Claims for Patent: 10,221,394

Summary for Patent: 10,221,394

Title:	In vitro model for a tumor microenvironment
Abstract:	Methods for mimicking a tumor microenvironment in vitro are provided. The methods comprise indirectly applying a shear stress upon at least one tumor cell type plated on a surface within a cell culture container. Methods for mimicking tumor metastasis and methods for testing drugs or compounds in such systems are also provided.
Inventor(s):	Wamhoff; Brian R. (Charlottesville, VA), Blackman; Brett R. (Charlottesville, VA), Figler; Robert A. (Earlysville, VA), Gioeli; Daniel G. (Charlottesville, VA), Simmers; Michael B. (Charlottesville, VA)
Assignee:	HemoShear, LLC (Charlottesville, VA)
Application Number:	15/483,010
Patent Claims:	1. An in vitro method of testing a drug or a compound for an effect on a tumor, the method comprising: (a) mimicking a tumor microenvironment in vitro, wherein mimicking the tumor microenvironment in vitro comprises: (i) adding a culture medium to a cell culture container; (ii) plating at least one tumor cell type on a first surface of a porous membrane within the cell culture container; (iii) indirectly applying a shear stress upon the at least one tumor cell type by applying a shear stress upon a second surface of the porous membrane, the shear stress resulting from flow of the culture medium induced by a flow device, the flow mimicking flow to which the tumor cells are indirectly exposed in vivo in the tumor microenvironment; and (b) adding a drug or a compound to the culture medium; wherein a change in the at least one tumor cell type, in the presence of the drug or the compound, indicates that the drug or the compound has an effect on the tumor. 2. The method of claim 1, wherein the porous membrane is suspended in the cell culture container such that the first surface is proximal and in spaced relation to a bottom surface of the cell culture container, thereby defining within the cell culture container a lower volume comprising the at least one tumor cell type and an upper volume comprising a second surface of the porous membrane, wherein the shear stress is applied upon the second surface of the porous membrane in the upper volume of the container. 3. The method of claim 1, further comprising: (i) depositing at least one extracellular matrix component on the first surface of the porous membrane and plating the at least one tumor cell type on the at least one extracellular matrix component; or (ii) suspending the at least one tumor cell type in a solution comprising at least one extracellular matrix component to create a suspension comprising the at least one tumor cell type and the at least one extracellular matrix component, and depositing the suspension on the first surface of the porous membrane; the porous membrane being suspended in the cell culture container such that the first surface is proximal and in spaced relation to a bottom surface of the cell culture container, thereby defining within the cell culture container a lower volume comprising the at least one extracellular matrix component and the at least one tumor cell type, and an upper volume comprising a second surface of the porous membrane, wherein the shear stress is applied upon the second surface of the porous membrane in the upper volume of the container. 4. The method of claim 1, further comprising plating endothelial cells on the second surface of the porous membrane and applying the shear stress upon the plated endothelial cells. 5. The method of claim 1, further comprising plating at least one stromal cell type on the second surface of the porous membrane and applying the shear stress upon the plated stromal cell type. 6. The method of claim 1, wherein upon application of the shear stress: (i) a change in the level of a marker of the tumor microenvironment in the at least one tumor cell type, as compared to the level of the marker in the at least one tumor cell type in the absence of the shear stress, confirms mimicking of the tumor microenvironment; or (ii) a change in the localization of a marker of the tumor microenvironment in the at least one tumor cell type, as compared to the localization of the marker in the at least one tumor cell type in the absence of the shear stress, confirms mimicking of the tumor microenvironment; or (iii) a change in the level of a marker of the tumor microenvironment in the culture medium, as compared to the level of the marker in the culture medium in the absence of application of the shear stress, confirms mimicking of the tumor microenvironment. 7. The method of claim 2, wherein the cell culture container further comprises inlets and outlets within the portions of the cell culture container defining the upper and lower volumes. 8. The method of claim 2, further comprising perfusing culture medium into and out of the upper volume and into and out of the lower volume. 9. The method of claim 1, further comprising plating at least one stromal cell type on the first surface of the porous membrane. 10. The method of claim 1, wherein the at least one tumor cell type comprises cells derived from a carcinoma, a sarcoma, a lymphoma, an adenosquamous carcinoma, a mixed mesodermal tumor, carcinosarcoma, a teratocarcinoma, or a combination thereof. 11. The method of claim 1, wherein the at least one tumor cell type is derived from a tumor of connective tissue, a tumor of endothelium or mesothelium, a tumor of lymphoid tissue, a tumor of muscle, a tumor of an epithelial tissue, a tumor of a neural tissue, a tumor of the amine precursor uptake and decarboxylation (APUD) system, a tumor of a neural crest-derived cell, a gonadal tumor, or a combination thereof. 12. The method of claim 1, wherein the at least one tumor cell type comprises cells derived from a tumor of the lung, breast, colon, rectum, prostate, bladder, bone, pancreas, liver, bile duct, ovary, testis, uterus, placenta, brain, cartilage, smooth muscle, striated muscle, membranous lining of a body cavity, fibrous tissue, blood vessel, lymph vessel, lymph node, adipose tissue, neurogenic connective tissue of the brain, kidney, pituitary gland, parathyroid, thyroid, bronchial lining, adrenalmedulla, stomach, large intestine, small intestine, carotid body, chemoreceptor system, skin, gall bladder, or a combination thereof. 13. The method of claim 1, wherein the at least one tumor cell type comprises primary tumor cells obtained from a subject by biopsy, tumor resection, blood draw, or a combination thereof. 14. The method of claim 1, wherein the at least one tumor cell type comprises tumor cells derived from a humanized mouse bearing a tumor derived from a human subject. 15. The method of claim 14, wherein the humanized mouse comprises a non-obese diabetic severe combined immunodeficiency (NOD SCID) mouse, a NOD/Shi-scid/IL-2R.gamma.null (NOG) mouse, or a NOD SCID IL-2R.gamma.knockout (NSG) mouse. 16. The method of claim 4, wherein the endothelial cells comprise: microvascular endothelial cells, macrovascular endothelial cells, endothelial progenitor cells, or a combination thereof; endothelial cells derived from a tumor; endothelial cells derived from an organ or tissue in which a tumor resides; endothelial cells derived from lung, breast, colon, rectum, prostate, bladder, bone, pancreas, liver, bile duct, ovary, testis, uterus, placenta, brain, cartilage, smooth muscle, striated muscle, a membranous lining of a body cavity, fibrous tissue, blood vessel, lymph vessel, lymph node, adipose tissue, neurogenic connective tissue of the brain, kidney, pituitary gland, parathyroid, thyroid, bronchial lining, adrenal medulla, stomach, large intestine, small intestine, carotid body, chemoreceptor system, skin, gall bladder, or a combination thereof; cells derived from inducible pluripotent stem cells (iPSC); or a combination of any thereof. 17. The method of claim 9, wherein the at least one stromal cell type comprises fibroblasts, immune cells, pericytes, inflammatory cells, or a combination thereof. 18. The method of claim 9, further comprising mixing the at least one stromal cell type with the at least one tumor cell type prior to plating. 19. The method of claim 9, wherein the method comprises sequentially plating the at least one tumor cell type and the at least one stromal cell type. 20. The method of claim 19, comprising plating the at least one stromal cell type and subsequently plating the at least one tumor cell type on the plated stromal cell type. 21. The method of claim 17, wherein at least one stromal cell type comprises the fibroblasts, the fibroblasts comprising human lung fibroblast cell line Hs888Lu. 22. The method of claim 1, wherein the method comprises culturing the cell type or cell types in the substantial absence of exogenously added extracellular matrix. 23. The method of claim 3, wherein the at least one extracellular matrix component comprises a collagen, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, an elastin, a fibronectin, a laminin, a vitronectin, decellularized extracellular matrix purified from a biological source, or a combination thereof. 24. The method of claim 1, wherein the culture medium comprises sera, blood, blood cells, a blood component, immune cells, conditioned culture medium, or a combination thereof. 25. The method of claim 24, wherein the sera, blood, blood cells, blood component, or immune cells are derived from a human or other animal. 26. The method of claim 25, wherein the sera, blood, blood cells, blood component, or immune cells comprise sera, blood, blood cells, a blood component, or immune cells derived from a mouse, rat, guinea pig, hamster, rabbit, cat, dog, monkey, cow, pig, horse, goat, sheep, bird, or fish. 27. The method of claim 24, wherein the immune cells comprise B cells, dendritic cells, granulocytes, innate lymphoid cells, megakaryocytes, monocytes, macrophages, natural killer cells, T cells, thymocytes, or a combination thereof. 28. The method of claim 24, wherein the blood cells comprise platelets, red blood cells, or a combination thereof. 29. The method of claim 24, wherein the blood component comprises a clotting factor, a lipoprotein, a triglyceride, or a combination thereof. 30. The method of claim 24, wherein the conditioned culture medium comprises conditioned culture medium from a culture comprising tumor cells, a culture comprising endothelial cells, a culture comprising a stromal cell type, or a combination thereof. 31. The method of claim 1, wherein the at least one tumor cell type comprises tumor cells derived from a subject's tumor and the method further comprises determining whether to administer the drug or the compound to the subject based on the results of the in vitro testing. 32. The method of claim 1, wherein the concentration of the drug or compound in the culture medium is within the concentration range of the drug or the compound that achieves the effect in vivo. 33. The method of claim 1, wherein the concentration of the drug or the compound in the culture medium is within the concentration range of the in vivo therapeutic C.sub.max for the drug or the compound. 34. The method of claim 33, wherein the concentration of the drug or the compound in the culture medium is approximately the same as the in vivo therapeutic C.sub.max for the drug or the compound. 35. The method of claim 1, wherein the concentration of the drug or the compound in the culture medium is about 2-fold to about 20-fold lower than the concentration range of the in vivo therapeutic C.sub.max for the drug or the compound. 36. The method of claim 35, wherein the concentration of the drug or the compound in the culture medium is about 5-fold to about 15-fold lower than the concentration range of the in vivo therapeutic C.sub.max for the drug or the compound. 37. The method of claim 35, wherein the concentration of the drug or the compound in the culture medium is about 10-fold lower than the concentration range of the in vivo therapeutic C.sub.max for the drug or the compound. 38. The method of claim 1, wherein the effect comprises a toxic effect, a protective effect, a pathologic effect, a disease-promoting effect, an inflammatory effect, an oxidative effect, an endoplasmic reticulum stress effect, a mitochondrial stress effect, an apoptotic effect, a necrotic effect, an autophagic effect, an immunogenic cell death effect, a ferroptotic effect, a remodeling effect, a proliferative effect, an effect on angiogenesis, an effect on the activity of a protein, or an effect on the expression of a gene. 39. The method of claim 1, wherein the drug comprises an anti-cancer agent. 40. The method of claim 39, wherein the anti-cancer agent comprises an alkylating agent, an anti-metabolite, an anti-tumor antibiotic, a topoisomerase inhibitor, a corticosteroid, an anti-microtubule agent, a kinase inhibitor, a pathway inhibitor, a differentiating agent, a hormone therapy, an immunotherapy, L-asparaginase, a chelating agent, an ATP mimetic, a biologic medical product, or a combination thereof. 41. The method of claim 1, wherein the method further comprises perfusing the drug or the compound into at least one of the upper volume and the lower volume. 42. The method of claim 1, wherein the shear stress applied upon the at least one tumor cell type is about 0.1 dynes/cm.sup.2 to about 200 dynes/cm.sup.2. 43. The method of claim 42, wherein the shear stress applied upon the at least one tumor cell type is about 0.1 dynes/cm.sup.2 to about 100 dynes/cm.sup.2. 44. The method of claim 1, wherein the shear stress is applied at a rate of about 1 sec.sup.-1 to about 1000 sec.sup.-1. 45. The method of claim 1, wherein the method comprises directly exposing the at least one tumor cell type to the drug or the compound. 46. The method of claim 1, wherein the method comprises indirectly exposing the at least one tumor cell type to the drug or the compound. 47. The method of claim 4, wherein upon application of the shear stress, a change in the level or localization of a marker of the tumor microenvironment in the endothelial cells, as compared to the level or localization of the marker in the endothelial cells in the absence of application of the shear stress, confirms mimicking of the tumor microenvironment. 48. The method of claim 9, wherein upon application of the shear stress, a change in the level or localization of a marker of the tumor microenvironment in the at least one stromal cell type, as compared to the level or localization of the marker in the at least one stromal cell type in the absence of application of the shear stress, confirms mimicking of the tumor microenvironment.

Details for Patent 10,221,394

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Recordati Rare Diseases, Inc.	ELSPAR	asparaginase	For Injection	101063	01/10/1978		2033-10-21