WO2019006136A1 - Organoïdes dérivés d'une cellule vésicale unique - Google Patents

Organoïdes dérivés d'une cellule vésicale unique Download PDF

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WO2019006136A1
WO2019006136A1 PCT/US2018/040041 US2018040041W WO2019006136A1 WO 2019006136 A1 WO2019006136 A1 WO 2019006136A1 US 2018040041 W US2018040041 W US 2018040041W WO 2019006136 A1 WO2019006136 A1 WO 2019006136A1
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Prior art keywords
bladder
cells
organoids
patient
agent
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PCT/US2018/040041
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English (en)
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Hatem Sabaawy
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Rutgers, The State University Of New Jersey
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    • 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
    • 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
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4742—Keratin; Cytokeratin

Definitions

  • Tissue stem cells maintaining the balance between normal differentiated cells and progenitor or stem cells is complex.
  • Adult stem cells provide regeneration of different tissues, organs, or neoplastic growth through responding to cues regulating the balance between cell proliferation, cell differentiation, and cell survival, with the later including balanced control of cell apoptosis, necrosis, senescence and autophagy.
  • Epigenetic changes which are independent of the genetic instructions but heritable at each cell division, can be the driving force towards initiation or progression of diseases.
  • Tissue stem cells are heterogeneous in their ability to proliferate, self-renew, and differentiate and they can reversibly switch between different subtypes under stress conditions. Tissue stem cells house multiple subtypes with propensities towards multi-lineage differentiation.
  • Hematopoietic stem cells for example, can reversibly acquire three proliferative states: a dormant state in which the cells are in the quiescent stage of the cell cycle, a homeostatic state in which the cells are occasionally cycling to maintain tissue differentiation, and an activated state in which the cells are cycling continuously.
  • the growth and regeneration of many adult stem cell pools are tightly controlled by these genetic and/or epigenetic responses to regulatory signals from growth factors and cytokines secreted through niche interactions and stromal feedback signals.
  • Bladder cancer is one of the leading causes of cancer-related death.
  • Bladder cancer can be classified into non-muscle invasive bladder cancer, which is more common and has a favorable prognosis, and muscle invasive bladder cancer, which is less prevalent but has a worse prognosis.
  • bladder cancer is one of the most expensive cancers to treat, primarily due to the considerable costs associated with long-term clinical management of patients with non-muscle invasive bladder cancer, as well as the costs associated with caring for patients after surgical removal of the bladder.
  • bladder cancer has been remarkably understudied relative to other cancers, and remains significantly under-represented by cancer models.
  • Muscle invasive bladder cancers have a relatively poor prognosis, with a 5-year survival of -50% for localized disease and -15% for metastatic tumors.
  • muscle invasive bladder cancer has the highest prevalence of ERBB2 mutations among the most common solid tumors, but currently there is no bladder cancer cell line available for preclinical studies that expresses the S310F ERBB2 mutant, which is the mutant allele found in >90% of ERBB2 mutant bladder tumors. This emphasizes the need for generation of new models, particularly models that represent the full spectrum of genomic alterations that occur in bladder cancer. There is also considerable evidence that chronic urinary track inflammation contributes to the development of bladder cancer.
  • Interstitial cystitis or painful bladder syndrome (PBS) is a chronic urinary condition characterized by bladder pain and urinary frequency and urgency. Its incidence has been estimated at 1.2/100,000 and is growing globally. Despite the tremendous clinical and quality of life implications associated with IC, no clear pathophysiology and etiologies are currently available. The value of cell cultures and particularly cultures of well-characterized cell types specific to disease entity cannot be overstated. The understanding of the molecular determinants as well as development of novel therapeutics has been limited by the lack of three-dimensional (3D) models that accurately characterize IC.
  • 3D three-dimensional
  • the present invention provides a method of making an organoid from a mammalian bladder tissue in vitro comprising: isolating cells from a mammalian bladder tissue to provide isolated cells; culturing the isolated cells in a differentiation medium for a time sufficient to enrich for stem cells and induce differentiation; and amplifying the cells by culturing in an extracellular matrix in an organoid medium for a time sufficient to produce organoids.
  • the invention provides an in vitro bladder organoid comprising urothelial cells, the organoid exhibiting endogenous three-dimensional organ architecture.
  • the in vitro bladder organoid is derived from a single epithelial cell of a bladder tissue, the organoid exhibiting endogenous three-dimensional organ architecture.
  • the invention provides an in vitro bladder organoid derived from primary bladder IC tissue, wherein the organoid comprises urothelial cells and exhibits endogenous three-dimensional organ architecture.
  • the invention provides an in vitro bladder organoid derived from primary bladder normal tissue, wherein the organoid comprises urothelial cancer cells and exhibits endogenous three-dimensional organ architecture.
  • the invention provides an in vitro bladder organoid derived from primary bladder cancer tissue, wherein the organoid comprises urothelial cells and exhibits endogenous three-dimensional organ architecture.
  • the invention provides a cell culture medium supplemented with epidermal growth factor (EGF), bovine pituitary extract (BPE), and Cholera Toxin.
  • EGF epidermal growth factor
  • BPE bovine pituitary extract
  • Cholera Toxin Cholera Toxin
  • the invention provides a cell culture medium supplemented with EGF, basic fibroblast growth factor (bFGF), BPE, and Cholera Toxin.
  • EGF EGF
  • bFGF basic fibroblast growth factor
  • BPE BPE
  • Cholera Toxin Cholera Toxin
  • the present invention provides a kit including a cell culture medium supplemented with EGF, BPE and Cholera Toxin, and a cell culture medium supplemented with EGF, bFGF, BPE and Cholera Toxin.
  • the invention provides a method for identifying agents having anticancer activity against bladder cancer cells including selecting at least one test agent, contacting a plurality of patient- specific bladder organoids derived from the patient's bladder cancer cell with the test agent, determining the number of bladder organoids in the presence of the test agent and the absence of the test agent, and identifying an agent having anticancer activity if the number or the growth of the organoid cells is less in the presence of the agent than in the absence of the agent.
  • the method provides a step of treating the patient with the agent identified as having anticancer activity against the patient- specific organoids but not against normal organoids.
  • a method for identifying agents having anticancer activity against bladder cancer cells can further include providing a mouse engrafted with bladder cancer cells from the patient and containing a tumor formed from the bladder cancer cells; administering the identified agent having anticancer activity to the mouse; and determining if the tumor size is reduced in the presence of the identified agent.
  • a method for identifying agents having anticancer activity against bladder cancer cells can further include providing a humanized mouse engrafted with components of a patient's immune system and bladder cancer cells from the patient and containing a tumor formed from the bladder cancer cells; administering the identified agent to the humanized mouse; and comparing the size of the tumor in the humanized mouse with components of a patient's immune system to the size of the tumor in the mouse in which the identified agent was administered; and determining if the size of the tumor in the humanized mouse with components of a patient's immune system is reduced relative to the size of the tumor in the mouse in which the identified agent was administered.
  • This and other embodiments can further include providing a humanized mouse engrafted with bladder cancer cells from the patient and containing a tumor formed from the bladder cancer cells; administering a control agent to the humanized mouse engrafted with bladder cancer cells from the patient; and comparing the size of the tumor in the humanized mouse engrafted with bladder cancer cells from the patient to the size of the tumor in the mouse in which the identified agent was administered; and determining if the size of the tumor in the mouse in which the identified agent was administered is reduced relative to the size of the tumor in the humanized mouse engrafted with bladder cancer cells from the patient.
  • the invention provides a method for identifying agents having regenerative activity for bladder IC cells including selecting at least one test agent, contacting a plurality of patient-specific bladder organoids derived from the patient's bladder IC cell with the test agent, determining the number of bladder organoids in the presence of the test agent and the absence of the test agent, and identifying an agent having regenerative activity if the number or the growth of the organoid cells is more in the presence of the agent than in the absence of the agent.
  • the method provides a step of treating the patient with the agent identified as having anticancer activity against the patient- specific organoids but not against normal organoids.
  • the present invention provides normal patient- specific bladder organoids, and methods of using such organoids for personalized therapies for bladder cancer, and IC or PBS.
  • the present invention provides immune humanized mice with implanted patient-specific bladder organoids, and methods of using such mice to identify personalized therapies for bladder cancer, IC or PBS, and other bladder disorders.
  • the organoids exhibit endogenous three-dimensional organ architecture. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides bladder organoids derived in vitro from normal and cancerous tissues, and methods of making and using such organoids, as well as cell culture media and kits.
  • certain growth factors in an in vitro environment containing extracellular matrix molecules in a 3-dimensional culture device may be used to make the organoids.
  • An organoid is a miniature form of a tissue that is generated in vitro and exhibits endogenous three-dimensional organ architecture. See, e.g., Cantrell and Kuo (2015) Genome Medicine 7:32-34.
  • the organoids of the present invention can be used, for example, to: a) determine genomic targets within tumors and prediction of response to therapies in preclinical and clinical trials; b) detect the activity of an anti-cancer agent by examining the number of surviving organoids after treatment; c) detect the activity of a proliferative agent by determining the number of proliferating cells within each organoid and determining gene expression profiling of relevant pathways; d) detect the activity of a urothelial regenerative agent by examining the number of organoids after treatment to restore the inner wall of the bladder, similar to the activity of pentosan polysulfate sodium currently used for treatment of IC; e) examine the specificity of agents targeting different cell types within organoids; f) determine the effects of chemotherapy and radiation;
  • the invention provides a method of making an organoid from a mammalian bladder tissue in vitro including: isolating cells from a mammalian bladder tissue to provide isolated cells; culturing the isolated cells in a differentiation medium for a time sufficient to enrich for stem cells and induce differentiation; and amplifying one or more of the cells by culturing in an extracellular matrix in an organoid medium for a time sufficient to produce organoids.
  • a time sufficient to induce differentiation can be examining morphological changes associated with differentiation.
  • the time sufficient to induce differentiation is from about 7 to about 28 days.
  • the time sufficient to induce differentiation is about 14 days.
  • the isolated cells are epithelial cells and/or mesenchymal cells. In one embodiment, a single bladder epithelial cell or mesenchymal cell is amplified.
  • the differentiation medium comprises keratinocyte serum- free medium (KSFM), EGF, BPE and Cholera Toxin.
  • KSFM is typically used at IX.
  • the concentration of EGF present in the differentiation medium may range from about 0.1- 100 mg/mL (e.g., 1 mg/mL, 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 21 mg/mL, 25 mg/mL, 50 mg/mL, 100 mg/mL, etc).
  • the concentration of BPE present in the differentiation medium may range from about 1-100 ⁇ g/mL (e.g., 1 ⁇ g/mL, 25 ⁇ g/mL, 50 ⁇ g/mL, 75 ⁇ g/mL, 100 ⁇ g/mL, etc.).
  • the concentration of Cholera Toxin present in the differentiation medium may range from about 1-100 ng/mL (e.g., 1 ⁇ g/mL, 25 ⁇ g/mL, 30 ⁇ g/mL, 35 ⁇ g/mL, 75 ⁇ g/mL, 100 ⁇ g/mL, etc.).
  • the differentiation medium comprises one or both of Penicillin (500-5000 Units/mL) and Streptomycin (50-500 ⁇ g/mL).
  • the differentiation medium comprises the following concentrations: KSFM (ThermoFisher Scientific) (about IX); EGF (about 20 mg/mL); BPE (about 50 ⁇ g/mL); Cholera Toxin (about 30 ng/mL); Penicillin (about 1000 Units/mL); and Streptomycin (about 100 ⁇ g/mL).
  • the differentiation medium may further comprise or be substituted with other supplements, growth factors, antibiotics, vitamins metabolites, and hormones, synthetic or natural with similar properties as known in the art.
  • the organoid medium includes KSFM, EGF, bFGF, BPE, and Cholera Toxin.
  • concentration of EGF present in the organoid medium may range from about 0.1-100 mg/mL (e.g., 1 mg/mL, 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 21 mg/mL, 25 mg/mL, 50 mg/mL, 100 mg/mL, etc).
  • the concentration of bFGF present in the organoid medium may range from about 0.1-100 mg/mL (e.g., 1 mg/mL, 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 75 mg/mL, 100 mg/mL, etc).
  • the concentration of BPE present in the organoid medium may range from about 1-100 ⁇ g/mL (e.g., 1 ⁇ g/mL, 25 ⁇ g/mL, 50 ⁇ g/mL, 75 ⁇ g/mL, 100 ⁇ g/mL, etc.).
  • the concentration of Cholera Toxin present in the organoid medium may range from about 1-100 ng/mL (e.g., 1 ⁇ g/mL, 25 ⁇ g/mL, 30 ⁇ g/mL, 35 ⁇ g/mL, 75 ⁇ g/mL, 100 ⁇ g/mL, etc.).
  • the organoid medium further comprises one or both of Penicillin (500-5000 Units/mL) and Streptomycin (50-500 ⁇ g/mL).
  • the organoid medium includes the following concentrations: KSFM at IX, approximately 20 mg/mL EGF, approximately 10 mg/mL bFGF, approximately 50 ⁇ g/mL BPE, approximately 30 ng/mL Cholera Toxin, approximately 1000 Units/mL Penicillin, and approximately 100 ⁇ g/mL Streptomycin.
  • the organoid medium may further include or be substituted with other supplements, growth factors, antibiotics, vitamins metabolites, and hormones, synthetic or natural with similar properties as known in the art.
  • the cells are from human bladder tissue, IC, and human primary bladder cancer tissue.
  • cells that may be used to make an organoid are human bladder stem-like cells.
  • Such cells are known in the art and may be identified and isolated using markers, for example, cytokeratin 5 (CK5), cytokeratin 7 (CK7), cytokeratin 8 (CK8), cytokeratin 14 (CK14), cytokeratin 18 (CK18), cytokeratin 20 (CK20), p63 and uroplakin.
  • markers for example, cytokeratin 5 (CK5), cytokeratin 7 (CK7), cytokeratin 8 (CK8), cytokeratin 14 (CK14), cytokeratin 18 (CK18), cytokeratin 20 (CK20), p63 and uroplakin.
  • the cells are positive for at least one marker selected from the group consisting of CK5, CK8, and CK7. In another embodiment, the cells are positive for CK5, CK14, CK18, CK20 and CK7.
  • Such cells may be identified and isolated by methods of cell sorting that are known in the art. For example, in one embodiment, the cells may be isolated by laser capture microdissection, cell sorting, or RNA sorting using methods known in the art, such as molecular beacons and the SmartFlareTM probe protocol (EMD Millipore).
  • the cells are obtained from surgically excised tissues by subjecting the tissues to mechanical dissociation, collagenase treatment, and filtration.
  • the method is performed with a commercially available extracellular matrix such as MatrigelTM.
  • extracellular matrix such as MatrigelTM.
  • Other natural or synthetic extracellular matrices are known in the art for culturing cells.
  • an extracellular matrix comprises laminin, entactin, and collagen.
  • the method is performed using a 3-dimensional culture device (chamber) that mimics an in vivo environment for the culturing of the cells, where preferably the extracellular matrix is formed inside a plate that is capable of inducing the proliferation of stem cells under hypoxic conditions.
  • 3-dimensional devices are known in the art. An example of such a device is disclosed by Bansal, N., et al.
  • the invention provides a bladder organoid.
  • the bladder is comprised of a specialized epithelium known as the urothelium, which is encapsulated by the lamina intestinal and is surrounded by a thick layer of smooth muscle that forms the bladder wall.
  • the urothelium includes three cell types: (i) basal cells, which are small cuboidal cells that express p63 and high molecular weight cytokeratins, such as CK5 and CK14; (ii) intermediate cells, which express p63, CK5, and CK14 at lower levels than basal cells; and (iii) superficial cells, also called “umbrella cells", which express uroplakin proteins and low molecular weight cytokeratins such as CK18 and CK20.
  • basal cells which are small cuboidal cells that express p63 and high molecular weight cytokeratins, such as CK5 and CK14
  • intermediate cells which express p63, CK5, and CK14 at lower levels than basal cells
  • superficial cells also called “umbrella cells”
  • stem cells in the basal layer that can drive the process of tissue repair.
  • the bladder organoids of the present invention resemble the structures of the primary tissue.
  • organoids recreate the human urothelium. Bladder tissue origin of organoids can be confirmed by detecting the expression of CK5, CK18, CK7 and uroplakin.
  • the invention provides a bladder organoid derived in vitro from primary bladder cancer tissue. Tumor heterogeneity can be efficiently modeled using the methods described to make an organoid, by mapping the diagnostic dominant clone and tumor subclones from each patient biopsy sample, generating organoids derived from each clone and defining the genetic signature of each clone.
  • a bladder organoid derived from primary bladder cancer tissue will generally maintain expression of bladder lineage- specific markers and the functional secretory profile of the original primary tissue.
  • a bladder organoid as described herein can be serially propagated, cryofrozen and regenerated and established as a model for cancer drug discovery and precision therapy.
  • the invention provides a bladder organoid derived in vitro from surgically excised tissues of tumors identified to express histopathological tissue specific and tumorigenic markers.
  • Single cells from these tissues may be isolated with non-contact laser capture microdissection or by RNA sorting, for example using SmartFlareTM probes to generate single cell organoids with known expression features.
  • organoids described herein exhibit endogenous three-dimensional organ architecture.
  • the invention provides a method for identifying agents having anticancer activity against bladder cancer cells from a patient(s) including selecting at least one test agent, contacting a plurality of patient- specific bladder organoids derived from the patient's bladder cancer cell with the test agent, determining the number of bladder organoids in the presence of the test agent and the absence of the test agent, and identifying an agent having anticancer activity if the number or growth of the organoids is less in the presence of the agent than in the absence of the agent.
  • the method provides a step of treating the patient with the agent identified as having anticancer activity against the patient- specific organoids.
  • a method for identifying agents having anticancer activity can further include providing a mouse engrafted with bladder cancer cells from the patient and containing a tumor formed from the bladder cancer cells; administering the identified agent having anticancer activity to the mouse; and determining if the tumor size is reduced in the presence of the identified agent.
  • a method for identifying agents having anticancer activity can further include providing a humanized mouse engrafted with components of a patient's immune system and bladder cancer cells from the patient and containing a tumor formed from the bladder cancer cells; administering the identified agent to the humanized mouse; and comparing the size of the tumor in the humanized mouse with components of a patient's immune system to the size of the tumor in the mouse in which the identified agent was administered; and determining if the size of the tumor in the humanized mouse with components of a patient's immune system is reduced relative to the size of the tumor in the mouse in which the identified agent was administered.
  • the humanized mice with the patient's immune system can be used to compare the effects of the identified agent (e.g., candidate therapeutic) on tumors in the presence or absence of immune cells to examine a potential role for combination with immunotherapy.
  • These methods can further include providing a humanized mouse (an immune-deficient control mouse) engrafted with bladder cancer cells from the patient and containing a tumor formed from the bladder cancer cells; administering a control agent to the humanized mouse engrafted with bladder cancer cells from the patient; and comparing the size of the tumor in the humanized mouse engrafted with bladder cancer cells from the patient to the size of the tumor in the mouse in which the identified agent was administered; and determining if the size of the tumor in the mouse in which the identified agent was administered is reduced relative to the size of the tumor in the humanized mouse engrafted with bladder cancer cells from the patient.
  • the identified agent can be confirmed as a successful treatment for cancer in the patient.
  • the invention provides a method for identifying agents, growth factors or peptides having urothelial regenerative activity against bladder IC cells including selecting at least one test agent, contacting a plurality of patient- specific bladder organoids derived from the patient's bladder IC cell with the test agent, determining the number of bladder organoids in the presence of the test agent and the absence of the test agent, and identifying an agent having regenerative activity if the number or growth of the organoids is more in the presence of the agent than in the absence of the agent.
  • the method provides a step of treating the patient with the agent identified as having regenerative activity against the patient- specific organoids.
  • the invention provides a method of selecting a personalized treatment for bladder cancer in a subject including: selecting at least one form of treatment, contacting a plurality of bladder organoids with the form of treatment, wherein the organoids are derived from bladder cancer cells from the subject, determining the number of bladder organoids in the presence of the treatment and the absence of the treatment, and selecting the treatment if the number or growth of the bladder organoids is less in the presence of the treatment than in the absence of the treatment.
  • Various types of therapy can then be examined using the organoids to determine therapy resistance before initiation, to tailor the therapy for each individual patient based on oncogenic driver expression in the organoids, as well as further study induced clonal selection processes that are the frequent causes of relapse.
  • subject refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • subject and patient are used interchangeably herein in reference to a human subject.
  • Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition.
  • the foregoing methods may be facilitated by comparing therapeutic effects in organoids derived from cancer cells and normal cells from the same patient.
  • normal organoids and cancer organoids derived from cells of the same patient can be assessed to determine genetic and epigenetic mutations and gene expression profiles that are cancer-specific, thereby allowing the determination of gene-drug associations and optimization of treatment.
  • Such comparisons also allow one to predict a therapeutic response and to personalize treatment in a specific patient.
  • clonally targeted therapies can be determined by testing the effect of a therapeutic agent on multiple organoids derived from subsequently determined dominant clones of bladder cancer cells identified in the tumor tissue from a patient, and comparing to the effect of the therapeutic agent on organoids derived from normal cells of the same patient.
  • the invention provides a cell culture (e.g., organoid) medium supplemented with EGF, bFGF, and BPE.
  • the invention provides a cell culture (e.g., organoid) medium supplemented with EGF, bFGF, BPE and Cholera Toxin.
  • the invention provides a cell culture (e.g., organoid) medium supplemented with EGF, bFGF, BPE, Cholera Toxin, Penicillin and Streptomycin.
  • the medium is a commercially available cell growth medium such as KSFM (Thermo Fisher Scientific).
  • kits to make an organoid from a single cell contains containers for a differentiation medium and an organoid medium as previously described.
  • the containers may also contain the necessary supplements (growth factors, antibiotics, hormones, vitamins, amino acids, and combinations thereof) for a differentiation medium and an organoid medium.
  • the kit may further include the necessary components for a 3- dimensional culture device, for example, plates, and/or materials for an extracellular matrix, e.g. MatrigelTM.
  • the kit may further contain a set of instructions to perform the methods of making an organoid from a single cell as previously described.
  • the present invention provides a mouse with an implanted patient- specific bladder organoid.
  • the mouse is a humanized mouse.
  • the mouse is a human immune system (HIS) -reconstituted mouse.
  • the mouse is non-obese diabetic (NOD)-Rag (-)- ⁇ chain (-) (NRG) mouse.
  • the mouse is an NSG immune-deficient PDX mouse.
  • HIS -reconstituted mice are known in the art and disclosed for example by Drake et al. (2012) Cell Mol Immunol 9:215-24 and Harris et al. (2013) Clinical and Experimental Immunology 174:402-413.
  • human stem cells from patient for example from a diagnostic bone marrow or blood sample or HLA- matched, are transplanted into neonatal NRG mice to engraft components of the patient's immune system.
  • Methods of making NSG immune-deficient PDX mice are also known in the art and disclosed for example by Ciamporcero et al. (2016) Oncogene 12: 1541-1553.
  • mice are later subjected to grafting with bladder organoids derived from bladder cells of the same patient subcutaneously, under the renal capsule or orthotopically in the mouse bladder.
  • the mice are useful for identifying new treatments, urothelial regeneration agents, assessing responses to therapy, and evaluating combination therapies.
  • H&E hematoxylin-eosin
  • Biopsy specimens were minced into 0.5 cubic mm pieces to increase the surface area and placed in uncoated plastic tissue culture dishes containing complete epithelial cell medium supplemented with growth factors and cytokines that support epithelial and mesenchymal cell growth, as developed for normal urothelial cells.
  • Epithelial cells were isolated by dispase enzyme treatment and single cells are subjected to 3D cell cultures with different mixtures of growth factors to determine the optimum growth conditions for normal, IC and cancer organoids.
  • Cystoscopic bladder biopsies from IC patients and cystectomy derived normal adjacent tissue (NAT) and bladder cells were utilized to determine best conditions for generating organoids.
  • a two-step methodology was employed comprising a first phase of adult bladder stem cell enrichment, conducted in a 2D setting (stage I), followed by a second phase of organoid 3D growth obtained in pure matrigel chambers (stage II).
  • stage I the optimum media to use was identified: KSFM medium with EGF (20 mg/mL), BPE (50 ⁇ g/mL), cholera toxin (30 ng/mL) and penicillin streptomycin that allowed enrichment of bladder stem cells within 14 days to allow the generation of organoids in the following phase II culture.
  • the growth of organoids in stage II from single cells occurred within 21 days.
  • Organoid cellular growth and molecular markers (detailed above) are identified, and measured using Q-PCR, IF (immunofluorescence) and immunohistochemistry (IHC) techniques.
  • the molecular markers of different cell types in bladder are identified and quantified, and the conditions that produce organoids with expression of molecular markers of epithelial and mesenchymal features are determined. In addition, the organoids are studied morphologically and by imaging. Identification of the molecular signature of IC/PBS, identification of candidate genes (by microarray and whole genome sequencing), and identification of urothelial-muscle cell interaction are pursued.
  • IC/PBS molecular biomarkers of IC/PBS
  • these therapies are evaluated in the IC/PBS organoid cultures once established to identify potential molecular subtypes and novel targeted therapies for IC/PBS.
  • Increased levels of these bladder biomarkers not only could serve a diagnostic purpose, but also allows one to quantify the level of response to therapy by monitoring the progress during the treatment period.
  • targeted therapies including pharmacological interventions (e.g. intravesical administration of peptides or liposomes) aimed at targeting urothelial receptor/ion channel expression may provide new strategies for the clinical management of IC and PBS.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des organoïdes dérivés d'une cellule unique, telle qu'une cellule cancéreuse vésicale, ainsi que des procédés et des compositions se rapportant à la production et à l'utilisation de ceux-ci, notamment un milieu de culture cellulaire permettant de produire lesdits organoïdes, et des procédés de traitement personnalisé du cancer de la vessie et de la cystite interstitielle (CI) ou du syndrome de la vessie douloureuse (SVD). L'invention concerne en outre une souris humanisée comprenant un organoïde vésical dérivé d'une cellule vésicale d'un patient.
PCT/US2018/040041 2017-06-28 2018-06-28 Organoïdes dérivés d'une cellule vésicale unique WO2019006136A1 (fr)

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EP3772538A1 (fr) * 2019-08-09 2021-02-10 Urosphere Procédés de différenciation de cellules souches épithéliales
CN112899230A (zh) * 2020-12-08 2021-06-04 武汉大学 一种膀胱癌类器官培养基及制备方法及应用
CN114075539A (zh) * 2020-08-18 2022-02-22 四川大学华西医院 构建原位原发膀胱癌动物模型的方法
CN115216446A (zh) * 2022-06-17 2022-10-21 成都诺医德医学检验实验室有限公司 一种尿液来源的膀胱癌肿瘤细胞类器官培养方法

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US20130274226A1 (en) * 2010-11-30 2013-10-17 The Board Of Trustees Of The University Of Illinois Silica nanoparticle agent conjugates
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3772538A1 (fr) * 2019-08-09 2021-02-10 Urosphere Procédés de différenciation de cellules souches épithéliales
WO2021028361A1 (fr) * 2019-08-09 2021-02-18 Urosphere Procédé de différenciation de cellules souches épithéliales
CN114075539A (zh) * 2020-08-18 2022-02-22 四川大学华西医院 构建原位原发膀胱癌动物模型的方法
CN114075539B (zh) * 2020-08-18 2023-09-08 四川大学华西医院 构建原位原发膀胱癌动物模型的方法
CN112899230A (zh) * 2020-12-08 2021-06-04 武汉大学 一种膀胱癌类器官培养基及制备方法及应用
CN112899230B (zh) * 2020-12-08 2022-07-08 武汉大学 一种膀胱癌类器官培养基及制备方法及应用
CN115216446A (zh) * 2022-06-17 2022-10-21 成都诺医德医学检验实验室有限公司 一种尿液来源的膀胱癌肿瘤细胞类器官培养方法

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