological sample.

At 334, the substrate is scanned to identify binding between the glycan biomarker and the antibody using optical circuitry. The scan by the optical circuitry can be used to identify the locations on the glass slide or other substrates that reveal discrete spots associated with the detection agent and that correspond to an antibody bound to the glycan biomarker, at 336. As a specific example, if the blood cell exhibits the glycan biomarker, the antibody binds to the glycan biomarker on the glass slide. A detection agent, such as an anti-human antibody, can be applied to the substrate. For example, the anti-human IgG antibody, which is fluorescently, enzymatically, or radioactively labeled and washed over the glass slide, binds to the antibody and results in a signal, such as fluorescent emission, when scanned by the optical circuitry.

In response to identifying antibodies bound to the glycan biomarker, at 338, the presence of CSCs can be detected. In some embodiments, detecting the presences can further include classifying the cell population. For example, CSCs can be distinguished from CTCs among the cell population based on size, morphology, and cryptological differences. In other embodiments, CSCs can be distinguished from benign cells responsive to the detected bound antibodies. Optionally, at 340, in response to the detected presences of CSCs (or in response to the identified antibodies bound to the glycan biomarker), the subject can be identified as having cancerous cells, and optionally, diagnosed with cancer. More specifically, the subject can be analyzed for having metastatic cancer.

In some specific embodiments, based on the assessment of the cell population, at 342, the CTCs and/or CSCs can be immunotyped. Immunotyping the CSCs and/or CTCs may be used to further refine the assessment of cancerous cells (and optionally, the diagnosis), at 340, such as indicating a stage or cell line of cancer. In various

embodiments, different cancer cell lines express different levels of the glycan biomarker. As an example and as further illustrated herein, two triple negative cell lines (BT-549 and MDA-MB-468) and two ER+PR+ cell lines (T-47D and MCF-7) are strongly HAE3 positive and Sk-BR-3 is intermediately HAE3 positive. Immunotyping the CSCs (and/or CTCs) can be based on the detected binding of the antibody including a percentage of cells that bind to the antibody (e.g., percentage of staining from the label of the detection agent) and an intensity of the signal associated with the binding (e.g., intensity of the staining from the label of the detection agent). More specifically, the immunotyping can be based on the percentage or level of CTCs and/or CSCs captured that express levels of the glycan biomarker above a threshold intensity (e.g., based on numeric values of 0, 1+, 2+, 3+ as illustrated by FIG. 5D). Biological samples having a percentage of cells that are bound to the antibody, e.g., percentage of stained cells, above a first threshold (e.g., 5%, 40%, 50%) and that have a signal intensity above a second threshold (e.g., 2+ or more, l+_or more) can be used to immunotype the CSCs and/or CTCs. As a specific example, a biological sample exhibiting triple negative cells lines can be associated with a percentage of stained cells of 50% or more with a signal intensity of 2+ or more, although embodiments are not so limited. For example, various different quantifications of the expression signal intensities can be used and embodiments are not limited to the numerical values of 0, 1+, 2+, and 3+.

Based on the immunotyping, at 344, candidate therapeutic agents can be identified. The candidate therapeutic agents can include antibodies identified as useful in therapy for the specific immunotype of CTCs and/or CSCs, Car-T cells, and immunotype-cytokine, among other types of specific therapy and/or agents. The term "therapeutic agent" is used herein interchangeably with the term "drug candidate". Optionally, at 346, the identified therapeutic agent(s) can be used to refine the treatment for the subject. For example, the revised treatment can include administering the therapeutic agent(s) to the subject, at 348.

In other more specific and related embodiments, the level of CSCs (and/or CTCs) can be assessed. The level of CSCs may be used to further refine the assessment of the metastatic potential of the cancerous cells (and optionally, the diagnosis). Additionally and/or alternatively, the levels of CSCs can be used to assess the efficacy of treatment for the user, to revise treatment for the user, and/or assess efficacy of a drug candidate. For example, the levels of CSCs can be compared to a previous level of CSCs and/or immunotype of CSCs which may have been determined prior to a particular treatment or earlier in time. Based on the comparison, an efficacy of treatment for the user (or of a drug candidate compound in general) can be provided. More specifically, if the CSC levels decreased after treatment, the treatment may be considered effective. By contrast, greater levels of CSCs after treatment may indicate an ineffective treatment. Optionally, based on the efficacy assessment, the treatment for the user can be revised. Example revisions can include adjusted dosages of drug compound(s), different drug compound(s), and additional treatments, among other revisions.

As a specific example, assessment of CSC levels and/or immunotype can be used to determine an efficacy of a drug candidate compound for treatment of cancer. The subject (and optionally many subjects) suspected of having cancer is administered an amount of a drug candidate compound. Biological samples are obtained from blood of the subject before and after treatment with the drug candidate compound, the biological samples comprising a cell population suspected of having CSCs. A physical interaction is caused between the biological samples and an antibody by exposing the biological samples to the antibody. A presence of CSCs in the biological sample is determined by identifying the antibodies bound to the glycan biomarker within the cell population and which are bound to a detection agent, and detecting a presence or absence of the glycan biomarker in the biological samples responsive to the identified antibody bound to the glycan biomarker. The cell population is analyzed to identify levels of CSCs and/or the immunotype of CSCs in the biological samples before treatment with the drug candidate compound compared to after treatment with the drug candidate compound. This process can be repeated for the same subject, many subjects, different candidate drugs, and/or different amounts of a specific drug candidate compound. The presence of a decreased number of CSCs after treatment compared to a number of CSCs before treatment can indicate a relative efficacy of the drug candidate compound in treating the cancer in the subject. In response to a decreased level of CSCs after treatment (and perhaps assessment of other side effects of the drug candidate compound), the patient may be given an increased dosage of the drug candidate, although embodiments are not so limited.

Although FIGs. 2-3 illustrate a biological sample including a blood sample, embodiment are not so limited. For example, the biological sample can include a tissue sample, such as a biopsy specimen from tissue of the subject.

In various embodiments, the biological sample can be exposed to the antibody in a variety of ways. For example, the biological sample can be immobilized on a substrate or the antibody can be immobilized on the substrate. The substrate can include a bead, a glass slide and/or a matrix (e.g., agar or matrigel). The immobilized biological sample or the antibody can then be exposed to the antibody or the biological sample, respectively. The antibody can be labeled with a detection agent, such as with a fluorescent label, or the antibody as bound to the glycan biomarker can be exposed to a second antibody that is labeled, such as a secondary antibody configured to bind to a different epitope of the antigen associated with the glycan biomarker or an anti-antibody configured to bind to an epitope of the antibody. In some embodiments, the substrate coated with the antibodies bound to the glycan biomarker is treated with a labeled anti-human (or other anti-organism) antibody. The substrate can be washed and tagged with fluorescent anti-human IgG antibody. The anti -human IgG antibody can bind to antibodies present on the substrate and which are bound to the glycan biomarker of the antigen that is coated on the substrate.

FIGs. 4A-4C illustrate example substrates for detecting the presence of the glycan biomarker for CSCs, in accordance with various embodiments. As illustrated, in some embodiments, the exposure of the biological sample to the antibody can be used to form an immuno-assay, such as an immuno-sandwich.

In some embodiments, as illustrated by FIGs. 4A and 4B, the biological sample can first be immobilized to a substrate. The immobilized biological sample is then exposed to the antibody. As illustrated by FIG. 4A, the antibody can be directly labeled with a detection agent. The substrate can be washed to remove unbound antibody substance and scanned to detect antibodies bound to the glycan biomarker (e.g., a glycan biomarker antigen that is expressed on the CSC and/or CTC cell surface). In other embodiments, the substrate is washed to remove unbound antibody substance and then further exposed to a detection agent, such as a labeled secondary/anti-antibody that binds to the antibody, as illustrated by FIG. 4B. The substrate is then washed (to remove unbound detection agent) and scanned to detect the antibody bound to the glycan biomarker.

In other embodiments, as illustrated by FIG. 4C, the antibody is immobilized to the substrate. The antibody is a first antibody that binds to a first epitope of the glycan biomarker (e.g., anti-glycan epitope 1), such as the antibodies CI, HAE3, and/or Gl. The immobilized antibody is then exposed to the biological sample. The substrate is then washed to remove unbound biological sample (which can be analyzed before or after to determine a total cell population in the biological sample and to assess the full population). A detection agent is applied to the substrate, such as a labeled secondary antibody, e.g., the illustrated labeled antibody 2, that is specific to a different epitope of the antigen associated with the glycan biomarker (e.g., anti-glycan epitope 2). After washing the substrate to remove unbound secondary antibody substances, the substrate is scanned to detect antibodies, e.g., antibody 1, bound to the glycan biomarker via the labeled antibody 2.

Although the embodiments of FIGs. 4A-4C illustrate a flat substrate, such as a glass substrate, embodiments are not so limited and can include a variety of different substrates, such as beads and arrays.

Embodiments in accordance with the present disclosure include detecting the presence of a glycan biomarker. Somewhat suprisingly, given the differences in size, morphology, and function, the glycan biomarker can be used to identify and/or immunotype CSCs and CTCs. The glycan biomarker can be an epitope of a blood group precursor antigen. More specifically, the glycan biomarker is O-core cryptic epitope of a blood group precursor antigen selected from the group consisting of: Tij II 20% fraction 2nd 10% (Tij II), OG 10% 2X (OG), and a combination thereof. The glycan biomarker includes one or more of polylactosamine chains and/or (other) oligosaccharide chains. Each of the polylactosamine chains and/or (other) oligosaccharide chains can have branches of Πβ (Gaipi,4GlcNAcpi,6), ip(Gaipi,3GlcNAcpi,6), and/or Πβ/Ιβ (Gal βΐ, 4/3GlcNAc β1,6) -moieties. More specifically, the glycan biomarker can include a plurality of chains selected from the group consisting of polylactosamine chains, oligosaccharide chains, and combinations thereof. Each of the plurality of chains can have branches selected from the group consisting of Πβ (Galβl,4GlcNAcβl,6),

iP(Gaipi,3GlcNAcpi,6), Πβ/Ιβ (Gal βΐ, 4/3GlcNAc β1,6) -moieties, and combinations thereof. The glycan biomarker can bind to an anti -tumor mAb, such as CI, Gl or HAE3. More Specific/Experimental Embodiments

In specific experimental embodiments, a glycan biomarker is used to detect the presence of CSCs and CTCs, which is expressed by various cancer tumor cells including ovarian and breast tumor cells. Using the above-described techniques, glycan biomarkers are detected using mAbs, such as CI and HAE3. In specific experiment embodiments, a variety of gly comic tools are used to uncover glycan biomarkers of CTCs and CSCs.

Gly comics tools are used to probe cell-surface glycan biomarkers of breast CTCs (bCTCs). Specifically, carbohydrate microarrays are applied to screen anti-tumor antibodies to identify those that are specific for tumor glycan biomarkers. A high-speed FAST scan is then applied to verify whether the identified targets are CTC-specific cell-surface biomarkers.

Exploring glycan biomarkers of bCTCs and breast cancer stem cells (bCSCs) is useful in tumor biomarker discovery. Although bCTCs and bCSCs are rare in blood, they play a key role in tumor metastasis. Detection of CTCs and CSCs can be used as a noninvasive "liquid biopsy" for tumor diagnosis and prognosis. Glycan biomarkers of bCTCs and bCSCs have unique value in BCa healthcare, especially in personalized therapy that targets specific immunotypes of BCa. Thus, a practical strategy to facilitate identification and characterization of potential glycan biomarkers of bCTC and bCSC, as described in accordance with various embodiments, is beneficial.

In various experimental embodiments, blood samples from five Stage IV metastatic breast cancer (MBCA) patients are characterized (as further illustrated and described in connection with FIGs. 9A-9B). Glycan biomarker gpCl positive CTCs are detected in all subjects; approximately 40% of bCTCs are strongly gpCl positive. Interestingly, the CTCs from a triple-negative breast cancer (TNBC) patient with multiple sites of metastasis are predominantly gpCl positive (92.5%, 37/40 CTCs). This demonstrates the feasibility of detecting CTC-glycan biomarkers using FAST-scan technology.

Cell-surface expression of gpCl in a panel of tumor cell lines is also characterized, in specific embodiments, by a gly can-specific flow cytometry assay. In a first set of 5 experiments, tumor cell lines of distinct tissue origin, including a BCA line, T-47D; a lung cancer (LCA) line, A549; a prostate cancer (PCA) line, PC3; and a skin-derived melanoma cell line, SKMEL-28, are examined. SKMEL-28 (melanoma) and PC3 (PCA) are negative, A549 (LCA) are weakly positive, but T-47D (BCA) are strongly positive. In the second set of staining, a panel of seven human BCA lines are examined. These include two estrogeni c ⁾ receptor-positive (ER+) and progesterone-receptor-positive (PR+) lines (T-47D and MCF- 7), one ER+ (SK-BR-3), and four triple-negative (TN) cancers that lacked the estrogen, progesterone, and Her2)/neu receptors (BT-549, Hs 578T, MDA-MB-231, and MDA-MB- 468). T-47D and MCF-7 are strongly positive, and SK-BR-3 are intermediately positive in gpCl expression. Notably, two TNBC lines, BT-549 and MDA-MB-468, are found to be 15 strongly gpCl positive. In contrast, the two remaining TNBC lines, Hs578T and MDA- MB-231, are negative.

Somewhat surprisingly, some BCA cell lines analyzed exhibit CSC-phenotype and potency in establishing a metastatic tumor in vivo. The TNBC line MDA-MB-231 is phenotypically CD44+/CD24- and is able to establish bone metastasis in nude mice.

20 MDA-MB-468 is phenotypically CD44+/CD24+ and is highly efficient in lung (but not bone) metastasis. MDA-MB-231 is gpCl-negative but the lung metastatic MDA-MB-468 and another TNBC line BT-549 are strongly positive in gpCl -expression. These findings shed light on the gly comics diversity of bCTCs/CSCs.

An anti -tumor gly can mAb CI serves as a key reference reagent for monitoring 25 tumor cell-surface expression of gpCl. Of note, the parental hybridoma cell line of CI, called HAE3, is raised against epiglycanin, the major sialomucin glycoprotein (around 500 kDa) of murine mammary adenocarcinoma TA3 cells. Additionally, this anti-murine carcinoma antibody exhibits strong cross-reactivity with a number of human epithelial tumors in tissues, including lung, prostate, bladder, esophageal, and ovarian cancers. This 30 cross-species tumor-binding profile indicates antibody recognition of a conserved tumor gly can biomarker that is co-expressed by both mouse- and human-derived epithelial cancers.

Carbohydrate microarrays are introduced to explore the potential natural ligands of CI and HAE3. For this purpose, a large collection of purified natural carbohydrate antigens are applied for the microarray screening. A number of blood group substances are spotted in this carbohydrate microarray together with a large collection of carbohydrate antigens to examine the antibody binding specificity. The antibodies CI and HAE3 selectively bond to a number of blood-group precursor antigens. These precursor substances are prepared to remove and are essentially devoid of most of the a-L-fucosyl end groups that are essential for blood group A, B, H, or Lewis active side chains but possess the internal domains or core structures of blood group substances. By contrast, these mAbs have no or minimal detectable cross-reactivity with blood group substances A, B, O, or Lewis antigens, or the large panel of other carbohydrate antigens spotted in the same array.

Selective detection of these blood group precursors from a large panel of blood group substances by the mAbs illustrate they are specific for a shared cryptic glyco-epitope of these precursor substances. This microarray finding is further validated by gly can- specific enzyme-linked immunosorbent assay (ELISA) and gly co-conjugate-based epitope competition assays.

FIGs. 5A-5D illustrate example tools used to identify gly can biomarkers of CTC and CSCs, in accordance with various embodiments. FIG. 5A illustrates an example of a gly can-array, in accordance with various embodiments, and as further described below. FIG. 5B illustrates an example of a gly can biomarker, in accordance with various embodiments.

FIG. 5C illustrates example optical circuitry, in accordance with various embodiments. The optical circuitry illustrated is a fiber optic scanner 550. The fiber optic scanner 550 can be a portion of an apparatus, such as the apparatus 102 illustrated in FIG. 1. The fiber optic scanner 550 can be in communication with processing circuitry to form an apparatus that can identify CSCs from a blood sample and can profile the cell population of the blood sample.

The fiber optic scanner 550 includes a light source (e.g., laser 556) to excite fluorescence located in a sample. The sample (e.g., blood sample) is immobilized or fixed to a substrate and can be held in place by a stage 553. In specific embodiments the light source is a laser 556, such as a 10 mW Argon laser that can excite fluorescence in labeled cells. The fluorescence can be collected in optics with a large (e.g., 50 mm) field-of-view. The field-of-view is enabled by an optic fiber bundle 554. The optic fiber bundle 554 can have asymmetric ends, in some embodiments, and the resolution of the fiber optic scanner 550 can be determined by the spot size of the light source. The emissions from the fluorescent probe can be filtered through dichroic filters before detection at imaging circuitry 557, such as a photomultiplier. The substrate, via the stage 553, can be moved orthogonally across the light scan path on the stage 553. The location of a fluorescently labeled cell is determined by the scan and the stage positions at the time of emission (and to an accuracy of ± 70 urn). For more specific and general information regarding an example FAST system, reference is made to Hsieh HB, Marrinucci D, Bethel K, et al, "High speed detection of circulating tumor cells", Biosensors and Bioelectronics, 2006; 21 : 1893-1899, and Krivacic RT, Ladanyi A, Curry DN, et al, "A rare-cell detector for cancer", Proc Natl Acad Sci U S A. 2004; 101 : 10501-10504, each of which are fully incorporated herein by reference.

The fiber optic scanner 550 illustrated can include a FAST system as implemented by SRI International, however embodiments are not so limited and other high speed scanning methods such as multispectral or hyperspectral imaging may be used. FAST was originally developed for the rapid detection of CTCs, including enabling high throughput scanning for fluorescently -labeled CTCs. Briefly, blood collected from patient and the red blood cells are lysed, and white cells are adhered and fixed to a pretreated glass slide and permeabilized for immunofluorescent labeling. After labeling, the slide is scanned, such as using laser-printing optics, an array of optical fibers that detects fluorescence emission from the cells.

CSCs and/or CTCs are considered the seeds of residual disease and distant metastases, and their characterization are useful for early detection biomarkers, and may guide treatment options. FAST technology is a high-throughput, high-sensitivity scanner to scan all nucleated cells for an unbiased detection of CSCs and/or CTCs on a planar substrate. The instrument enables rapid location of CSCs and/or CTCs without the need for special enrichment, so its sensitivity is not degraded through, e.g., EpCAM targeted antibody enrichment. Because the sample preparation protocol does not distort cell morphology, CSCs and/or CTCs are located on a planar surface and CSC and/or CTC imaging is of high fidelity, which leads to improved specificity. FAST also enables the simultaneous (multiplexed) analysis of multiple protein, cytogenetic, and molecular biomarkers at a single CTC and CSC level.

Once the cancerous (CSC or CTC tumor) cells are identified, further investigation can examine the characteristics of single cells by immunohistochemistry and other analyses such as fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and single nucleotide polymorphism (SNP) analysis. In various embodiments, the apparatus including the fiber optic scanner 550 and the processing circuitry can include additional circuitry. For example, the apparatus can include a server for storage of data sets, intemal network connecting instrumentation control and database, and computer software for instrument control and data management (sometimes herein referred to as "processing circuitry" for ease of reference).

FIG. 5D illustrates an example of a resulting immunotype of CTCs and CSCS from a scan of blood samples using optical circuitry, in accordance with various embodiments. The gpCl positive CTCs are stained in green in the background of the DAPI-blue labeling of white blood cells (e.g., large circles in the top of FIG. 5D) and co-stained by an anti- cytokeratin (CK) antibody in red (e.g., the large circles in the bottom of FIG. 5D).

FIG. 6 illustrates an example of a blood group substance with a conserved O-glycan core, in accordance with various embodiments. More specifically, FIG. 6 illustrates the common blood group precursor core structure highlighted. The four types of branched structures illustrate the potential complexity of the internal portion of the carbohydrate moiety of blood group substances, which is proposed based on extensive immunochemical characterization of blood group substances.

As previously described, the glycan biomarker can include chains selected from the group consisting of polylactosamine chains, oligosaccharide chains, and combinations thereof. Each of the chains has branches selected from the group consisting of Πβ

(Gaipi,4GlcNAcpi,6), ip(Gaipi,3GlcNAcpi,6), Πβ/Ιβ (Gal βΐ, 4/3GlcNAc β1,6) - moieties, and combinations thereof.

In some specific embodiments, the gp ^ci -based blood group precursor epitopes are characteristically composed of a plurality of oligosaccharide chains with branches of Πβ (Gaipi,4GlcNAcpi,6) and/or Ιβ (Gaipi,3GlcNAcpi,6) moieties without fucosylation. The fucosyl epitopes are essential for forming blood group A, B, H, or Le antigens; the terminal nonreducing β-galactoside epitopes of Ιβ and/or Πβ are crucial for preserving the conserved gpCl epitope (s) of BCA. The intemal chain of blood group precursor may also express targetable biomarkers.

Tumor-associated overexpression of blood-group-related autoantigens is not limited to BCA. As recently reported, the natural ligand of a PCA-specific mAb F77 is blood group H, which is built on a 6-linked branch of a poly-N-acetyllactosamine backbone. Overexpression of gpF77 in PCA may reflect increased blood group H expression together with up-regulated expression of branching enzymes. HAE3 and CI differ from F77 in glycan binding specificities and tumor-binding profiles. Unlike F77, which is blood-group- H specific and stains the PCA cell line PC3, HAE3 and CI have neither reactivity with blood group H nor the cell surface targets of PC3. Taken together, these illustrate that epithelial tumor expression of blood-group substance-related autoantigens. This further illustrates the potential of this class of carbohydrate-based immunological targets for tumor vaccine development and targeted immunotherapy.

Materials and Methods

Patient Samples

CTCs analyzed are from patients undergoing treatment for metastatic breast cancer at the City of Hope Cancer Center. Blood samples are collected and used under protocols approved by the Institutional Review Boards of the City of Hope Cancer Center, Palo Alto Research Center, and SRI International. All patients gave their written, voluntary, informed consent (www.clinicaltrials.gov: NCT01048918 and NCT00295893). Patient demographics and clinical characteristics are described in the results section.

Table 1. Dataset from a Carbohydrate Microarray Analysis of MAb CI

Carbohydrate antigens Fluorescence intensity (INT) Microarray scores (Log2-INT)

Concentrations (μ^/μΙ) ID# N Mean StDev ^a Ag./ESg. Mean StDev f- Test (p value)

Man2-PAA (0.002) 1 6 237 31 1.00 7.8737 0.1803 0.914545639

Man2-PAA (0.01) 2 6 231 19 0.98 7.8443 0.1178 0.555761263

Man2-PAA (0.05) 3 6 239 30 1.01 7.8892 0.1736 0.956085932

Man2-PAA (0.25) 4 6 265 20 1.12 8.0462 0.1106 0.032956982

Man5-BSA (0.002) 5 6 248 10 1.05 7.9536 0.0576 0.088125398

Man5-BSA (0.01) 6 6 237 15 1.00 7.8824 0.0873 0.97480637

Man5-BSA (0.05) 7 6 232 27 0.98 7.8443 0.1619 0.687266729

Man5-BSA (0.25) 8 6 234 24 0.99 7.8572 0.1563 0.786298278

"Man9-BSA (0.002) 9 6 321 25 1.36 8.3162 0.1109 0.000876789

Man9-BSA (0.01) 10 6 286 20 1.21 8.1551 0.1009 0.00212074

Man9-BSA (0.05) 11 6 320 17 1.35 8.3180 0.0782 3.98574E-06

Man9-BSA (0.25) 12 6 387 36 1.64 8.5863 0.1348 6.48251E-05

P-Man (0.002) 13 6 245 15 1.04 7.9332 0.0870 0.353052118

P-Man (0.01) 14 6 252 15 1.07 7.9755 0.0872 0.115549225

P-Man (0.05) 15 6 259 20 1.10 8.0088 0.1125 0.093583925

P-Man (0.25) 16 6 259 29 1.10 8.0016 0.1430 0.258706274 LAM (0.002) 17 6 242 10 1.03 7.9172 0.0596 0.393531249

LAM (0.01) 18 6 239 14 1.01 7.8944 0.0817 0.83690219

LAM (0.05) 19 6 251 27 1.06 7.9600 0.1531 0.423657402

LAM (0.25) 20 6 240 23 1.02 7.9004 0.1399 0.821364692

OR (0.05) 21 6 220 23 0.93 7.7703 0.1495 0.200031297

OR (0.25) 22 6 225 13 0.95 7.8081 0.0828 0.166498725

ASOR (0.05) 23 6 233 20 0.99 7.8605 0.1242 0.714768394

ASOR (0.25) 24 6 219 16 0.93 7.7697 0.1048 0.09026433

AGOR (0.05) 25 6 230 15 0.98 7.8412 0.0916 0.493370387

AGOR (0.25) 26 6 232 12 0.98 7.8553 0.0759 0.59077204

Tij II (0.002) 27 6 240 17 1.02 7.9012 0.0997 0.784186197

Tij II (0.01) 28 6 247 27 1.04 7.9331 0.1558 0.594715802

Tij II (0.05) 29 6 326 30 1.38 8.3403 0.1354 0.001294538

Tij II [0.25) 30 6 1908 409 8.08 10.8562 0.3151 5.40788E-06 aGal-BSA (0.05) 31 6 284 17 1.20 8.1450 0.0864 0.000573103 aGal-BSA (0.25) 32 6 359 25 1.52 8.4831 0.0998 7.30853E-06

IM3-BSA (0.002) 33 6 251 13 1.06 7.9660 0.0699 0.144584279

IM3-BSA (0.01) 34 6 239 19 1.01 7.8940 0.1198 0.877710292

IM3-BSA (0.05) 35 6 262 21 1.11 8.0259 0.1133 0.071497953

IM3-BSA (0.25) 36 6 253 12 1.07 7.9773 0.0742 0.113684458

LD7 (0.02) 37 6 241 27 1.02 7.9002 0.1542 0.85762395

LD7 (0.1) 38 6 237 12 1.00 7.8843 0.0732 0.994141248

B1299S (0.02) 39 6 232 26 0.98 7.8470 0.1604 0.657503756

B1299S (0.1) 40 6 240 17 1.02 7.9012 0.1008 0.768999737

B1355S (0.02) 41 6 232 18 0.98 7.8517 0.1077 0.599367191

B1355S (0.1) 42 6 241 20 1.02 7.9074 0.1182 0.729147575

Dex2000K (0.02) 43 6 244 14 1.03 7.9277 0.0829 0.431623112

Dex2000K (0.1) 44 6 237 16 1.00 7.8816 0.0986 0.965736843

N279 (0.02) 45 6 218 20 0.92 7.7614 0.1345 0.134677298

N279 (0.1) 46 6 247 24 1.05 7.9343 0.1270 0.588883272

Levari (0.02) 47 6 239 25 1.01 7.8887 0.1590 0.95570906

Levari (0.1) 48 6 240 16 1.02 7.9032 0.0922 0.740106425

E. coli 0111:B4 (0.02) 49 6 224 30 0.95 7.7952 0.1949 0.38369683

E. coli 0111:B4 (0.1) 50 6 232 16 0.98 7.8516 0.0984 0.570865809

E. coli K92 (0.02) 51 6 233 16 0.99 7.8608 0.0976 0.683969572

E. coli K92 (0.1) 52 6 245 15 1.04 7.9347 0.0884 0.359544745

K. pneumoniae (0.02) 53 6 237 24 1.01 7.8800 0.1486 0.962786162

K. pneumoniae (0.1) 54 6 238 19 1.01 7.8888 0.1210 0.94198138 S. typhi (0.02) 55 6 247 22 1.05 7.9424 0.1222 0.411190903

S. typhi (0.1) 56 6 243 19 1.03 7.9146 0.1081 0.666740193

PnSIV (0.02) 57 6 231 23 0.98 7.8408 0.1457 0.603461225

PnSIV (0.1) 58 6 234 17 0.99 7.8625 0.1087 0.740270948

Background 96 236 23 7.8840 0.1522

aAg./Bg: Ratio of mean fluorescence intensity of antigen and mean background.

bPositive detections are highlighted with bold italics. A positive score is given if the mean fluorescence intensity value of an antibody activity for a given antigen preparation (antigen spot# = 6) is significantly higher than the mean of background spots (n = 96) with a p-value < 0.01.

Carbohydrate Antigens, Anti-glycan Antibodies, and Tumor Cell Lines

Carbohydrate antigens for carbohydrate microarray analysis are listed in Table 1.

Antibody CI (IgM) is produced in specific experimental embodiments by cell line HAE3- CI, which is a subclone of the parent murine hybridoma, HAE3. Tumor cell lines used include lung (A549)- and breast (T47D and SKBR3)-derived epithelial tumor cell lines.

Both T47D and SKBR3 are derived from metastatic sites in breast cancer patients. All tumor cell lines are acquired from the American Type Culture Collection (ATCC),

Manassas, VA.

Antigen preparations and key references: Man2-PAA: Manal,2Man- polyacrylamide, this report; Man5-BSA: (Man5GlcNAc2Asn)n-BSA, Wang, et al, Drug Dev Res. 75, 172 (2014); Man9-BSA: (Man9GlcNAc2Asn)n-BSA, Wang, et al, Drug Dev Res. 75, 172 (2014); P-Man: Yeast phosphomannan polysaccharide, NRRL B-2448, Kabat et al., J. Exp. Med. 164, 642 (1986); LAM: Lipoarabinomannan from

Mycobacterium tuberculosis Aoyama-B, this report; OR: Orosomucoid (al-acid glycoprotein), Wang and Lu, Physiol Genomics 18(2), 245 (2004); ASOR: Asialo- orosomucoid-expressing Tri/m-II glyco-epitopes, Wang & Lu, Physiol Genomics 18(2), 245 (2004); AGOR: Agalacto-orosomucoid-expressing Tri/m-Gn glyco-epitopes, Wang & Lu, Physiol Genomics 18(2), 245 (2004); Tij II: Blood group precursor Tij II 20%fr. 2nd 10%, Maisonrouge-McAuliffe & Kabat, Arch. Biochem. Biophys. 175, 71(1976); aGal- BSA: Galal,3Gaipi, 4Glc i-BSA, this report; IM3-BSA: Isomaltotriose-BSA, Zopf et al, Methods Enzymol. 50, 163 (1978); LD7: a(l→6)dextran, Linear chains, Wang et al, Nat Biotechnol 20(3):275-81, (2002); B1299S: a(l→6)dextran, NRRL B-1299S, Wang et al, Nat Biotechnol 20(3):275-81, (2002); B1355S: a(l→3)(l→6)dextran, NRRL B-1355S, Wang et al, Nat Biotechnol 20(3):275-81, (2002); Dex-2000K: Fluorescein isothiocyanate- conjugated dextrans (2,000 kDa), Wang et al., Nat Biotechnol 20(3):275-81, (2002); N279: a(l→6)dextran, NRRL N279, Wang et al, Nat Biotechnol 20(3):275-81, (2002); Levan: Levan purified from preparation of B-512-E dextran, Kabat et al, J. Exp. Med. 164, 642 (1986); E. coli 0111 :B4: Lipopolysaccharides from Escherichia coli Oi l 1 :B4 L 2630, Sigma-Aldrich Co., St Louis, MO; E. coli. K92: E. coli K92 polysaccharide, Kabat et al, J. Exp. Med. 164, 642 (1986); K. pneumoniae: Lipopolysaccharides from Klebsiella pneumoniae L4268, Sigma-Aldrich Co., St Louis, MO; S. typhi LPS: Lipopolysaccharides from Salmonella enterica serotype typhimurium L7261, Sigma-Aldrich Co., St Louis, MO; Pn SIV: Pneumococcus type IV soluble polysaccharide, Kabat et al, J. Exp. Med. 164, 642 (1986); OG: Blood group precursor substance OG 10% 2X, Vicari & Kabat, J. Immunol. 102, 821 (1969); Feizi et al, J. Exp. Med. 133, 39 (1971); EPGN: Asialo-epiglycanin, produced by murine mammary adenocarcinoma TA3 cells, Codington et al., Biochemistry, 11, 2559 (1972).

Carbohydrate Microarrays and ELISA

Carbohydrate antigens of various structural compositions are dissolved in phosphate-buffered saline (PBS) (glycoprotein conjugates) or saline (polysaccharides) and spotted onto SuperEpoxy 2 Protein slides (Array It Corporation, Sunnyvale, CA) by a high- precision robot designed to produce cDNA microarrays (Cartesian Technologies PIXSYS 5500C). Immediately before use, the printed microarray slides are washed in IX PBS at room temperature for 5 minutes and blocked with 1% bovine serum albumin (BSA)-PBS at room temperature for 30 minutes. They are incubated at room temperature with CI (IgM) antibody at 5.0 μg/mL in 1% (wt/vol) BSA in PBS containing 0.05% (wt/vol) NaN3 and 0.05% (vol/vol) Tween 20. An R-phycoerythrin (R-PE)-conjugated affinity-purified F(ab') fragment of goat anti-mouse IgM secondary antibody preparation (Rockland

Immunochemicals, Inc., Limerick, PA) is applied at 2.0 μg/mL to reveal the Cl-specific staining signal. The stained slides are rinsed five times with PBS with 0.05% (vol/vol) Tween 20, air-dried at room temperature, and then scanned for fluorescent signal using a ScanArray5000A Microarray Scanner (PerkinElmer Life Science). The SAS Institute JMP-Genomics software package (http://www.jmp.com/) is used for further microarray data standardization and statistical analysis. Results of the microarray assay are shown as microarray scores, i.e., means of the log2 -transformed fluorescent intensities (MFIs) of multiple detections of a given antigen preparation. Gly can-specific ELISA can also be performed following a standard protocol.

FIG. 7A-7B illustrates example experimental results of a carbohydrate microarray used to identify gly can biomarkers using antibody HAE3, in accordance with various embodiments. As previously described, a large collection of purified natural carbohydrate antigens are applied for carbohydrate microarray construction. Blood group substance reference reagents (Kabat 1956) used include Cyst 9 and Cyst 14, A active; Beach phenol insoluble, B active; Hog, H active; JS phenol insoluble, H and Leb active, and N-l 20% from the second 10%, Lea active. A number of blood group precursor references, including OG, Tij II, Beach PI, and McDon PI (29#-32#), are spotted in this carbohydrate microarray. These precursor substances are prepared to remove most of the a-L-fucosyl end groups that are essential for blood group A, B, H, or Lewis active side chains, but possess the internal domains or core structures of blood group substances. A large panel of other autoantigens and microbial polysaccharides are also spotted in the same microarrays to critically examine the antibody binding specificity. A preparation of HAE3-reactive human carcinomaassociated antigen (HCA) serves as a positive control for this assay (Li et al. 2004).

FIG. 7A is a graph illustrating the HAE3 binding signal (red column) as plotted with corresponding local background reading (blue column) as an overlay plot. Each data point represents the mean of triplicate detections; these are shown in the FIG. 7B microarray image with the number of positive antigens labeled. Each error bar is constructed using one standard deviation from the mean. As illustrated, HAE3 is strongly positive with HCA (1# and 2#) as expected. This antibody selectively binds to four blood group precursor antigens, Beach PI (29#), McDon PI (30#), Tij II (31#), and OG (32#). By contrast, HAE3 has no detectable crossreactivity with blood group substances A, B, O, or Lewis antigens, or the large panel of other carbohydrate antigens spotted in the same array.

Flow Cytometry Analysis to Examine Tumor Cell Surface Expression ofHAE3 ⁺ Glyco-Epitopes

FIGs. 8A-8B illustrate example experimental results showing expression of surface tumor biomarkers, in accordance with various experimental embodiments. As illustrated by FIGs. 8A-8B, various experimental embodiments further examined whether the HAE3+ glyco-epitopes are expressed as cell-surface tumor biomarkers. To ensure the observed cross-species antigenic reactivities are not owing to the unexpected presence of oligoclonal populations in the original HAE3 hybridoma cell line, HAE3 is subcloned to produce an antibody from a single clone, HAE3-C1 (CI). Antibody CI is verified by carbohydrate microarrays and a gly can-specific ELISA to be highly specific for a conserved O-glycan cryptic glyco-epitope gpCl in human blood group precursors.

In an example set of experiments, a panel of four tumor cell lines are screened by cell surface staining in flow cytometry. These include (a) a BCa line, T-47D, which is selected owing to the fact that breast cancer patients are found to produce substances in circulation that are highly effective in inhibiting AE3-binding of epiglycanin; (b) a lung cancer (LCa) line, A549, which is known to produce an HAE3-positive substance in cell culture; (c) a prostate cancer (PCA) line, PC3, which is found to express a blood group B- related F77 glyco-epitope; and (d) a melanoma cell line SKMEL-28, which is derived from skin but not epithelial tissue.

As shown in FIG. 8A, melanoma SKMEL-28 and prostate cancer PC3 are negative for HAE3. The A549 lung cancer cell line was weakly positive. By contrast, the breast cancer cell line T-47D are strongly positive in HAE3-cell surface staining. Given these results, the flow cytometry analysis is extended to a panel of seven human breast cancer cell lines, including two estrogen receptor positive (ER+) and progesterone receptor positive (PR+) lines (T-47D and MCF-7), one ER+ (SK-BR-3), and four triple-negative (TN) cancers that lack the estrogen, progesterone, and Her2)/neu receptors (BT-549, Hs 578 T, MDA-MB-231, and MDA-MB-468). FIG. 8B shows that two ER+PR+ lines, T- 47D and MCF-7, and two triplenegative lines, BT-549 and MDA-MB-468, are HAE3 strongly positive. SK-BR-3 is intermediately positive. By contrast, the two remaining triple-negative cell lines, Hs578T and MDA-MB-231, are HAE3 negative.

Detection of Glycan Biomar er -Positive bCTC/CSC in Stage IV Breast Cancer Patients

With antibody CI as a key probe, it can be determined that gpCl is applicable for detection and immunotyping analysis of CTCs/CSCs in patients with metastatic breast cancer. In a pilot clinical case study, blood samples from five Stage IV breast cancer patients are characterized the F AST-scan technology.

FIGs. 9A-9B illustrate example experimental results from characterizing blood samples from five Stage IV breast cancer patients, in accordance with various

embodiments. As previously illustrated by FIG. 5D, CTCs captured from the Stage IV breast cancer patients can be scored as 3+, 2+, 1+, and 0, left to right in the graph. Four representative bCTCs are shown in which the epithelial-derived cells are labeled by anti- cytokeratins (CK) antibodies in red, and the gpCl positive cells are stained in green in the background of the DAPIblue labeling of white blood cells. FIG. 9 A and FIG. 9B show that all subjects characterized have gpCl-positive CTCs. Approximately 40% of CTCs captured in these patients express higher levels (2+ and 3+) of the gpCl biomarkers; gpCl- positive and -negative CTCs are found to co-exist in four subjects. Notably, a triple- negative patient (ID# 189370) produced predominantly gpCl-positive CTCs (37 of 40 CTCs) with 50% scored gpCl 2+/3+. In this patient, metastatic tumors are seen in multiple sites, including bone, liver, and skin, which is indicative of the presence of gp ^ci+ CSCs in the CTCs population.

Carbohydrate Microarrays Detect Glyco-Determinants in Human Carcinoma- associated Antigen (HCA)

FIGs. 10A-10D illustrate example experimental results of use of carbohydrate microarrays for detecting glycan biomarkers using antibodies Gl and HAE3, in accordance with various embodiments. As shown in FIGS. 10A-10D and Table 2, mAb Gl and AE-3 are highly reactive with three glycoconjugates (ovarian cyst glycoproteins) designated Beach PI insol., Tij 2 20% fir.10%, and Ogunsheye 10% 2x but that they are marginally reactive with neoglycoconjugates that display T and Tn glyco-epitopes. The antibody binding signals elicited by these three glycoproteins are in the range elicited by HCA and the original immunogen, epiglycanin (EPGN). Thus, Gl and AE-3 are specific for the glyco-epitopes that are shared among the five glycoproteins. The three strongly positive ovarian glycoproteins are known for their expression of antigenicities associated with the O-glycan core and backbone domains such as Ii antigens, or Lewis antigens. Therefore, Gl and AE-3 are directed to glyco-epitopes associated with the complex backbone sequences rather than T/Tn structures as previously postulated. This blood group precursor epitope is referred to herein as gpCl and/or gp ^ci .

More specifically, FIGs. 10A-10D illustrate results from a set of carbohydrate microarrays used to examine the binding of anti-HCA mAb Gl and HAE3, sometimes interchangeably referred to as AE3. Forty eight glycoproteins and neoglycoconjugates are spotted in triplicates and in two dilutions to yield a total of 288 microspots per microarray slide. Images of microarrays stained with: lectin Helix pomatia agglutinin (HP A), as illustrated by FIG. 10A, which is highly cross-reactive with Gal/GalN Ac-terminated glyco- epitopes and serves as a reagent for monitoring efficacy of immobilization of Gal- containing glycoconjugates; Anti-mouse IgM alone, as illustrated by FIG. 10B; mAb

HAE3, as illustrated by FIG. I OC; and Gl, as illustrated by FIG. 10D.

Table 2, as illustrated below, shows relative reactivities of mAb HAE3 and Gl with blood group substances and their precursors, which is measured as ratio of mean

fluorescent intensity to mean background. The positive reactivities are highlighted in bold. MAb Gl and HAE3 were provided by Egenix, Inc. (Rochester, NY). In Table 2, only 12 of the glycoconjugates tested are shown as the rest are negative as statistically measured with cut-off of around ratio 1.5, illustrating the specificities of Gl and HAE3.

Table 2: Carbohydrate Microarray Characterization of Blood Group Substance Binding Reactivities of Anti-HCA mAbs HAE3 and Gl .

Ciirbo-microspots Anti-MS IgM ! j mAb AE3/Anti-MS IglU 1 mAb G WAnti-MS IgM

Glycoconjugates Class i ID j InUBk.* ! InUBk. InUBk.

N-1 10% 2X B-5 1.17 1.14 1.14

N-1 l04 NaOH I Le ^a j B-6 1.15 1.16 1.16

Beach P1 ψ insol. I B, li i B-7 1.17 2.11 3.46

Cyst 9 ψ sol. A B-8 1.16 1.17 1.15

Tij 2 20%f. 2nd 10% li B-9 1.18 7.29 6.89

Ogunsheye 10% 2X li C-8 1.16 11.8 18.83

Hog H C-9 1.17 1.19 1.22

Beach ψ insol. B C-10 1.15 1.26 1.31

J. S φ insol. A C-11 1.17 1.14 1.16

HCA D-7 1.16 8.3 10.27

HCA 1 : 5 Dil D-8 1.16 2.49 2.87

Tn-Antigen HAS Tn H-8 1.14 1.17 1.17

T-Antgen HAS T H-9 1.19 1.18 1.15

FITC-dextran i Ctrl-FITC i H-12 58.66 53.12 39.2

Int/Bk... Ratio of mean flurescence intensity to meai background. Antibody Gl Recognizes Triple Negative Breast Tumor Cell (including CSC-like cell) -associated differential expression of gp ^ci

FIG. 1 1 illustrates example experimental results of staining cell lines using antibody

Gl and CSC biomarkers, in accordance with various embodiments. In accordance with various experimental embodiments, antibody Gl is shown to recognize differential expression of gpCl among triple negative breast tumor cell lines (TNBC), including CSCs.

A panel of TNBC lines are FACS stained using mAb Gl and CSC markers CD44 and CD24. As illustrated by FIG. 11, two of the three CSC-like TNBC lines (CD44+CD24-), MDAMB-231 and BT549, are strongly gp ^ci+ .

More specifically, FIG. 11 illustrates that antibody Gl recognizes differential expression of gpCl among TNBC lines. Gl or an isotype control mAb 9.14.7 was applied at l.C^g per staining in combination with anti-CD44 and anti-CD24. The isotype control signal is in a first color (e.g., red) contour plot, Gl-staining signal is in a second color (e.g., blue) contour plot. As illustrated by bottom two rows of FIG. 11 (labelled CD44 and CD24), MDAMB-231 and BT549 are stained strongly gpCl+, MDAMB-46 is intermediate positive, while Hs578T is weakly stained. As illustrated by the circled areas of the top row of FIG. 11, MDAMB-231 (A), Hs578T (B) and BT549 (C) are characteristically CSC cells with a CD44+CD24- main population. By contrast, also illustrated by the two circles areas in the top row of FIG. 11 related to MDAMB-468, MDAMB-468 is composed of two sub populations, CD44+CD24+ (Dl, subset 2) and CD44-CD24+ (Dl, subset 1).

It is noteworthy that more than 1 million global cases of BCa are diagnosed each year and approximately 15% are triple negative. Owing to the lack of an effective therapeutic target, a younger age at onset, and early metastatic spread, patients suffering triple-negative BCa often have poor prognoses and clinical outcomes. Use of gpCl for diagnosis of and/or treatment of the triple-negative BCa can provide a variety of benefits. For example, the O-core cryptic glycan biomarker can be used for immunotype-enhanced precision diagnosis and prognosis of BCa and targeted immunotherapy against BCa metastasis.

Although tumor-associated abnormal glycosylation has been recognized for years, identifying glycan biomarkers of CSCs and CTCs remains technically challenging.

Embodiments described herein include a practical approach to overcome this difficulty. Conceptually, the approach utilizes the fact that the immune systems of many animal species are able to recognize subtle changes in sugar moieties displayed by cells or soluble antigens and produce specific antibodies for abnormally expressed tumor glycan biomarkers. Experimentally, anti-tumor mAbs are first screened using carbohydrate microarrays to identify those that are specific for glycan biomarkers. Subsequently, it is determined whether the selected mAbs are specific for the cell-surface glycan biomarkers using flow cytometry and FAST-scan technology. Finally, the new antibody is used to probe to monitor CSC and/or CTC-expression of corresponding glycan biomarkers in advanced breast cancer patients. This approach is generally useful for exploring potential glycan biomarkers of CSCs and CTCs of epithelial cancers. The identified glycan biomarkers are demonstrated as successfully identifying CSCs and CTCs in blood samples, which can be used to monitor expression overtime, monitor treatment efficacy, and efficacy of drug candidates.

Although the embodiments illustrated by the various experimental embodiments describe identification of CSCs within a cell population of a blood sample of a human, embodiments are not so limited. For example, in various embodiments, CSCs can be detected and used for treatment of other organisms, such as various vertebrates and/or mammals including dogs, cats, horses, livestock, birds, fish, etc. The blood sample used is from the specific organism. Further, the antibodies used as targets are not limited to those identified herein and can include a variety of antibodies.

Terms to exemplify orientation, such as on top, onto, within, may be used herein to refer to relative positions of elements as shown in the figures. It should be understood that the terminology is used for notational convenience only and that in actual use the disclosed structures may be oriented different from the orientation shown in the figures. Thus, the terms should not be construed in a limiting manner.

Various embodiments are implemented in accordance with the underlying

Provisional Application (Ser. No. 62/447,654), entitled "Unraveling Sugar Chain

Signatures of the 'Seeds' of Tumor Metastasis", filed January 18, 2017, to which benefit is claimed and is fully incorporated herein by reference. For instance, embodiments herein and/or in the provisional application (including the appendices therein) may be combined in varying degrees (including wholly). Reference may also be made to the

experimental teachings and underlying references provided in the underlying provisional application. Embodiments discussed in the provisional application are not intended, in any way, to be limiting to the overall technical disclosure, or to any part of the claimed disclosure unless specifically noted.

As illustrated, various modules and/or other circuit-based building blocks (shown in the immediately preceding figure) may be implemented to carry out one or more of the operations and activities described herein, and/or shown in the block-diagram-type figures. In such contexts, these modules and/or building blocks represent circuits that carry out one or more of these or related operations/activities. For example, in certain of the

embodiments discussed above, one or more modules and/or blocks are discrete logic circuits or programmable logic circuits configured for implementing these

operations/activities, as in the circuit modules/blocks (e.g., the cell picking circuitry, processing circuitry, optical circuitry, and fluorescent microscope) shown above. In certain embodiments, the programmable circuit is one or more computer circuits programmed to execute a set (or sets) of instructions (and/or configuration data). The instructions (and/or configuration data) can be in the form of firmware or software stored in and accessible from a memory (circuit). As an example, first and second modules/blocks include a combination of a CPU hardware-based circuit and a set of instructions in the form of firmware, where the first module/ block includes a first CPU hardware circuit with one set of instructions and the second module/block includes a second CPU hardware circuit with another set of instructions.

Various embodiments described above, and discussed in the provisional application may be implemented together and/or in other manners. One or more of the items depicted in the present disclosure and in the underlying provisional application can also be implemented separately or in a more integrated manner, or removed and/or rendered as inoperable in certain cases, as is useful in accordance with particular applications. For example, the particular structures illustrated as shown and discussed may be replaced with other structures and/or combined together in the same apparatus. As another example, the methods illustrated by FIGs. 2 and 3 and can be implemented using the apparatus illustrated by FIG. 1. Further, the methods described by FIGs. 2-3 can be implemented together, separately, and/or using various combinations of the steps described there in. In view of the description herein, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the present disclosure.