WO2017079632A1 - Modèles de xenogreffes de ctc dérivées d'un patient - Google Patents

Modèles de xenogreffes de ctc dérivées d'un patient Download PDF

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WO2017079632A1
WO2017079632A1 PCT/US2016/060646 US2016060646W WO2017079632A1 WO 2017079632 A1 WO2017079632 A1 WO 2017079632A1 US 2016060646 W US2016060646 W US 2016060646W WO 2017079632 A1 WO2017079632 A1 WO 2017079632A1
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cancer
ctcs
subject
cells
ctc
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Leland W.K. Chung
Ruoxiang Wang
Gina Chia-Yi CHU
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Cedars-Sinai Medical Center
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to oncology and medicine.
  • Pancreatic ductal adenocarcinoma is highly malignant and has the lowest survival of any human cancer. Extensive metastasis and therapeutic resistance are the two major contributors to its dismal prognosis. The mechanisms by which PDAC cells successfully spread and metastasize are largely unknown, and molecular events underlying its resistance to therapeutics remain undefined.
  • FIG 1 A organoid aggregate growth of CTC847 (200x), an ex vivo expanded CTC culture as 3-D spheroids from a prostate cancer patient blood sample.
  • FIG IB upper row, Representative H&E images of prostate CTC induced subcutaneous and intraosseous tumors and kideney metastasis (400x magnification).
  • H&E assay of tumorigenicity and metastatic potential of CTC847 was determined by CTC-PDX tumor formation via s.c. (left) and intra-femoral (middle) inoculation. The bone tumor caused many secondary metastases in many organs including the kidney (right panel).
  • FIG 1C Ex vivo cultured CTCs containing metastasis-initiating cell (MIC) properties (MIC-CTCs) transformed and reprogrammed non-tumorigenic prostate epithelial cells, DC-1, derived from primary prostate tumor to express MIC genes examined by qRT-PCR.
  • MIC metastasis-initiating cell
  • DC-1 cells were co-cultured with PCa CTCs in 3-D suspension co-culture for 4 days before DC-1 cells were isolated from the co- culture by FACS analysis and antibiotic selection.
  • DC-1 is a non-tumorigenic "dormant" epithelial cell line from a prostate tumor specimen. After co-culture with CTC847 in 3-D in mixture for 5 passages, DC-1 became tumorigenic in athymic mouse (not shown) and started to express elevated prostate cancer biomarkers indicative of EMT, sternness and neuroendocrine differentiation as detected by qRT-PCR.
  • Figure 2 depicts, in accordance with various embodiments of the invention, schematic diagram of the study for biomarkers of PDAC metastasis and therapeutic resistance.
  • FIG. 3 depicts, in accordance with various embodiments of the invention, gene fusion nomination for the discovery of driver gene fusions in individual samples.
  • ETV1 and ERG gene fusions as driver mutations in certain cancers positive for ETS gene rearrangement (Palanisamy N., et al. Nat Med 2010; 16(7):793-798).
  • FIG 4A-4D depicts, in accordance with various embodiments of the invention, ex vivo CTC expansion as 3-D spheroids and the CTC-PDX model. Representative results are shown.
  • FIG 4A A matched pair of normal pancreatic tissue and a PaCa tumor specimens were cultured ex vivo for 14 days. Appearance of CTC-like cells in PaCa tumor culture was recorded.
  • FIG 4B A packed blood sample from the same PaCa patient was subjected to ammonium chloride hemolysis to isolate peripheral blood mononuclear cells (PBMCs), which were subjected to ex vivo culture as 3-D spheroids (1 ⁇ 10 6 /ml) for 8 weeks with a unique protocol developed in our laboratory.
  • PBMCs peripheral blood mononuclear cells
  • FIG 4C Pancreatic mesenchymal stromal/stellate cells in the tumor microenvironment promote CTC growth and survival.
  • Conditioned medium (CM) from a culture of the cancer-associated pancreatic mesenchymal cells was used (at 1 :4 dilution) to treat a CTC culture. Growth rate of the cells were determined by counting the cells with an automatic cell counter.
  • FIG 4D Establishment of CTC-PDX model.
  • i.f. intra-femoral
  • o.t. orthotopic pancreas
  • FIG 5A-5D depicts, in accordance with various embodiments of the invention, characterization of the CTC-PDX model. Characteristics of the CTC-PDX-752 tumor formation and metastasis are shown.
  • FIG 5A The CTC-PDX model was established by inoculating ex vivo expanded CTCs as 3-D spheroids (2> ⁇ 10 6 /site) to NSG mice via different routes to induce rapid PDX tumor formation. Both subcutaneous and intra-femoral (i.f.) inoculation resulted in multiple organ metastases and rapid animal death ( ⁇ 80 days). Following i.f. inoculation, the CTCs formed bone tumors, which caused soft tissue metastasis in kidneys and spleen.
  • i.f. intra-femoral
  • FIG 5B Demonstrated by microCT scan, the CTC bone tumor growth resulted in osteolytic lesion.
  • FIG 5C Examined with H&E stain, the CTC-PDX tumors bore similar histopatological features of PaCa (in situ PDAC patient tumor - 400 ⁇ ). Intra-femoral tumor growth caused osteolytic lesion.
  • FIG 5D The s.c. CTC-PDX tumors displayed biomarker expression similar to clinical PaCa, as determined by immunohistopathologic stain (200x). Similarity between patient PDAC and CTC-PDX tumors validated with a PDAC panel of biomarkers at the Department of Pathology, VAGLA. CTC-PDX tumors show a typical CK7+/CK20-/panCK+/CA19-9+ stain pattern (200x).
  • FIG. 6 depicts, in accordance with various embodiments of the invention, using CTC-PDX model to investigate PaCa progression and metastasis.
  • CTCs were dually tagged with luciferase and green fluorescence protein (GFP) reporters and were inoculated o.t to mouse pancreas head (2 ⁇ 10 5 /site), to track CTC-PDX growth and metastasis by bioluminescence imaging.
  • GFP green fluorescence protein
  • FIG 7A-7B depicts, in accordance with various embodiments of the invention, effects of anti-tumor agents in PaCa models.
  • a near infrared ( R) IR-783-gemcitabine conjugate in human pancreatic MiaPaCa II and BX-PC3 tumor cell models.
  • FIG 7B In a PaCa inhibition study, IR-783-gemcitabine was given 10 weeks after tumor cell inoculation. IR-783-gemcitabine markedly inhibited MiaPaCa II tumor growth, and improved dramatically animal survival.
  • FIG 8A-8H depicts, in accordance with various embodiments of the invention, metastasis-initiating cell (MIC) signatures and functions in CTCs from human patients.
  • FIG 8A Morphology of CTCs.
  • FIG 8B CTC protein expression detected by the mQDL method.
  • FIG 8C In vitro expanded human CTCs consist of both MIC and non-MIC cells.
  • FIG 8D Percent MICs in CTCs.
  • FIG 8E Cultured CTCs as 3-D spheroids from prostate, pancreas, and kidney cancers express MIC markers. Cultured CTCs of prostate, pancreas, and kidney cancers express MIC markers with elevated EMT, neuroendocrine (NE), and stem cell markers detected by western blot analysis.
  • MIC metastasis-initiating cell
  • FIG 8F MIC marker expression of longitudinal PCa CTC samples from one of the CRPC patients before and after therapeutic interventions detected by western blot analysis.
  • Ex vivo cultured CTCs as 3-D spheroids express EMT, stem and NE phenotype.
  • FIG 8G Ex vivo cultured CTCs containing MIC properties (MIC- CTCs) transformed and reprogrammed non-tumorigenic prostate epithelial cells, DC-1, derived from primary prostate tumor to express MIC proteins examined by western blot analysis.
  • DC-1 cells were co-cultured with PCa CTCs in 3-D suspension co-culture for 4 days before DC-1 cells were isolated from the co-culture by FACS analysis and antibiotic selection.
  • CTCs with MIC phenotype reprogram indolent DC-1 cells.
  • FIG 8H MIC-CTCs also up-regulates CK13 expression in DC-1 cells during the reprogramming process.
  • FIG 9A-9F depicts, in accordance with various embodiments of the invention, ex vivo cultured human CTCs from PC patients form tumors and metastases in mice.
  • FIG 9A Representative image of prostate CTC-induced intrafemoral tumors and kidney metastases.
  • FIG 9B Representative microCT scan of prostate CTC bone tumors with osteolytic lesions.
  • FIG 9C Kaplan-Meier survival curves of mice bearing prostate cancer CTCs.
  • FIG 9D Kaplan-Meier survival curves of mice bearing pancreatic, renal, and breast cancer CTCs.
  • FIG 9E Representative bioluminescence (BLI) images of mice bearing prostate orthotopic CTC- derived xenograph (CDX) tumors and metastases.
  • BBI bioluminescence
  • PCa CTCs (847) were tagged with luciferase and implanted into the prostate of the NSG mice, which were monitored for tumor formation by BLI imaging once a week.
  • FIG 9F Representative bioluminescence images of mice bearing luciferase-tagged pancreatic CTC (752) metastases via intracardiac inoculation.
  • Figure 10 depicts, in accordance with various embodiments of the invention, that cultured CTCs induced in vitro osteoclastogenesis of mouse osteoclast precursors upon co- culture.
  • Graph depicts the quantification of mature osteoclasts (nuclei > 3) induced by cultured CTCs of prostate and pancreas cancer.
  • FIG 11A-11B depicts, in accordance with various embodiments of the invention, STR DNA fingerprint authentication of a CTC line (CTC-752-S) established from a PDAC patient blood sample.
  • FIG 11 A Identical polymorphic alleles are detected between CD45+ PBMCs from the same patient and FIG 11B) cells of the newly established CTC-752-S line.
  • An additional allele appearing in the D19S433 locus (lower right panel) of the CTC-782-S is suggestive of genomic instability, a common observation in malignant cells and not affecting the authentication.
  • Figures 12 depicts, in accordance with various embodiments of the invention, representative IHC staining of CK14 and CK13 in ex vivo expanded CTC organoids of pancreatic and prostate cancer (top panels, 400x magnification) and representative IHC staining of CK13 in pancreatic and prostate cancer-derived CDX tumors (lower panels, 200x magnification).
  • Figure 13 depicts, in accordance with various embodiments of the invention, a time course of CTC culture morphology.
  • Figure 14 depicts, in accordance with various embodiments of the invention, detection of the epithelial marker EpCAM on expanded CTCs by flow cytometry.
  • Figure 15 depicts, in accordance with various embodiments of the invention, CTC counts in primary prostate cancer patients and castration-resistant prostate cancer cases. Results obtained from culturing l x lO 7 peripheral blood mononucleated cells in defined medium for 21 days.
  • Various embodiments of the present invention provide for a method of establishing a personalized model for a cancer, comprising: isolating circulating tumor cells (CTCs) from a biological sample from a subject with the cancer; expanding the CTCs as 3-D spheroids ex vivo; inoculating the ex vivo expanded CTCs into a non-human animal, thereby generating a patient-derived xenograft non-human animal; and establishing the patient-derived xenograft non-human animal as the personalized model for cancer detection and therpy.
  • CTCs circulating tumor cells
  • the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer.
  • pancreatic cancer can be pancreatic ductal adenocarcinoma.
  • the biological sample can be a liquid biopsy or tissue biopsy from the subject with cancer.
  • a liquid biopsy can be a blood sample from the subject with cancer.
  • the biological sample can be obtained from the subject with cancer before, during, and/or after therapeutic treatment.
  • CTCs may be isolated from the blood sample by separating the blood cells and plasma to obtain packed nucleated blood cells.
  • the packed blood cells may be lysed using ammonium chloride hemolysis to obtain a cell pellet comprising CTCs.
  • the cells from the cell pellet may be cultured in a defined medium to form spherical organoid aggregates.
  • expanding CTCs ex vivo can result from culturing cells from the biological sample to form spherical organoid aggregates.
  • the expanded CTCs can be inoculated orthotopically, intrafemorally or intraosseosly, intracardiac, or subcutaneously.
  • the non-human animal can be a rodent, mouse, rat, rabbit or guinea pig.
  • the method further comprises tagging the CTCs with a lentiviral reporter construct that expresses green fluorescent protein, red fluorescent protein, luciferase, or a combination thereof.
  • bioluminescence imaging can be used to monitor and track the tagged CTCs in the tumor.
  • the method further comprises co-innoculating PaSCs with the expanded CTCs into a non-human animal.
  • MIC-CTCs are inoculated into a non-human animal.
  • Various embodiments of the present invention provide for a method of identifying one or more drugs to treat a cancer, comprising: providing a personalized model for a cancer, wherein the model is the personalized model established herein; administering one or more drugs to the model; and detecting a therapeutic response in the model and identifying the drug as being therapeutically effective to the cancer, or detecting no therapeutic response in the model and identifying the drug as being therapeutically ineffective to the cancer.
  • the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer.
  • pancreatic cancer can be pancreatic ductal adenocarcinoma.
  • the therapeutic response can be inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited cancer cell invasion, inhibited cancer cell mobility, inhibited cancer cell differentiation, promoted cancer cell death, inhibited cancer progression, inhibited cancer metastasis, or improved animal survival, or a combination thereof.
  • the method further comprises instructing the subject with cancer to receive the one or more drugs identified to treat the subject's cancer.
  • Various embodiments of the present invention provide for a method of identifying a subject that has cancer, as having resistance or not to a drug, comprising: providing a personalized model for a cancer; administering one or more drugs to the model; and detecting resistance in the model and identifying the subject as having resistance to the drug, or detecting no resistance in the model and identifying the subject as having no resistance to the drug.
  • the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer.
  • pancreatic cancer can be pancreatic ductal adenocarcinoma.
  • the personalized model for cancer can be the model established herein.
  • resistance to a drug can be detected when there is uninhibited cancer cell proliferation, uninhibited cancer cell growth, uninhibited cancer cell invasion, uninhibited cancer cell mobility, uninhibited cancer cell differentiation, diminished cancer cell death, uninhibited cancer progression, uninhibited cancer metastasis, a decline in animal survival, or a combination thereof.
  • no resistance to a drug can be detected when there is inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited cancer cell invasion, inhibited cancer cell mobility, inhibited cancer cell differentiation, inhibited cancer cell death, inhibited cancer progression, inhibited cancer metastasis, improved animal survival, or a combination thereof.
  • Various embodiments of the present invention provide for a method of identifying MIC-CTCs in a sample from a subject, comprising: obtaining a biological sample from a subject; assaying for metastasis-initiating cell - circulating tumor cells (MIC-CTCs), and identifying MIC-CTCs in a sample when mesenchymal, stem, neuroendocrine markers, or a combination thereof, are detected.
  • MIC-CTCs metastasis-initiating cell - circulating tumor cells
  • the cancer is pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In various other embodiments, pancreatic cancer is pancreatic ductal adenocarcinoma.
  • the mesenchymal, stem and neuroendocrine markers comprise RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDH1,SDF1, PR1, Lin28b, and/or SYP.
  • the biological sample is a liquid biopsy or tissue biopsy from the subject.
  • the mesenchymal, stem, neuroendocrine markers are assayed by mQDL.
  • identifying MIC-CTCs in the biological sample obtained from a subject establishes the subject has cancer.
  • Various embodiments of the present invention provide for a method of establishing a model for a cancer, comprising: isolating circulating tumor cells (CTCs) from a subject with the cancer; expanding the CTCs as 3-D spheroids ex vivo, thereby establishing the ex vivo expanded CTCs as the model for the cancer.
  • CTCs circulating tumor cells
  • a composition comprising circulating tumor cells (CTCs) isolated from a subject with a cancer.
  • the cancer is pancreatic cancer or prostate cancer.
  • the CTCs are isolated from a biological sample from the subject.
  • the biological sample is a liquid biopsy of the cancer.
  • the CTCs are expanded ex vivo.
  • the CTCs are inoculated into a non-human animal.
  • CTCs circulating tumor cells isolated from a subject with a cancer.
  • the CTCs are inoculated subcutaneously, intrafemorally, orthotopically, or intraosseously, or a combination thereof.
  • the non- human animal is a rodent, mouse, rat, rabbit or guinea pig. DETAILED DESCRIPTION OF THE INVENTION
  • the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).
  • the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, reverse, alleviate, ameliorate, inhibit, lessen, slow down or stop the progression or severity of a symptom or condition.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease, disorder or medical condition is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Also, “treatment” may mean to pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.
  • “Beneficial results” or “desired results” may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition, decreasing morbidity and mortality, and prolonging a patient's life or life expectancy.
  • "beneficial results” or “desired results” may be alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized (i.e., not worsening) state of pancreatic cancer, delay or slowing of pancreatic cancer, and amelioration or palliation of symptoms associated with pancreatic cancer.
  • Diseases may include, but are in no way limited to any form of malignant neoplastic cell proliferative disorders or diseases. Examples of such disorders include but are not limited to cancer and tumor.
  • a “cancer” or “tumor” as used herein refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems, and/or all neoplastic cell growth and proliferation.
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are dormant tumors or micrometastasis. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • the term “invasive” refers to the ability to infiltrate and destroy surrounding tissue. Melanoma is an invasive form of skin tumor.
  • cancer refers to a cancer arising from epithelial cells.
  • cancer include, but are not limited to, nervous system tumor, brain tumor, nerve sheath tumor, breast cancer, colorectal cancer, colon cancer, rectal cancer, bowel cancer, carcinoma, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen- independent prostate cancer.
  • brain tumor examples include, but are not limited to, benign brain tumor, malignant brain tumor, primary brain tumor, secondary brain tumor, metastatic brain tumor, glioma, glioblastoma, glioblastoma multiforme (GBM), medulloblastoma, ependymoma, astrocytoma, pilocytic astrocytoma, oligodendroglioma, brainstem glioma, optic nerve glioma, mixed glioma such as oligoastrocytoma, low-grade glioma, high-grade glioma, supratentorial glioma, infratentorial glioma, pontine glioma, meningioma, pituitary adenoma, and nerve sheath tumor.
  • GBM glioblastoma multiforme
  • medulloblastoma medulloblastoma
  • Nervous system tumor or nervous system neoplasm refers to any tumor affecting the nervous system.
  • a nervous system tumor can be a tumor in the central nervous system (CNS), in the peripheral nervous system (PNS), or in both CNS and PNS.
  • Examples of nervous system tumor include but are not limited to brain tumor, nerve sheath tumor, and optic nerve glioma.
  • administering refers to the placement of an agent, composition and/or drug into a subject by a method or route which results in at least partial localization of the agents or composition at a desired site.
  • Route of administration may refer to any administration pathway known in the art, including but not limited to oral, topical, aerosol, nasal, via inhalation, anal, intra-anal, peri-anal, transmucosal, transdermal, parenteral, enteral, or local.
  • Parenteral refers to a route of administration that is generally associated with injection, including intratumoral, intracranial, intraventricular, intrathecal, epidural, intradural, intraorbital, infusion, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravascular, intravenous, intraarterial, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.
  • the agent or composition may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.
  • the agent or composition can be in the form of capsules, gel capsules, tablets, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release.
  • the agent or composition can be in the form of aerosol, lotion, cream, gel, ointment, suspensions, solutions or emulsions.
  • agent or composition may be provided in a powder form and mixed with a liquid, such as water, to form a beverage.
  • “administering” can be self-administering. For example, it is considered as “administering" that a subject consumes a composition as disclosed herein.
  • biological sample or “sample” or “liquid biopsy” as used herein denotes a sample taken or isolated from a biological organism, e.g., a tumor sample from a subject.
  • exemplary biological samples include, but are not limited to, cheek swab; mucus; whole blood, blood, serum; plasma; bone marrow aspirate, urine; saliva; semen; lymph; fecal extract; sputum; intestinal fluids or aspirate, and stomach fluids or aspirate, cerebral spinal fluid (CSF), other body fluid or biofluid; cell sample; tissue sample; tumor sample; tumor cells in blood circulation (circulating tumor cells (CTCs)) and/or tumor biopsy etc.
  • CSF cerebral spinal fluid
  • sample also includes a mixture of the above-mentioned samples.
  • sample also includes untreated or pretreated (or pre-processed) biological samples.
  • liquid biopsy refers to any liquid sample obtained from a subject.
  • a sample can comprise one or more cells from the subject.
  • a sample can be a tumor cell sample, e.g. the sample can comprise cancerous cells, cells from a tumor, and/or a tumor biopsy.
  • the sample can be a blood sample which comprises of cancerous cells.
  • the subject is mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.
  • the methods described herein can be used to treat domesticated animals and/or pets.
  • “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, rabbits and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., pancreatic cancer) or one or more complications related to the condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.
  • a subject can also be one who has not been previously diagnosed as having a condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for a condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • a subject can be one who exhibits one or more symptoms for a condition or one or more complications related to the condition or a subject who does not exhibit symptoms.
  • a "subject in need" of diagnosis or treatment for a particular condition can be a subject suspected of having that condition, diagnosed as having that condition, already treated or being treated for that condition, not treated for that condition, or at risk of developing that condition.
  • statically significant refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p- value.
  • Described herein are methods of establishing a patient-derived xenograft, using CTC cells isolated from a subject to obtain a personalized model of cancer.
  • CTCs are the culprit cell pool for tumor spreading and metastasis.
  • CTCs exist in a minute fraction among vast numbers of normal cells in a given clinical blood sample. Repeated investigation in a CTC preparation is nearly impossible due to the rarity of this cell type in the blood.
  • the inventors have developed an ex vivo CTC expansion protocol. Expanding CTCs ex vivo is necessary for reproducible examination of their genomic makeups and behaviors in vitro in culture or in vivo as patient-derived xenografts (PDXs).
  • PDXs patient-derived xenografts
  • CTC-PDX With conventional PDX modeling, pieces of patient tumor are implanted directly to athymic mice for tumor formation. Conventional PDX suffers from inherent drawbacks including extremely low tumor formation rates in mice and less tumor progression and metastasis. CTCs are in dynamic equilibrium with tumor cells at the primary and metastatic sites, thus reflecting the state of the in situ tumor in real time.
  • the advantages of CTC-PDX over the conventional PDX include: 1) unlike PDX, CTC-PDX can be studied repeatedly in culture and in mice; 2) CTC-PDX tumor can metastasize in the mouse host, whereas PDXs rarely metastasize; and 3) multiple CTC-PDXs can be established with longitudinally acquired patient samples at return visits, to monitor tumor progression, metastasis and therapeutic resistance.
  • the present invention is based, at least in part, on these findings.
  • the present invention addresses the need in the art for methods of obtaining reproducible examination of the genomic makeup and behavior of a tumor in a subject, in vitro in culture or in vivo as patient-derived xenografts (PDXs) established from live CTCs, to study cancer metastasis and aid in biomarker discovery for predicting survival and therapeutic resistance in PDAC patients.
  • PDXs patient-derived xenografts
  • Various embodiments of the present invention provide for a method of establishing a personalized model for a cancer, comprising: isolating circulating tumor cells (CTCs) from a biological sample from a subject with the cancer; expanding the CTCs as 3-D spheroids ex vivo; inoculating the ex vivo expanded CTCs into a non-human animal, thereby generating a patient-derived xenograft from CTCs in a non-human animal; and establishing the patient- derived xenograft non-human animal as the personalized model for the cancer.
  • CTCs circulating tumor cells
  • the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer.
  • pancreatic cancer can be pancreatic ductal adenocarcinoma.
  • the biological sample can be a liquid biopsy or tissue biopsy from the subject with cancer.
  • a liquid biopsy can be a blood sample from the subject with cancer.
  • the biological sample can be obtained from the subject with cancer before, during, and/or after therapeutic treatment.
  • CTCs may be isolated from the blood sample by separating the blood cells and plasma to obtain packed blood cells.
  • the packed blood cells may be lysed using ammonium chloride hemolysis to obtain a cell pellet comprising CTCs.
  • the cells from the cell pellet may be cultured in a defined medium to form spherical organoid aggregates.
  • expanding CTCs ex vivo can result from culturing cells from the biological sample to form spherical organoid aggregates.
  • the expanded CTCs can be inoculated orthotopically, intrafemorally, intracardiac, intraosseosly, or subcutaneously.
  • the non- human animal can be a rodent, mouse, rat, rabbit or guinea pig.
  • the method further comprises tagging the CTCs with a lentiviral reporter construct that expresses green fluorescent protein, red fluorescent protein, luciferase, or a combination thereof.
  • bioluminescence imaging can be used to monitor and track the tagged CTCs in the tumor.
  • the construct of the invention encoding the luciferase or the fluorescent proteins may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859 - 1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801805.
  • the DNA construct may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or Saiki et al., Science 239 (1988), 487 - 491.
  • the DNA construct of the invention may be inserted into a vector which may be any vector which may conveniently be subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector can be a viral vector.
  • the vector is a lentiviral vector.
  • Suitable fluorescent proteins include, but are not limited to, Y66H, Y66F, EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, ECFP, CyPet, Y66W, mKeima- Red, TagCFP, AmCyanl, mTFPl, S65A, Midoriishi Cyan, Wild Type GFP, S65C, TurboGFP, TagGFP, S65L, Emerald, S65T (Invitrogen), copGFP (SABiosciences), EGFP (Clontech), Azami Green (MBL), ZsGreenl (Clontech), TagYFP (Evrogen), EYFP (Clontech), Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellowl (Clontech), Kusabira Orange (MBL), mOrange
  • Luciferases are proteins which react with a suitable substrate to produce light as one of the reaction products. Luciferases catalyze the oxygen oxidation of an organic molecule, i.e., a luciferin (such as aldehydes, benzothiazoles, imidazolopyrazines, tetrapyrroles and flavins). Luciferases that use coelenterazine (an imidazoloyrazine derivative) as a substrate to produce luminescence include luciferases from the species Renilla, Gaussia, Metridia and Obelia. The amount of light produced by a bioluminescent reaction can be measured and used to determine the presence of or amount of luciferase in a sample.
  • the term "luciferase” refers to a naturally occurring or mutant luciferase.
  • bioluminescence refers to the emission of light from a cell tagged with a fluorescent protein or resulting from a reaction catalyzed by a luciferase. Bioluminescence can be measured using luminometer or imaging systems. In various embodiments, CTC bioluminescence can be assessed in vivo. In various other embodiments, CTC bioluminescence can be assessed in vitro.
  • the method further comprises co-innoculating PaSCs with the expanded CTCs into a non-human animal.
  • MIC-CTCs are inoculated into a non-human animal.
  • MIC-CTCs are inoculated either alone or together with a dormant cell, DC- 1 , into a non-human animal.
  • the personalized model for cancer of the invention is useful for the screening of agents or treatment modalities, (e.g., anti-cancer drugs, antibodies, combination of drugs, or administration regimens), having efficacy in cancer therapy.
  • agents or treatment modalities e.g., anti-cancer drugs, antibodies, combination of drugs, or administration regimens
  • host rodents are inoculated with CTC cells and, following a time interval sufficient to allow development of a tumor, the host is then administered with the tested agent or treatment modality and the therapeutic effect of such agent or treatment can then be evaluated, for example by determining the animals' median survival time or by assessing tumorigenic cellular characteristics, such as, but not limited to tumor size, angiogenesis, cell proliferation, cellular migration, cellular differentiation, apoptosis and cancer metastasis.
  • Various embodiments of the present invention provide for a method of identifying one or more drugs to treat a cancer, comprising: providing a personalized model for a cancer, wherein the model is the personalized model established herein; administering one or more drugs to the model; and detecting a therapeutic response in the model and identifying the drug as being therapeutically effective to the cancer, or detecting no therapeutic response in the model and identifying the drug as being therapeutically ineffective to the cancer.
  • the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer.
  • pancreatic cancer can be pancreatic ductal adenocarcinoma.
  • the therapeutic response can be inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited angiogenesis in a tumor, inhibited cancer cell invasion, inhibited cancer cell mobility, inhibited cancer cell differentiation, promoted cancer cell death, inhibited cancer progression, inhibited cancer metastasis, or improved animal survival, or a combination thereof.
  • the method further comprises instructing the subject with cancer to receive the one or more drugs identified to treat the subject's cancer.
  • Various embodiments of the present invention provide for a method of identifying a subject that has cancer, as having resistance or not to a drug, comprising: providing a personalized model for a cancer; administering one or more drugs to the model; and detecting resistance in the model and identifying the subject as having resistance to the drug, or detecting no resistance in the model and identifying the subject as having not having resistance to the drug.
  • the cancer can be pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer.
  • pancreatic cancer can be pancreatic ductal adenocarcinoma.
  • the personalized model for cancer can be the model established herein.
  • resistance to a drug can be detected when there is uninhibited cancer cell proliferation, uninhibited cancer cell growth, uninhibited cancer cell invasion, uninhibited cancer cell mobility, uninhibited cancer cell differentiation, diminished cancer cell death, uninhibited cancer progression, uninhibited cancer metastasis, a decline in animal survival, or a combination thereof.
  • no resistance to a drug can be detected when there is inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited cancer cell invasion, inhibited cancer cell mobility, inhibited cancer cell differentiation, inhibited cancer cell death, inhibited cancer progression, inhibited cancer metastasis, improved animal survival, or a combination thereof.
  • MIC-CTCs in a sample from a subject comprising: obtaining a biological sample from a subject; assaying for metastasis-initiating cell - circulating tumor cells (MIC-CTCs), identifying MIC-CTCs in a sample when mesenchymal, stem, neuroendocrine markers, or a combination thereof, are detected.
  • MIC-CTCs metastasis-initiating cell - circulating tumor cells
  • the mesenchymal, stem and neuroendocrine markers comprise RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP.
  • detecting the mesenchymal, stem and neuroendocrine markers comprises detecting 1 or combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and/or 24 of the markers disclosed herein.
  • the cells assayed for are cells with metastasis- initiating cell properties within the pool of circulating tumor cells.
  • identifying MIC-CTCs in the biological sample obtained from a subject and establishes that the subject has cancer.
  • the subject identified with MIC-CTCs in the biological sample may have a poor prognosis and can be treated and closely.
  • the biological sample is obtained from a subject with cancer.
  • the cancer is pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer.
  • pancreatic cancer is pancreatic ductal adenocarcinoma.
  • the biological sample can be obtained before, during and/or after treatment.
  • the biological sample is a liquid biopsy or tissue biopsy from the subject.
  • the mesenchymal, stem, neuroendocrine markers are assayed by mQDL.
  • the mesenchymal, stem, neuroendocrine markers assayed for can be compared to a reference value.
  • the reference value can depend on the type of disease or condition that will be determined. Different types of diseases and conditions may have different reference values.
  • the reference value can be established from biological samples from a healthy subject or the same subject with samples obtained from different time of disease progression. For example, if the biological sample is a blood sample, then the reference value can be obtained from the blood sample of a healthy subject or the same subject with samples obtained from different time of the disease progression.
  • the reference value to be used to compare with the expression value of the subject will typically be from the same tissue, cell, and/or location in the cell.
  • RANKL protein expression level in a cell is measured for the subject, it will be compared to RANKL protein expression level in a cell obtained from a healthy control sample(s) or the same subject with samples obtained from different time of disease progression.
  • the reference value used can typically be from control samples having known disease states and survival times.
  • Various embodiments of the invention provide for a method of treating prostate cancer in a subject, comprising obtaining the results of an analysis of a level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP in a subject; and administering a treatment when the level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP is increased compared to a healthy individual.
  • the subject with an increased RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP level is indicative of a MIC-CTC phenotype.
  • the analysis can be accomplished by multiplex quantum dot labeling (mQDL), qRT-PCR, western blotting, fluorescence in situ hybridization (FISH), immunohistochemistry and/or in situ hybridization.
  • Various embodiments of the invention provide for a method of treating prostate cancer in a subject, comprising: requesting the results of an analysis of a level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP in a subject; and administering a treatment to the subject when the level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP is increased compared to a healthy individual.
  • the subject with an increased RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDH1,SDF1, NPRl, NSE, Lin28b, and/or SYP level is indicative of a MIC-CTC phenotype.
  • the analysis can be accomplished by multiplex QD-based labeling (mQDL), qRT-PCR, western blotting, fluorescence in situ hybridization (FISH), immunohistochemistry and/or in situ hybridization.
  • Various embodiments of the invention provide for a method of treating prostate cancer in a subject, comprising administering a treatment to the subject which has been determined to have an increased level of RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDH1,SDF1, NPRl, NSE, Lin28b, and/or SYP compared to a healthy individual.
  • the subject with an increased RANKL, Vimentin, FOXM1, FOXA2, c- Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N- Cad, ALDH1,SDF1, NPRl, NSE, Lin28b, and/or SYP level is indicative of a MIC-CTC phenotype.
  • the subject identified with increased RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDH1,SDF1, NPRl, NSE, Lin28b, and/or SYP in the biological sample may be treated.
  • the subject with a MIC-CTC phenotype is treated.
  • the analysis can be accomplished by multiplex quantum dot labeling (mQDL), qRT-PCR, western blotting, fluorescence in situ hybridization (FISH), immunohistochemistry and/or in situ hybridization.
  • the subject is treated by administering a drug that interrupts MIC-non-MIC cell communication. In various embodiments, the subject is treated by administering a therapeutically effective amount of a drug that targets MIC-CTCs along with a pharmaceutically acceptable excipient.
  • the targeting of MIC-CTCs can be accomplished by modifying or modulating
  • gene regulation is modified by inhibiting or inducing the MIC-CTCs associated genes and causing an over-expression or under- expression of the genes, mentioned above.
  • the modifications can occur in transcriptional initiation, RNA processing and/or during post-translation. In certain embodiments, the modifications can occur at the transcription and/or the translational level.
  • the modifications at the transcriptional level can include, but are not limited to the administration of siRNA or shRNA to add or remove the gene.
  • the modifications at the translational level can include, but are not limited to phosphorylation, methylation, acetylation and/or the use of the respective inhibitors and gene silencing and/or gene induction can occur through translational modifications.
  • the genes can be modified by altering upstream and/or downstream effectors of the genes.
  • the genes, the upstream effectors and/or the downstream effectors can be up-regulated and/or down-regulated.
  • RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N- Cad, ALDH1,SDF1, PR1, NSE, Lin28b, and/or SYP targeting can occur through the activation and/or deactivation of receptors and/or ligands.
  • the activation and/or deactivation of receptors and/or ligands can inhibit and/or induce gene binding.
  • the activation and/or deactivation of receptors and/or ligands can modulate the growth and behaviors of cancer cells and can increase or reverse their MIC-CTCs characteristics which include, but are not limited to cell growth, invasion, migration and metastasis.
  • the subject is treated by administering a cancer therapeutic.
  • the cancer therapeutic includes but it not limited to heptamethine carbocyanine near-infrared ( R) dye-drug conjugate.
  • the subject is treated by administering the one or more drugs identified using the personalized model established herein.
  • “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.
  • Various embodiments of the present invention provide for a method of identifying a subject with cancer, comprising: obtaining a biological sample from a subject; assaying for metastasis-initiating cells (MIC)-CTCs, activated PaSCs, macrophages or a combination thereof; and identifying the subject with cancer when MIC-CTCs, activated PaSCs, macrophages or a combination thereof are detected.
  • MIC-CTCs metastasis-initiating cells
  • activated PaSCs macrophages or a combination thereof
  • the reciprocal activation of MIC-CTCs by activated PaSCs is assayed.
  • Various embodiments of the present invention provide for a method of identifying a subject with cancer, comprising: obtaining a biological sample from a subject; assaying for metastasis-initiating cells (MIC)-CTCs, activated PaSCs, macrophages or a combination thereof; and identifying the subject with metastatic cancer when MIC-CTCs, activated PaSCs, macrophages or a combination thereof are detected.
  • MIC metastasis-initiating cells
  • the cancer is pancreatic cancer, pancreatic ductal adenocarcinoma, prostate cancer, kidney cancer or breast cancer. In various other embodiments, pancreatic cancer is pancreatic ductal adenocarcinoma. In various embodiments, MIC-CTCs are identified by assaying for mesenchymal, stem and neuroendocrine markers.
  • the mesenchymal, stem and neuroendocrine markers comprise RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and/or 24 of these markers are identified.
  • the assaying for metastasis-initiating cells (MIC)-CTCs, activated PaSCs, and/or macrophages can be accomplished by multiplex QD-based labeling (mQDL), qRT-PCR, western blotting, fluorescence in situ hybridization (FISH), immunohistochemistry and/or in situ hybridization.
  • mQDL multiplex QD-based labeling
  • FISH fluorescence in situ hybridization
  • immunohistochemistry immunohistochemistry and/or in situ hybridization.
  • the subject identified with cancer can be treated, as discussed above.
  • Markers used for the detection of macrophages include, but are not limited to CD1 lb, F4/80, CD68, CSF1R, MAC2, CDl lc, LY6G, LY6C, IL-4Ra, and/or CD163. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of these markers are identified. Markers used for the detection of pancreatic stellate cells (PaSCs) include, but are not limited to a-SMA and/or collagen I.
  • Nucleic acid or protein samples derived from diseased and non-diseased cells of a subject that can be used in the methods of the invention can be prepared by means well known in the art. For example, surgical procedures or needle biopsy aspiration can be used to collect diseased samples from a subject. In some embodiments, it is important to enrich and/or purify the diseased tissue and/or cell samples from the non-diseased tissue and/or cell samples. In other embodiments, the diseased tissue and/or cell samples can then be microdissected to reduce the amount of non-diseased tissue contamination prior to extraction of genomic nucleic acid or gene expression RNAs for use in the methods of the invention. Such enrichment and/or purification can be accomplished according to methods well-known in the art, such as fine needle aspiration or biopsy, laser capture microdissection, fluorescence activated cell sorting, and immunological cell sorting.
  • a biological sample can comprise one or more cells from the subject.
  • a sample can be a tumor cell sample, e.g. the sample can comprise cancerous cells, cells from a tumor, and/or a tumor biopsy.
  • the sample can be a blood sample which comprises of cancerous cells.
  • the biological sample comprises MIC-CTCs, activated PaSCs, macrophages.
  • the biological sample can be assayed by various methods. These methods include but are not limited to multiplex QD-based labeling (mQDL), diaminobenzidine (DAB) immunohistochemical methods, fluorescent immunohistochemical methods, ELISA methods, Western blotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR). These methods and systems also include but are not limited to enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, flow cytometry, fluorescence in situ hybridization (FISH), radioimmuno assays, and affinity purification. Examples of ELISAs include but are not limited to indirect ELISA, sandwich ELISA, competitive ELISA, multiple and portable ELISA.
  • mQDL multiplex QD-based labeling
  • DAB diaminobenzidine
  • FISH fluorescence in situ hybridization
  • affinity purification examples include but are not limited to indirect ELISA, sandwich ELISA, competitive ELISA, multiple and portable ELISA.
  • assaying the biological sample comprises using multispectral quantitative imaging analysis. In certain embodiments, assaying the biological sample comprises using multiplex quantum dot labeling. This method is quantitative in comparison to the conventional method for assaying the samples to determine expression levels in tissues, which uses the intensity of IHC staining scored based on a combined intensity and percentage positive scoring cells as previously reported by De Marzo et al. (De Marzo AM, Knudsen B, Chan-Tack K, Epstein JL E-cadherin expression as a marker of tumor aggressiveness in routinely processed radical prostatectomy specimens. Urology 53(4):707-713, 1999).
  • the analysis of gene expression levels may involve amplification of an individual's nucleic acid by the polymerase chain reaction.
  • Use of the polymerase chain reaction for the amplification of nucleic acids is well known in the art (see, for example, Mullis et al. (Eds.), The Polymerase Chain Reaction, Birkhauser, Boston, (1994)).
  • nucleic acid means a polynucleotide such as a single or double-stranded DNA or RNA molecule including, for example, genomic DNA, cDNA and mRNA.
  • nucleic acid encompasses nucleic acid molecules of both natural and synthetic origin as well as molecules of linear, circular or branched configuration representing either the sense or antisense strand, or both, of a native nucleic acid molecule.
  • Quantitative amplification involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction.
  • Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis is described in Ginzonger, et al. (2000) Cancer Research 60:5405-5409.
  • the known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene.
  • Fluorogenic quantitative PCR may also be used in the methods of the invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green.
  • ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.
  • LCR ligase chain reaction
  • a DNA sample suitable for hybridization can be obtained, e.g., by polymerase chain reaction (PCR) amplification of genomic DNA, fragments of genomic DNA, fragments of genomic DNA ligated to adaptor sequences or cloned sequences.
  • Computer programs that are well known in the art can be used in the design of primers with the desired specificity and optimal amplification properties, such as Oligo version 5.0 (National Biosciences).
  • PCR methods are well known in the art, and are described, for example, in Innis et al., eds., 1990, PCR Protocols: A Guide to Methods And Applications, Academic Press Inc., San Diego, Calif. It will be apparent to one skilled in the art that controlled robotic systems are useful for isolating and amplifying nucleic acids and can be used.
  • the nucleic acid samples derived from a subject used in the methods of the invention can be hybridized to arrays comprising probes (e.g., oligonucleotide probes) in order to identify RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPRl, NSE, Lin28b, and/or SYP and in instances wherein a housekeeping gene expression is also to be assessed, comprising probes in order to identify housekeeping genes.
  • probes e.g., oligonucleotide probes
  • the probes used in the methods of the invention comprise an array of probes that can be tiled on a DNA chip (e.g., SNP oligonucleotide probes).
  • Hybridization and wash conditions used in the methods of the invention are chosen so that the nucleic acid samples to be analyzed by the invention specifically bind or specifically hybridize to the complementary oligonucleotide sequences of the array, preferably to a specific array site, wherein its complementary DNA is located.
  • the complementary DNA can be completely matched or mismatched to some degree as used, for example, in Affymetrix oligonucleotide arrays.
  • the single-stranded synthetic oligodeoxyribonucleic acid DNA probes of an array may need to be denatured prior to contact with the nucleic acid samples from a subject, e.g., to remove hairpins or dimers which form due to self-complementary sequences.
  • Optimal hybridization conditions will depend on the length of the probes and type of nucleic acid samples from a subject.
  • General parameters for specific ⁇ i.e., stringent hybridization conditions for nucleic acids are described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012); Ausubel et al., eds., 1989, Current Protocols in Molecules Biology, Vol. 1, Green Publishing Associates, Inc., John Wiley & Sons, Inc., New York, at pp. 2.10.1- 2.10.16.
  • Exemplary useful hybridization conditions are provided in, e.g., Tijessen, 1993, Hybridization with Nucleic Acid Probes, Elsevier Science Publishers B. V. and Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Calif.
  • DNA arrays can be used to determine the expression levels of genes, by measuring the level of hybridization of the nucleic acid sequence to oligonucleotide probes that comprise complementary sequences.
  • oligonucleotide probes i.e., nucleic acid molecules having defined sequences
  • probes i.e., nucleic acid molecules having defined sequences
  • a set of nucleic acid probes, each of which has a defined sequence is immobilized on a solid support in such a manner that each different probe is immobilized to a predetermined region.
  • the set of probes forms an array of positionally-addressable binding (e.g., hybridization) sites on a support.
  • Each of such binding sites comprises a plurality of oligonucleotide molecules of a probe bound to the predetermined region on the support.
  • each probe of the array is preferably located at a known, predetermined position on the solid support such that the identity (i.e., the sequence) of each probe can be determined from its position on the array (i.e., on the support or surface).
  • Microarrays can be made in a number of ways, of which several are described herein. However produced, microarrays share certain characteristics, they are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other.
  • the microarrays are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions.
  • the microarrays are preferably small, e.g., between about 1 cm 2 and 25 cm 2 , preferably about 1 to 3 cm 2 .
  • both larger and smaller arrays are also contemplated and may be preferable, e.g., for simultaneously evaluating a very large number of different probes.
  • Oligonucleotide probes can be synthesized directly on a support to form the array.
  • the probes can be attached to a solid support or surface, which may be made, e.g., from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, gel, or other porous or nonporous material.
  • the set of immobilized probes or the array of immobilized probes is contacted with a sample containing labeled nucleic acid species so that nucleic acids having sequences complementary to an immobilized probe hybridize or bind to the probe. After separation of, e.g., by washing off, any unbound material, the bound, labeled sequences are detected and measured. The measurement is typically conducted with computer assistance.
  • DNA array technologies have made it possible to determine the expression level of RA KL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N- Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP and housekeeping genes, as mentioned above.
  • high-density oligonucleotide arrays are used in the methods of the invention. These arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface can be synthesized in situ on the surface by, for example, photolithographic techniques (see, e.g., Fodor et al., 1991, Science 251 :767- 773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91 :5022-5026; Lockhart et al., 1996, Nature Biotechnology 14: 1675; U.S. Pat. Nos.
  • Another method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al. (1995, Science 270:467-470).
  • Other methods for making microarrays e.g., by masking (Maskos and Southern, 1992, Nucl. Acids. Res. 20: 1679-1684), may also be used.
  • oligonucleotides ⁇ e.g., 15 to 60-mers
  • the array produced can be redundant, with several oligonucleotide molecules corresponding to each informative locus of interest ⁇ e.g., SNPs, RFLPs, STRs, etc.).
  • One exemplary means for generating the oligonucleotide probes of the DNA array is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (Froehler et al., 1986, Nucleic Acid Res. 14:5399-5407; McBride et al., 1983, Tetrahedron Lett. 24:246-248). Synthetic sequences are typically between about 15 and about 600 bases in length, more typically between about 20 and about 100 bases, most preferably between about 40 and about 70 bases in length.
  • synthetic nucleic acids include non-natural bases, such as, but by no means limited to, inosine.
  • nucleic acid analogues may be used as binding sites for hybridization.
  • An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., 1993, Nature 363 :566-568; U.S. Pat. No. 5,539,083).
  • the hybridization sites ⁇ i.e., the probes are made from plasmid or phage clones of regions of genomic DNA corresponding to SNPs or the complement thereof.
  • the size of the oligonucleotide probes used in the methods of the invention can be at least 10, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length.
  • hybridization stringency condition e.g., the hybridization temperature and the salt concentrations, may be altered by methods that are well known in the art.
  • the high-density oligonucleotide arrays used in the methods of the invention comprise oligonucleotides corresponding to RANKL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD 133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, NPR1, NSE, Lin28b, and/or SYP and housekeeping genes, as mentioned above.
  • the oligonucleotide probes may comprise DNA or DNA "mimics" ⁇ e.g., derivatives and analogues) corresponding to a portion of each informative locus of interest ⁇ e.g., SNPs, RFLPs, STRs, etc.) in a subject's genome.
  • the oligonucleotide probes can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone.
  • Exemplary DNA mimics include, e.g., phosphorothioates.
  • For each SNP locus, a plurality of different oligonucleotides may be used that are complementary to the sequences of sample nucleic acids.
  • a single informative locus of interest ⁇ e.g., SNPs, RFLPs, STRs, etc.
  • a single informative locus of interest ⁇ e.g., SNPs, RFLPs, STRs, etc.
  • about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more different oligonucleotides can be used.
  • Each of the oligonucleotides for a particular informative locus of interest may have a slight variation in perfect matches, mismatches, and flanking sequence around the SNP.
  • the probes are generated such that the probes for a particular informative locus of interest comprise overlapping and/or successive overlapping sequences which span or are tiled across a genomic region containing the target site, where all the probes contain the target site.
  • overlapping probe sequences can be tiled at steps of a predetermined base interval, e. g. at steps of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases intervals.
  • the assays can be performed using arrays suitable for use with molecular inversion probe protocols such as described by Wang et al. (2007) Genome Biol. 8, R246.
  • cross-hybridization among similar probes can significantly contaminate and confuse the results of hybridization measurements.
  • Cross-hybridization is a particularly significant concern in the detection of S Ps since the sequence to be detected ⁇ i.e., the particular SNP) must be distinguished from other sequences that differ by only a single nucleotide.
  • Cross-hybridization can be minimized by regulating either the hybridization stringency condition and/or during post- hybridization washings.
  • the probes used in the methods of the invention are immobilized ⁇ i.e., tiled) on a glass slide called a chip.
  • a DNA microarray can comprises a chip on which oligonucleotides (purified single-stranded DNA sequences in solution) have been robotically printed in an (approximately) rectangular array with each spot on the array corresponds to a single DNA sample which encodes an oligonucleotide.
  • the process comprises, flooding the DNA microarray chip with a labeled sample under conditions suitable for hybridization to occur between the slide sequences and the labeled sample, then the array is washed and dried, and the array is scanned with a laser microscope to detect hybridization.
  • the maximum number of RA KL, Vimentin, FOXM1, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP or the housekeeping genes being probed per array is determined by the size of the genome and genetic diversity of the subject's species.
  • DNA chips are well known in the art and can be purchased in pre-5 fabricated form with sequences specific to particular species. In other embodiments, SNPs and/or DNA copy number can be detected and quantitated using sequencing methods, such as "next-generation sequencing methods" as described further above.
  • the protein, polypeptide, nucleic acid, fragments thereof, or fragments thereof ligated to adaptor regions used in the methods of the invention are detectably labeled.
  • the detectable label can be a fluorescent label, e.g., by incorporation of nucleotide analogues.
  • Other labels suitable for use in the present invention include, but are not limited to, biotin, iminobiotin, antigens, cofactors, dinitrophenol, lipoic acid, olefinic compounds, detectable polypeptides, electron rich molecules, enzymes capable of generating a detectable signal by action upon a substrate, and radioactive isotopes.
  • Radioactive isotopes include that can be used in conjunction with the methods of the invention, but are not limited to, 32P and 14C.
  • Fluorescent molecules suitable for the present invention include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, texas red, 5 'carboxy -fluorescein (“FAM”), 2', 7'-dimethoxy-4', 5'-dichloro-6- carboxy-fluorescein (“JOE”), N, N, N', N'-tetramethyl-6-carboxy-rhodamine (“TAMRA”), 6-carboxy-X-rhodamine (“ROX”), HEX, TET, IRD40, and IRD41.
  • Fluorescent molecules which are suitable for use according to the invention further include: cyamine dyes, including but not limited to Cy2, Cy3, Cy3.5, CY5, Cy5.5, Cy7 and FLUORX; BODIPY dyes including but not limited to BODIPY-FL, BODIPY-TR, BODIPY- TMR, BODIPY-630/650, and BODIPY-650/670; and ALEXA dyes, including but not limited to ALEXA-488, ALEXA-532, ALEXA-546, ALEXA-568, and ALEXA-594; as well as other fluorescent dyes which will be known to those who are skilled in the art.
  • Electron rich indicator molecules suitable for the present invention include, but are not limited to, ferritin, hemocyanin and colloidal gold.
  • Two-color fluorescence labeling and detection schemes may also be used (Shena et al., 1995, Science 270:467-470). Use of two or more labels can be useful in detecting variations due to minor differences in experimental conditions (e.g., hybridization conditions). In some embodiments of the invention, at least 5, 10, 20, or 100 dyes of different colors can be used for labeling. Such labeling would also permit analysis of multiple samples simultaneously which is encompassed by the invention.
  • the labeled nucleic acid samples, fragments thereof, or fragments thereof ligated to adaptor regions that can be used in the methods of the invention are contacted to a plurality of oligonucleotide probes under conditions that allow sample nucleic acids having sequences complementary to the probes to hybridize thereto.
  • the hybridization signals can be detected using methods well known to those of skill in the art including, but not limited to, X-Ray film, phosphor imager, or CCD camera.
  • the fluorescence emissions at each site of a transcript array can be, preferably, detected by scanning confocal laser microscopy.
  • a separate scan, using the appropriate excitation line, is carried out for each of the two fluorophores used.
  • a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see Shalon et al. (1996) Genome Res. 6, 639-645).
  • the arrays are scanned with a laser fluorescence scanner with a computer controlled X-Y stage and a microscope objective. Sequential excitation of the two fluorophores is achieved with a multi-line, mixed gas laser, and the emitted light is split by wavelength and detected with two photomultiplier tubes.
  • Such fluorescence laser scanning devices are described, e.g., in Schena et al. (1996) Genome Res. 6, 639-645.
  • a fiber-optic bundle can be used such as that described by Ferguson et al. (1996) Nat. Biotech. 14, 1681-1684.
  • the resulting signals can then be analyzed to determine the expression of RA KL, Vimentin, FOXMl, FOXA2, c-Myc, Max, AP4, CgA, NSE, CK13, CD133, CD44, Nanog, Oct4, SOX2, c-Met, E-Cad, N-Cad, ALDHl, SDF1, PR1, NSE, Lin28b, and/or SYP and the reference genes, using computer software.
  • the amplification can comprise cloning regions of genomic DNA of the subject.
  • amplification of the DNA regions is achieved through the cloning process.
  • expression vectors can be engineered to express large quantities of particular fragments of genomic DNA of the subject (Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4 th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012)).
  • the amplification comprises expressing a nucleic acid encoding a gene, or a gene and flanking genomic regions of nucleic acids, from the subject.
  • RNA pre-messenger RNA
  • RNA pre-messenger RNA
  • the genomic DNA, or pre-RNA, of a subject may be fragmented using restriction endonucleases or other methods. The resulting fragments may be hybridized to SNP probes.
  • a DNA sample of a subject for use in hybridization may be about 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1000 ng of DNA or greater.
  • methods are used that require very small amounts of nucleic acids for analysis, such as less than 400 ng, 300 ng, 200 ng, 100 ng, 90 ng, 85 ng, 80 ng, 75 ng, 70 ng, 65 ng, 60 ng, 55 ng, 50 ng, or less, such as is used for molecular inversion probe (MTP) assays.
  • MTP molecular inversion probe
  • the resulting data can be analyzed using various algorithms, based on well-known methods used by those skilled in the art.
  • CTCs exist in a minute fraction among vast numbers of normal cells in a given clinical blood sample. Repeated investigation in a CTC preparation is nearly impossible due to the rarity of this cell type in the blood. Expanding CTCs ex vivo is thus necessary for reproducible examination of their genomic makeups and behaviors in vitro in culture or in vivo as patient-derived xenografts (PDXs).
  • PDXs patient-derived xenografts
  • the present invention provides a composition, comprising circulating tumor cells (CTCs), grown as 3-dimensional (3-D) spheroids, isolated from a subject with a cancer.
  • CTCs circulating tumor cells
  • the CTCs are cultured and/or expanded ex vivo.
  • the CTCs are inoculated into a non-human animal.
  • the composition is used as a model of the cancer.
  • the CTCs and a buffer solution are inoculated in the non- human animal.
  • the buffer solution can include, but is not limited to, PBS, TBS, TAPS, bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, and/or MES.
  • the buffer solution is PBS.
  • the CTCs, a buffer solution and a gelatinous protein mixture are inoculated in the non-human animal.
  • the gelatinous protein mixture is Matrigel and/or Geltrex. In various embodiments, the gelatinous protein mixture is Matrigel.
  • a clinically useful number of CTCs are obtained through the CTC expansion protocol.
  • the amount of cells obtained can range between lxl0 2 to lOxlO 2 , lxlO 3 to 10xl0 3 , lxl0 4 to lOxlO 4 , lxlO 5 to lOxlO 5 , lxlO 6 to lOxlO 6 , lxlO 7 to lOxlO 7 , lxlO 8 to lOxlO 8 cells or a combination thereof.
  • the present invention also provides a method of establishing a model for a cancer, comprising isolating circulating tumor cells (CTCs) from a subject with the cancer; culturing and/or expanding the CTCs as 3-D spheroids ex vivo, thereby establishing the ex vivo cultured and/or expanded CTCs as the model for the cancer.
  • CTCs circulating tumor cells
  • the present invention provides a non-human animal inoculated with circulating tumor cells (CTCs) isolated from a subject with a cancer.
  • CTCs circulating tumor cells
  • the CTCs are inoculated subcutaneously, intrafemorally, orthotopically, or intraosseously, or a combination thereof.
  • the CTCs are cultured and/or expanded ex vivo.
  • the non-human animal is a rodent, mouse, rat, rabbit or guinea pig. In certain embodiments, the non-human animal is used as a model of the cancer.
  • the present invention also provides a method of establishing a model for a cancer, comprising: isolating circulating tumor cells (CTCs) from a subject with the cancer; culturing and/or expanding the CTCs as 3-D spheroids ex vivo; and inoculating the ex vivo cultured and/or expanded CTCs into a non-human animal, thereby establishing the non -human animal as the model for the cancer.
  • CTCs circulating tumor cells
  • the cancer is pancreatic cancer or prostate cancer.
  • the CTCs are isolated from a biological sample from the subject.
  • the biological sample is a liquid biopsy of the cancer.
  • the biological sample is cheek swab; mucus; whole blood, blood, serum; plasma; urine; saliva; semen; lymph; fecal extract; sputum; other body fluid or biofluid; cell sample; tissue sample; tumor sample; tumor biopsy, or their combinations.
  • the present invention provides a method of identifying a drug as being therapeutically effective or ineffective to a cancer, comprising: providing a model for the cancer; administering the drug to the model; and detecting a therapeutic response in the model and identifying the drug as being therapeutically effective to the cancer, or detecting no therapeutic response in the model and identifying the drug as being therapeutically ineffective to the cancer.
  • the model is a composition, comprising circulating tumor cells (CTCs) isolated from a subject with the cancer.
  • the composition is cultured in RPMI1640, 10% FBS and antibiotics.
  • the antibiotics include but are not limited to, actinomycin D, ampicillin, carbenicillin, cefotaxcime, fosmidomycin, gentamicin, kanamycin, neomycin, penicillin streptocysin, polymyxin C and/or streptomycin.
  • the antibiotic are penicillin streptocysin, penicillin and/or streptocysin.
  • the composition comprises CTCs which can range between lxlO 2 to lxlO 9 cells/ml. In some embodiments, the composition comprises lxlO 6 cells/ml.
  • the CTCs are tagged with a fluorescent protein or a luciferase, as discussed above.
  • the model is a non-human animal inoculated with circulating tumor cells (CTCs) isolated from a subject with the cancer.
  • CTCs circulating tumor cells
  • the non-human model is a rodent, mice, rat, rabbit or guinea pig.
  • the method is used for screening drugs.
  • the therapeutic response is inhibited cancer cell proliferation, inhibited cancer cell growth, inhibited cancer cell invasion, inhibited cancer cell mobility, promoted cancer cell differentiation, promoted cancer cell death, inhibited cancer progression, inhibited cancer invasion, inhibited cancer metastasis, or improved animal survival, or a combination thereof.
  • the present invention provides a method, comprising: providing a model for a cancer, wherein the model is a composition comprising circulating tumor cells (CTCs) isolated from a subject with the cancer; administering a drug to the model; and detecting a therapeutic response in the model and instructing the subject to receive the drug to treat the cancer, or detecting no therapeutic response in the model and instructing the subject not to receive the drug to minimize exposure to side effects associated with the drug.
  • this method is used for selecting appropriate drugs for individual cancer patients.
  • this method is used for personalizing treatments for individual cancer patients.
  • the present invention provides a method, comprising: providing a model for a cancer, wherein the model is a non-human animal inoculated with circulating tumor cells (CTCs) isolated from a subject with the cancer; administering a drug to the model; and detecting a therapeutic response in the model and instructing the subject to receive the drug to treat the cancer, or detecting no therapeutic response in the model and instructing the subject not to receive the drug to minimize exposure to side effects associated with the drug.
  • this method is used for selecting appropriate drugs for individual cancer patients.
  • this method is used for personalizing treatments for individual cancer patients.
  • the present invention provides a method of identifying a subject as having resistance or not to a drug, wherein the subject has a cancer, comprising: providing a model for the cancer; administering the drug to the model; and detecting resistance in the model and identifying the subject as having resistance to the drug, or detecting no resistance in the model and identifying the drug as having no resistance to the drug.
  • the model is a composition, comprising circulating tumor cells (CTCs) isolated from the subject.
  • the model is a non-human animal inoculated with circulating tumor cells (CTCs) isolated from the subject.
  • the subject is a human.
  • the subject is a mammalian subject including but not limited to human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse and rat.
  • Typical dosages of an effective amount of the drug can be in the ranges recommended by the manufacturer where known therapeutic molecules or compounds are used, and also as indicated to the skilled artisan by the in vitro responses in cells or in vivo responses in animal models. Such dosages typically can be reduced by up to about an order of magnitude in concentration or amount without losing relevant biological activity.
  • the actual dosage can depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of relevant cultured cells or histocultured tissue sample, or the responses observed in the appropriate animal models.
  • the drug may be administered once a day (SID/QD), twice a day (BID), three times a day (TDD), four times a day (QID), or more, so as to administer an effective amount of the drug to the subject, where the effective amount is any one or more of the doses described herein.
  • the drug is administered at about 0.001-0.01, 0.01-0.1, 0.1- 0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600- 700, 700-800, 800-900, or 900-1000 mg/kg, or a combination thereof.
  • the drug is administered at about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10- 20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800- 900, or 900-1000 mg/m 2 , or a combination thereof.
  • the drug is administered once, twice, three or more times. In some embodiments, the drug is administered 1-3 times per day, 1-7 times per week, 1-9 times per month, or 1-12 times per year. Still in some embodiments, the drug is administered for about 1-10 days, 10-20 days, 20-30 days, 30-40 days, 40-50 days, 50-60 days, 60-70 days, 70-80 days, 80-90 days, 90-100 days, 1-6 months, 6-12 months, or 1-5 years.
  • “mg/kg” refers to mg per kg body weight of the subject
  • mg/m 2 refers to mg per m 2 body surface area of the subject.
  • the drug is administered to a human.
  • the effective amount of the drug is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 ⁇ g/kg/day, or a combination thereof.
  • the effective amount of the drug is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 ⁇ g/m 2 /day, or a combination thereof.
  • the effective amount of the drug is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200- 300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg/kg/day, or a combination thereof.
  • the effective amount of the drug is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg/m 2 /day, or a combination thereof.
  • ' ⁇ g/kg/day" or “mg/kg/day” refers to ⁇ g or mg per kg body weight of the subject per day
  • ' ⁇ g/m 2 /day” or “mg/m 2 /day” refers to ⁇ g or mg per m 2 body surface area of the subject per day.
  • the drug may be administered using the appropriate modes of administration, for instance, the modes of administration recommended by the manufacturer for each of the drug.
  • various routes may be utilized to administer the drug of the claimed methods, including but not limited to intratumoral, intravascular, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, aerosol, nasal, via inhalation, oral, transmucosal, transdermal, parenteral, implantable pump or reservoir, continuous infusion, enteral application, topical application, local application, capsules and/or injections.
  • the retinoid agonist is administered intracranially, intraventricularly, intrathecally, epidurally, intradurally, topically, intravascularly, intravenously, intraarterially, intratumorally, intramuscularly, subcutaneously, intraperitoneally, intranasally, or orally.
  • examples of the drug include, but are not limited to, Temozolomide, Actinomycin, Alitretinoin, All-trans retinoic acid, Azacitidine, Azathioprine, Bevacizumab, Bexatotene, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cetuximab, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Erlotinib, Etoposide, Fluorouracil, Gefitinib, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Ipilimumab, Irinotecan, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Ocrelizuma
  • the amount of cells inoculated can range between lxlO 6 to 2xl0 6 cells, 2xl0 6 to 3xl0 6 cells, 3xl0 6 to 4xl0 6 cells, 4xl0 6 to 5xl0 6 cells, 5xl0 6 to 6xl0 6 cells, 6xl0 6 to 7xl0 6 cells, 7xl0 6 to 8xl0 6 cells, 8xl0 6 to 9xl0 6 cells, or 9xl0 6 to lOxlO 6 cells or a combination thereof.
  • the amount of cells inoculated is 2xl0 6 CTC cells per 50 ⁇ 1 phosphate buffer saline (PBS) mixed with equal volume of Matrigel.
  • the inoculated animal is maintained for a period of months for tumor growth and metastasis. In various embodiments, the animal is maintained for 1-2 months, 2-3 months, 3-4 months, 4-5 months, 5-6 months, 6-8 months, or 8-12 months. In some embodiments, the animal is maintained for 4 months.
  • the CTC cells can be inoculated orthotopically, intrafemorally, intracardiac, intraosseosly, or subcutaneously.
  • the animal can be inoculated multiple times with CTC cells.
  • the animal receives 2, 3, 4 or 5 inoculations of CTC cells.
  • the inoculation can be administered simultaneously, consecutively, or subsequently in a series of administrations.
  • the CTCs inoculated can be from samples obtained before, during, and/or after therapeutic treatment.
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
  • PDAC cases can be categorized into 3 clinical phases: 1) primary localized resectable tumor; 2) locally advanced unresectable disease; and 3) metastatic disease. All cases of PDAC are eventually metastatic, which accounts for the poor prognosis. Accumulating evidence indicates that military service per se may be a risk factor for PDAC, since military personnel and veterans have a higher incidence of PDAC, and veteran patients are more frequently seen with unresectable diseases. There is a 40% increased risk overall and a 90% increased risk in men between ages 48-52, indicating a association of PDAC oncogenesis with military service. Two major risk factors of the disease are alcohol consumption and smoking, which leads to higher PDAC incidence among veterans.
  • pancreatic stellate cells PaSCs
  • a salient feature of PDAC histopathology is a marked desmoplastic reaction by actively proliferating PaSCs and macrophages. Activated PaSCs and macrophages participate in modulating the malignant behavior of the tumor, and their contribution to the development of therapeutic resistance is currently unknown.
  • CTCs are the culprit cell pool for tumor spreading and metastasis.
  • CTCs As a metastatic cancer cell type readily available from liquid biopsy, CTCs exist in a minute fraction among vast numbers of normal cells in a given clinical blood sample. Though CTCs are being tested widely for diagnosis and prognosis, repeated investigation in a CTC preparation is nearly impossible due to the rarity of this cell type in the blood. Expanding CTCs as 3-D spheroids ex vivo is thus necessary for reproducible examination of their genomic makeups and behaviors in vitro in culture or in vivo as patient-derived xenografts (PDXs).
  • PDXs patient-derived xenografts
  • CTC-PDX With conventional PDX modeling, pieces of patient tumor are implanted directly to athymic mice for tumor formation. Conventional PDX suffers from inherent drawbacks including extremely low tumor formation rates in mice and less tumor progression and metastasis. CTCs are in dynamic equilibrium with tumor cells at the primary and metastatic sites, thus reflecting the state of the in situ tumor in real time.
  • the advantages of CTC-PDX over the conventional PDX include: 1) unlike PDX, CTC- PDX can be studied repeatedly in culture and in mice; 2) CTC-PDX tumor can metastasize in the mouse host; and 3) multiple CTC-PDXs can be established with longitudinally acquired patient samples at return visits, to monitor metastasis and therapeutic resistance.
  • MICs function as "drivers” to recruit and reprogram dormant cells as an integral part of the tumor (Chu GC et al., Endocr Relat Cancer 2014;21(2):311-326).
  • Bystander cell R&R may be one source of cancer progression and metastasis. Identification and characterization of MICs within CTCs has important implications for the study of cancer metastasis, and could aid novel biomarker discovery for predicting survival and therapeutic resistance in PDAC patients.
  • the targeted therapeutic agent is a heptamethine carbocyanine near-infrared (NIR) dye-drug conjugate targeting tumor cells specifically with the tumor-homing NIR heptamethine carbocyanine dye.
  • NIR near-infrared
  • cancer-associated stromal cells grew faster than their normal counterparts.
  • cancer-associated stromal culture could boost CTC growth (Wang RX, American Pancreatic Association 2015 Annual Meeting, Loews Coronado Bay, San Diego, November 4-7 2015), suggesting that some of the established stromal cells produced soluble paracrine factors.
  • Molecular profiling analyses expedite biomarker discovery and are used to characterize CTCs as a new cancer cell type.
  • Palanisamy et al. has completed many molecular profiling projects, and comprehensively characterized prostate cancer PDXs in collaboration with Dr. Nora Navonne from MDACC (Palanisamy et al, American Association for Cancer Research Annual Meeting 2013, Washington, DC, April 6-10, 2013).
  • NGS Next Generation Sequencing
  • FISH fluorescence in situ hybridization
  • FISH procedures have been standardized for formalin-fixed paraffin embedded (FFPE) tissues and FISH probes have been developed for the 27 ETS family genes and PTEN under two and four color schemes for simultaneous detection of ERG and PTEN status in the same nucleus.
  • FISH detection of candidate genes we can generate probes using bacterial artificial chromosome (BAC) clones on FFPE tissues from CTC-PDX tumor and biopsy tissues.
  • BAC bacterial artificial chromosome
  • PaSC and CTC factors that mediate PDAC metastasis and therapeutic resistance are identified, including extracellular matrices and soluble factors secreted immune cells.
  • the effects of therapeutics and their transport carriers are evaluated in PaSCs and CTCs in an immune-deficient PDAC mouse model.
  • Blood and tumor biopsy samples from PDAC patients are obtained from CSMC and VAGLA, which is the referral center for veteran PDAC patients.
  • Patient grouping is based on clinical diagnosis and subtyping at the time of blood sampling. For patients undergoing surgery, matched pairs of normal and cancerous specimens along with a blood sample from the same patient are collected. Tissue specimens are kept at -80°C. IRB approved written informed consent is used to collect packed blood cells and fresh tissue specimens. We conduct 3-D spheroid CTC expansion ex vivo. Fresh samples of packed blood cells, usually 2-3 ml in volume, are transported on ice.
  • Red blood cells are removed by hemolysis and whole PBMCs are cultured at l x l0 6 /ml in a specified medium for 4 weeks to obtain outgrowth of CTCs in organoid aggregates with unlimited proliferation potential (Figure 4B).
  • CTCs are expanded to 2> ⁇ 10 8 cells for animal inoculation, karyotyping, preparation of genomic DNA, total RNA, and whole cell lysates for molecular analyses.
  • CTCs virulence of the CTCs reflects the malignant potential of the disease.
  • the behavior of ex vivo expanded CTCs therefore, may serve as a measure of tumor status in situ in the patient.
  • the modeling is complemented by additionally analyzing longitudinally cultured CTCs from blood samples of 4 returning patients from the on- going clinical trial. They are sampled every 3 months for at least 3 consecutive time points. Behavior of the CTCs is correlated to status of patient disease progression, including the extent of metastasis and responsiveness to therapy. These CTCs are invaluable for elucidating the relationship of genomics, epigenetics, and gene expression with the mechanisms of cancer metastasis and therapeutic resistance.
  • cancer cells could transform host cells and a subpopulation of cancer cells, MICs, could recruit and reprogram "dormant" bystander cells in the tumor microenvironment.
  • RNASeq analysis of the reprogrammed cells revealed that transcription factors c-Myc and FOXMl were obligatory for R&R and for activated expression of EMT, stem and neuroendocrine markers ( Figures 1 and 8).
  • FACS fluorescence-activated cell sorting
  • PaSCs from the co-culture are tested in co-inoculation for chimeric CTC- PDX tumor formation. Compared to CTC-PDX without PaSCs, accelerated tumor formation and metastasis can reflect the activation status of the PaSCs.
  • CTCs are shown to recruit and re- program bystander pancreatic epithelial or stromal cells, three modes of cancer-stromal interaction are examined. First, we determine whether R&R occurs through intercellular communication, using gene expressional and behavioral changes in bystander cells as read- out.
  • CM Conditioned media
  • CM from CTC culture is subjected to EV enrichment by ultracentrifugation, as we have reported (Morello M. et al., Cell cycle 2013; 12(22):3526- 3536). Both enriched EVs in ⁇ ⁇ concentration and EV-depleted CM is used to treat bystander epithelial cells and PaSC monolayers, with the same protocol described above, to observe changes in growth rate and behavior. After treatment with CM for 5 passages, any acquisition of tumorigenicity in treated cells is examined by xenograft tumor formation.
  • enriched EVs from CTC culture medium are processed by miRNeasy kit (Qiagen) for miRNA isolation, which are used in RNASeq analysis to determine the identity of prevalent non-coding RNA species, which are studied further to evaluate their role in R&R, following our reported method (Josson S. et al., Clinical cancer research: an official journal of the American Association for Cancer Research 2014;20(17):4636-4646, Josson S.
  • the non-coding RNA is overexpressed via lentiviral infection to bystander PaSCs. Altered behavior of the overexpressing cells is assayed in vitro in cell culture and in vivo in chimeric CTC-PDX xenograft tumor formation.
  • CTCs recruit and re-program bystander cells through epigenetic mechanisms. Besides paracrine communication, we have found that cancer cells can fuse to bystander cells to cause epigenetic reprogramming.
  • CTCs are tagged with RFP (with neor selection) and subjected to co-culture under 3-D conditions with PaSCs for spontaneous CTC-PaSC fusion by our reported protocol (Wang R. et al., PloS one 2012;7(8):e42653).
  • the remaining stromal monolayer is treated with G418 (200 ⁇ g/ml) for 3 days to remove the PaSCs that are not involved in cell fusion.
  • CTC-PaSC hybrids are subjected to functional analyses in in vitro cell culture and by chimeric CTC-PDX tumor formation.
  • the hybrids are also examined for signs of epigenetic modification (e.g., histone modifications, genomic methylation and imprinting).
  • NIR dye-gemcitabine conjugates We assessed the clinical usefulness of the experimental agent in mice. CTC inoculated mice are maintained till tumor size reaches 300 mm 3 in volume, and then treated with the test agent disclose herein, to assess whether the treatment can eradicate previously formed PDAC tumors in mice. NIR dye and gemcitabine are used separately as a control treatment for NIR dye- gemcitabine conjugates.
  • NIR dye-gemcitabine conjugate is highly effective agents promoting the death of PDAC cells in culture and as tumors in mice.
  • PaSCs promote PDAC metastasis and therapeutic resistance, with strong stroma reaction associated with worse outcome.
  • Secretion of large amounts of extracellular matrix proteins by PaSCs is responsible for the fibrotic characteristics of PDAC and for enhanced cancer cell survival.
  • the authors observed an association between fewer activated PaSCs and worse patient survival. As described below, our approach distinguishes these seemly opposite effects by comparing the effects of normal PaSCs to those derived from advanced PDAC.
  • CTCs in suspension aggregate growth are co-cultured with PaSC monolayer for 72 hours.
  • activation status of the PaSCs is assayed by a-smooth muscle actin (a-SMA) expression.
  • Proliferation of the treated CTCs is measured and apoptosis is detected.
  • EMT and invasion are assessed by measuring its markers, E- cadherin, N-cadherin and vimentin, by western blot and Matrigel invasion assays.
  • Cancer sternness is assessed by detecting stem cell markers of CD133, sox2 and Nanog and by colony formation assays.
  • target CTCs or PaSCs are examined for loss of hENTl protein by western blot to identify cells lacking hENTl expression. These cells are infected with a lentiviral construct for hENTl overexpression to make the cells re-gain sensitivity toward treatment. Potential synergism between the two new agents is assessed by controlling doses of the agents in CTC-PaSC co-culture. Validate the effects of newly developed anti-tumor agents
  • CTC-PDX models extensive necropsy is done following euthanasia. Pancreatic tumor size is measured and metastatic lesions in abdominal and thoracic cavities identified. Tissues are histopathologically assessed and immunostained for aSMA and PCNA and CK- 19 to identify activated PaSCs and proliferating CTC cancer cells. Comparative analysis is used to detect changes in cancer stem cell marker expression with our established protocol.
  • PDAC biomarkers Genomic abnormalities and expressional aberrations are identified and validated as PDAC biomarkers. Actionable and "driver” biomarkers are selected as tools for PDAC diagnosis and therapeutic evaluation.
  • CTCs in PDAC patients are readily available "liquid biopsy" materials. Due to the lack of collaborative clinical and basic research, however, comprehensive characterization of this specified tumor cell type has not been attempted. Given the rarity of CTCs in patient blood samples, we pioneered an approach for successful expansion of CTCs and generation of CTC-PDX. This approach overcomes several technical limitations in obtaining high quality DNA and RNA from CTCs, which are urgently needed for reproducible molecular studies to avoid artifacts inherent to conventional genomic and expressional assay methods.
  • Abnormality is determined by comparative analysis of normal tissue, primary and metastatic PDAC specimens, cultured CTCs, CTC-PDX tumors and chimeric CTC-PDX tumors of the same patient.
  • the innovative approaches and unique capabilities of this study are the first to generate an integrated molecular signature for PDAC tumor heterogeneity, metastatic potential, drug sensitivity and treatment follow-up.
  • a stepwise strategy is used to identify novel genomic abnormalities associated with metastasis and therapeutic resistance in PDAC patients and CTC-PDX mouse models.
  • Each ex vivo expanded CTC sample is analyzed successively with: 1) SKY assay; 2) aCGH comparison; and 3) NGS analysis.
  • Whole genome sequencing with NGS technology is conducted by NantHealth Systems (Los Angeles, CA).
  • genomic DNA from the respective samples for aCGH analysis to assess genomic fidelity from CTC to CTC-PDX, and in comparison to primary and metastatic tumor tissues.
  • the aCGH assay is an essential tool to assess the genomic changes.
  • representative CTC cultures are subjected to mutation screening in CTC, CTD-PDX and biopsy materials by whole genome sequencing using NGS for comprehensive molecular profiling of abnormalities at the nucleotide level.
  • NGS data are transferred for biostatistics and bioinformatics analysis. Given the unbiased nature of NGS analysis, we can identify new druggable genomic mutations on a personalized basis.
  • RNASeq high-throughput transcriptome sequencing
  • transcripts for further study, stringent statistical analysis is used to examine the RNASeq data to identify transcripts that are differentially expressed between samples. Differential expression of top outliner candidate transcripts is validated with alternative methods of RT -PCR, western blot, mQDL, or RNA-ISH assays. Subsequently, extensive literature search and bioinformatics are used to identify transcripts that may play causal role in PDAC metast asis and therapeutic resistance.
  • Biomarkers in ex vivo expanded CTCs are recognized by stringent statistical analysis and subjected to further characterization in three studies. First, we confirm the association of biomarkers with CTCs and CTC-PDX models. Second, the biomarkers are validated for correlation with clinical PDAC metastasis and therapeutic resistance. Finally, we retrospectively test the application of selected biomarkers in PDAC diagnosis, treatment evaluation and disease prognosis. As biomarkers could be in the form of genomic abnormalities or transcriptional aberrations, corresponding methods are used for validation, which we have previously reported (Wang R. et al., Biochemical and biophysical research communications 2009;389(3):455-460, Wang R.
  • Genomic PCR is tested extensively as a convenient and preferred tool for detecting biomarkers of genomic abnormalities. After confirmation on ex vivo expanded CTCs, optimal PCR settings are established and tested for detection of CTCs directly from blood samples, first from the blood of mice bearing CTC-PDX tumors, then from clinical blood samples of PDAC patients at CSMC and VAGLA. Clinical PDAC specimens from the BioBank at CSMC are examined retrospectively with our established methods (Wang R. et al., Biochemical and biophysical research communications 2009;389(3):455-460, Wang R. et al., Clinical cancer research: an official journal of the American Association for Cancer Research 2007; 13(20):6040-6048 and Wang R.
  • the specimens are also used in FISH assays as a supplementary to confirm the PCR detection. Sensitivity and specificity of the detection are critically analyzed by stringent statistical examination.
  • RT-PCR may be used in the initial phase, differential expression of coding genes is confirmed by western blot, especially for biomarkers of gene fusion.
  • Immune-based techniques such as enzyme-linked immunosorbent assay (ELISA), FACS, mQDL and IHC is used depending on subcellular localization of the protein.
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescence-activated cell sorting
  • mQDL mQDL
  • IHC is used depending on subcellular localization of the protein.
  • Biomarkers of non-coding RNA and miRNA are confirmed with special real-time PCR as we reported previously (Josson S. et al., Oncogene 2015;34(21):2690-2699, Josson S. et al., Prostate 2008;68(15): 1599-1606 and Gururajan M. et al., Int Immunol 2010;22(7):583- 592).
  • Their validation as candidate markers for clinical PDAC detection is tested first with CTC-PDX tumor specimens and then with PDAC specimens by FISH.
  • Plasma samples from CSMC BioBank are used for retrospective validation with our established PCR method (Morello M. et al., Cell cycle 2013; 12(22):3526-3536).
  • SKY analysis is applied to all CTC cultures to detect gross chromosomal changes. Whether a consensus can be made and whether gross abnormalities can be exploited as biomarkers are determined with clinical specimens.
  • representative CTC cultures are subjected to aCGH to identify smaller genomic abnormalities. These abnormalities are evaluated as biomarkers with clinical specimens.
  • selected CTCs are subjected to NGS analysis to obtain a comprehensive view of genomic abnormalities at the nucleotide level.
  • Next generation paired-end transcriptome sequencing is a well-established platform, particularly the Illumina sequencing platform. This is the most economical thoroughfare to a full understanding of the genomic base for PDAC metastasis and therapeutic resistance.
  • ARID 1 A AT -rich interactive domain-containing protein 1 A
  • DC-1 a non-tumorigenic human prostate cancer cell line established in our own laboratory
  • ERBB2 a member of the epidermal growth factor (EGF) receptor family
  • FGFR1 a member of the fibroblast growth factor receptor family
  • JQ1 an inhibitor of the BET family of bromodomain proteins
  • Ki67 a cellular marker protein for proliferation
  • RAF rapidly accelerated fibrosarcoma protein RAF 1 a proto-oncoprotein of the RAF family
  • Sox2 a member of the Sox family of transcription factors
  • Pancreatic cancer often spread rapidly to form metastatic tumors.
  • CTCs tumor cells
  • stromal cells from liquid and tissue biopsies as source materials to examine the pathobiology and therapeutic responses of CTCs from PaCa patients.
  • Peripheral blood mononuclear and pancreatic stromal cells were isolated respectively from the whole blood and PaCa tumor biopsies from the same patient.
  • Ex vivo expansion of CTCs and stromal cells was performed by standardized outgrowth protocols and characterized. The tumorigenicity of CTCs and their responses to pancreatic stromal fibroblasts were assessed.
  • Therapeutic responses of CTCs and CTC-derived patient xenografts CTC-PDXs
  • CTCs grew as spherical organoids with high proliferative activity, which was not observed in healthy donor blood cultures.
  • CTC-PDX tumors expressed PaCa markers and exhibited local invasive and metastatic behaviors when inoculated orthotopically or intraosseously but not subcutaneously.
  • CTC growth was enhanced by stromal conditioned media.
  • CTC-PDX tumors share malignant features with in situ tumors of the patients, and can be used as a reproducible model for the study of PaCa metastasis to facilitate personalized oncological research on PaCa diagnosis, treatment and prognosis.
  • CTCs and CTC-PDX models can be used to study the mechanism of PaCa progression, may be used as tools for testing therapeutic agents, and can facilitate application in personalized oncology.
  • Circulating tumor cells (CTCs) from patient's blood represent a promising noninvasive liquid biopsy compartment useful for prognosis and diagnosis of prostate cancer (PC) patients. Since CTCs reflect the pool of tumor cells originated from the primary as well as metastatic sites, molecular and phenotypic characterizations of CTCs allow us understand the biology and metastatic process. The challenges of CTC research are a small number of PC cells in blood, the difficulties of repeating molecular and phenotypic assays, and validating these results with patients' clinical outcome. We established here an in vitro CTC expansion protocol, evaluated gene expression and behavioral profiles of CTCs, and correlated these results with the clinical status of PC patients.
  • the cultured CTCs were used for the development of CTC-Derived patient Xenografts (CDXs) and for the study of CTCs with MIC phenotype that recruit and reprogram non-tumorigenic PC cells to participate in tumorigenesis.
  • CDXs CTC-Derived patient Xenografts
  • CTCs post-RBC lysed peripheral blood mononuclear cells (PBMCs) as the source materials.
  • PBMCs peripheral blood mononuclear cells
  • the ability of in vitro CTC growth, in vivo tumor formation and distant metastases are determined and compared between PC patients and within PC patients with PBMCs harvested at various time points of hormonal/ chemotherapy.
  • CTCs are genetically tagged with GFP, RFP or Luc for ease of detection of local tumor growth and distant dissemination in mice and for subsequent tumor cell derivation for molecular analyses using microarray, qRT-PCR, western blots and in vitro behavioral assays including migration, invasion and anchorage-independent 3-D growth.
  • CTC-PDX models in mice from PC patients with two patients longitudinally before, during and after therapeutic intervention. Some but not all models expressed PC-associated biomarkers, androgen receptor, PSA and PSMA. Within the same patient, CTC-PDXs models established post therapy had increased tumor growth, invasiveness, metastasis and earlier death than prior therapy in mice.
  • MICs metastasis-initiating cells
  • E neuroendocrine
  • CTCs with MIC phenotype are shown to promote tumorigenic and metastatic potential in primary non-tumorigenic PC cells.
  • MICs can be developed as a novel diagnostic, prognostic and therapeutic targets.
  • PBMCs collected from 3 prostate cancer patients were used to establish 10 CDXs in mice.
  • CDXs were found to express variable levels of prostate epithelial and cancer-associated biomarkers, including EpCAM, AR, PSA and PSMA.
  • Metastasis- initiating cells are identified in CTCs, by the mQDL method, demonstrating the overexpression of genes associated with mesenchymal-, stem-, and neuroendocrine cells.
  • CDXs consist of MIC and non-MIC cells in which cellular interactions occur between tumorigenic/metastatic (MIC) and non-tumorigenic (non-MIC) populations of cells in the tumors.
  • MIC tumorigenic/metastatic
  • non-MIC non-tumorigenic
  • MIC can recruit and reprogram non-MIC or indolent prostate cancer cells to participate in tumorigenic and metastatic processes.
  • Therapeutic interruption of MIC -non-MIC communication reduced tumor burden and the aggressiveness of prostate cancer cells in mice.
  • Circulating tumor cells represent an important component of liquid biopsy, which have the potential of predicting cancer metastasis and therapeutic responsiveness.
  • the number, genomic alteration, and phenotypic feature of CTCs could offer unique insights of cancer plasticity and virulence.
  • Such studies are limited by the recovery of adequate number of live CTCs for thorough evaluation since the number of CTCs in blood samples is often small. This deficiency can be overcome by expanding CTCs in ex vivo culture prior to biochemical and molecular characterization.
  • Peripheral blood samples in the form of packed blood cells after removal of plasma, were scavenged from clinical laboratory. Mononuclear cells were isolated following ammonium chloride hemolysis and cultured in defined medium, which was formulated in our laboratory. Surface epithelial marker stains were used to detect CTC expansion, and cytotoxicity assays were conducted to evaluate the sensitivities of cultured CTCs to chemotherapeutic or differentiation agents.
  • a CTC-like population can be cultured consistently from patient's blood samples, while the presence of epithelial marker presence supports the cancer origin of the expanded CTCs. Further optimization of the ex vivo culture protocol could provide unique opportunities for reliable characterizations of the genomics, gene expression, and behavior of the CTCs. Without being bound to any particular theory, results of this study could lead to improved patient care in predicting disease progression and selecting effective therapy, at an individual basis for cancer targeting.

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Abstract

La présente invention concerne des méthodes d'établissement d'un modèle personnalisé pour le cancer utilisant des cellules tumorales circulantes provenant du sujet. La présente invention concerne également des méthodes d'identification d'un ou plusieurs médicaments pour le traitement du cancer et d'identification de la pharmacorésistance chez un sujet atteint d'un cancer, à l'aide du modèle personnalisé pour le cancer.
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US10174289B2 (en) 2014-05-28 2019-01-08 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
WO2020017948A1 (fr) * 2018-07-17 2020-01-23 Institute For Medical Research Cellules isolées de carcinome nasopharyngé et dérivés préparés à partir de ces dernières
US10781425B2 (en) 2010-05-06 2020-09-22 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
EP3730941A1 (fr) * 2019-04-23 2020-10-28 Institut Jean Paoli & Irène Calmettes Procédé de détermination d'un gradient moléculaire d'agressivité tumorale de référence pour un adénocarcinome canalaire du pancréas
KR20210012318A (ko) * 2019-07-24 2021-02-03 덕성여자대학교 산학협력단 암 오가노이드 및 내피 집락 세포를 이용한 대장암 이종이식 동물 모델 및 그 제조방법
US11066650B2 (en) 2016-05-05 2021-07-20 Children's Hospital Medical Center Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same
CN114480250A (zh) * 2020-11-12 2022-05-13 四川大学华西医院 构建原位原发胃癌动物模型的方法
US11584916B2 (en) 2014-10-17 2023-02-21 Children's Hospital Medical Center Method of making in vivo human small intestine organoids from pluripotent stem cells
US11738095B2 (en) 2007-07-13 2023-08-29 Emory University Cyanine-containing compounds for cancer imaging and treatment
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US11738095B2 (en) 2007-07-13 2023-08-29 Emory University Cyanine-containing compounds for cancer imaging and treatment
US10781425B2 (en) 2010-05-06 2020-09-22 Children's Hospital Medical Center Methods and systems for converting precursor cells into intestinal tissues through directed differentiation
US10174289B2 (en) 2014-05-28 2019-01-08 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
US11053477B2 (en) 2014-05-28 2021-07-06 Children's Hospital Medical Center Methods and systems for converting precursor cells into gastric tissues through directed differentiation
US11584916B2 (en) 2014-10-17 2023-02-21 Children's Hospital Medical Center Method of making in vivo human small intestine organoids from pluripotent stem cells
US11066650B2 (en) 2016-05-05 2021-07-20 Children's Hospital Medical Center Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same
US11767515B2 (en) 2016-12-05 2023-09-26 Children's Hospital Medical Center Colonic organoids and methods of making and using same
WO2020017948A1 (fr) * 2018-07-17 2020-01-23 Institute For Medical Research Cellules isolées de carcinome nasopharyngé et dérivés préparés à partir de ces dernières
WO2020216722A1 (fr) * 2019-04-23 2020-10-29 Institut Jean Paoli & Irene Calmettes Procédé de détermination d'un gradient moléculaire d'agressivité tumorale de référence pour un adénocarcinome canalaire du pancréas
EP3730941A1 (fr) * 2019-04-23 2020-10-28 Institut Jean Paoli & Irène Calmettes Procédé de détermination d'un gradient moléculaire d'agressivité tumorale de référence pour un adénocarcinome canalaire du pancréas
KR20210137940A (ko) * 2019-07-24 2021-11-18 덕성여자대학교 산학협력단 암 오가노이드 및 내피 집락 세포를 이용한 대장암 이종이식 동물 모델 및 그 제조방법
KR102475776B1 (ko) 2019-07-24 2022-12-08 덕성여자대학교 산학협력단 암 오가노이드 및 내피 집락 세포를 이용한 대장암 이종이식 동물 모델 및 그 제조방법
KR102324038B1 (ko) 2019-07-24 2021-11-10 덕성여자대학교 산학협력단 암 오가노이드 및 내피 집락 세포를 이용한 대장암 이종이식 동물 모델 및 그 제조방법
KR20210012318A (ko) * 2019-07-24 2021-02-03 덕성여자대학교 산학협력단 암 오가노이드 및 내피 집락 세포를 이용한 대장암 이종이식 동물 모델 및 그 제조방법
CN114480250A (zh) * 2020-11-12 2022-05-13 四川大学华西医院 构建原位原发胃癌动物模型的方法
CN114480250B (zh) * 2020-11-12 2023-09-08 四川大学华西医院 构建原位原发胃癌动物模型的方法

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