Methods of primary tissue culture and drug screening using autologous serum and fluids
11667894 · 2023-06-06
Assignee
Inventors
Cpc classification
C12N2509/00
CHEMISTRY; METALLURGY
C12N2533/90
CHEMISTRY; METALLURGY
C12N5/0694
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides methods for culturing primary cells and tissues from a subject in the presence of the subject's own serum, ascites or pleural effusion fluid. Methods of treating cancer, and screening for the effectiveness or toxicity of drugs are also provided herein.
Claims
1. A method for the primary culturing of cancer cells from the ascites or pleural effusion fluid of a subject, using the following steps in the order shown: A. obtaining the fluid from the subject; B. isolating the cells from the fluid while retaining the cell-free fluid; C. filtering the cell-free fluid to prevent protein clotting; D. building a three dimensional autologous structure by a) mixing one or more matrix proteins with the filtered, cell-free fluid at a ratio of approximately 1:1 to make an autologous matrix, b) coating the surface of a culture well or plate with the autologous matrix, E. seeding the isolated cells from step B onto the coated surface of step D; and F. culturing the seeded cells in a medium consisting essentially of 100% cell-free fluid (v/v) from step C.
2. A method for culturing circulating tumor cells (CTC) from the peripheral blood of a subject, using the following steps in the order shown: A. obtaining blood from the subject; B. isolating a cell population containing both CTC and the peripheral blood mononuclear cells (PBMC) from the subject's blood, while retaining the serum; C. building a three dimensional autologous structure by a) mixing one or more matrix proteins with the serum from step B at a 1:1 ratio to make an autologous matrix, b) coating the surface of a culture well or plate with the autologous matrix, D. seeding the cells from step B onto the coated surface of step C, E. culturing the seeded cells in a medium consisting essentially of 100% serum (v/v) from the subject.
3. The method of claim 1 or 2 wherein the cells are isolated using density gradient centrifugation.
4. The method of claim 1 or 2 wherein the thickness of the layer coating the well or plate is approximately one to four millimeters.
5. The method of claim 1 or 2 wherein the one or more matrix proteins comprises laminin, elastin, collagen, and heparin sulfate proteoglycans.
6. A method for the concurrent testing of the toxicity and effectiveness of candidate cancer therapies to select the treatment that is most suitable for a particular subject, comprising: A. culturing cells from the subject according to the method of claim 1 or 2, depending on the type of cells: B. obtaining blood from the subject, isolating PBMC from the blood by density gradient centrifuge, and maintaining the isolated PBMC in a medium consisting essentially of 100% serum (v/v) from the subject; C. applying the candidate therapy in parallel to the culture of step A and to the PBMC from step B, following the same treatment protocol; D. assessing the effectiveness of the candidate treatment against the cancerous cells in the culture from step A, and measuring the toxicity of the treatment to the PBMC from step B; E. selecting the therapy having the highest effectiveness against the cancer, and an acceptable toxicity to the PBMC from the subject.
7. The method of claim 6, step C wherein the candidate therapy comprises, but is not limited to, any chemical agent, targeted gene therapy product, immunotherapy product, radiation procedure or other anti-cancer regimen, applied singly or concurrently.
8. The method of claim 6, step D, wherein the effectiveness and toxicity of the candidate therapy is measured using one or more cell viability assays.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
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DETAILED DESCRIPTION
(10) The ex vivo culturing technique of the present invention use the patient's body fluid or serum to culture the patient's own cells or tissues, such as tumor tissue or cancer cells. It can be used to culture solid tumors, tumors from malignant effusion (ascites and pleural fluids) and PBMC as well as CTCs. Because this technique creates an external environment that mimics the tumor's internal condition (inside of a patient), it makes it possible, for the first time, to grow a tumor ex vivo not just as tumor cells but as a tissue, with a high rate of success. This provides a reliable platform for precision, individualized medicine and is expected to greatly improve the accuracy of drug-sensitivity test for an individual patient.
(11) In some embodiments, one difference of this technique compared with traditional cell cultures is that tumor cells and their autologous surrounding tissue cells survive and grow together in their own body solutions, which enables them to reconstruct the tumor's original histopathology structures. Because of this similarity in the tumor's in vivo and ex vivo microenvironments, no change in immunophenotypes was found in tumor tissues before and after cultures.
(12) The applications of this invention include, but are not limited to:
(13) (1) supporting the study of cancer as a complete tissue in lab research, instead of cancer cells alone (current status). It provides a new platform for scientists to study the cellular communications of tumor-to-tumor and tumor-stromal cells in real-time.
(14) (2) improving the accuracy of drug-sensitivity tests. Since the tumor grows in a microenvironment similar to its original condition, the reaction to medical treatment in this culture system should more correctly predict the real therapeutic response of the tumor in the patient. In addition, because tumor tissues can survive in the culture for at least 8-10 days, it makes it possible to functionally profile a living tumor's reactions after exposure to drug(s).
(15) (3) enabling the comparison of the toxicity of tumor cells and PBMCs side-by-side to a given drug. Since “normal” PBMCs also survive in this system for at least 4-5 days, drug-sensitivity testing can be simultaneously conducted on patient's tumor cells and PBMCs to compare their toxicities to the same given drug(s). This procedure will prevent or greatly reduce negative side-effects to cancer patients of potential therapies.
(16) (4) increasing the CTC population for treatment and study. The rare population of CTC can be efficiently expanded in this culture system. Since they are living and proliferating in their own serum, they can also be used for functional tests and drug sensitivity tests.
(17) (5) providing a new platform for other types of pre-clinical treatment tests. Since no anti-cancer regimes, including various targeted therapies (e.g. gene-therapy or immunotherapy), can fit all cancer patients, they should be tested prior to use in a particular patient. This culture system can also be employed for these tests, to improve the accuracy and efficacy of individualized/personalized treatment.
(18) (6) studying, and testing of therapies for, non-cancerous diseases. Because “normal” stromal cells (existing in tumor surrounding tissue) including endothelial, fibroblast, mesothelial and cartilage, can also survive in this system for 7-10 days, this technique makes it possible to conduct studies and treatment tests on non-cancer diseases.
(19) Reference will now be made in detail to embodiments of the invention which, together with the drawings and the following examples, serve to explain the principles of the invention. These embodiments describe in sufficient detail to enable those skilled in the art to practice the invention, and it is understood that other embodiments may be utilized, and that structural, biological, and chemical changes may be made without departing from the spirit and scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
(20) For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, un-recited elements or method steps. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of” and/or “consisting of.” As used herein, the term “about” means at most plus or minus 10% of the numerical value of the number with which it is being used.
(21) Culturing Solid Tissue in Autologous Serum
(22) In one embodiment, the invention provides a method of culturing solid tissue from a subject, comprising i) obtaining the solid tissue from the subject; ii) obtaining serum from the subject; and iii) culturing the tissue in a three-dimensional biocompatible matrix in the presence of a media comprising at least 50% serum (v/v) from the subject.
(23) The solid tissue that can be cultured according to the methods of the invention is not limiting. In some embodiments, the solid tissue comprises normal healthy tissue. In some embodiments, the solid tissue comprises diseased tissue. In some embodiments, the solid tissue comprises tissue isolated from a tumor. In some embodiments, the tissue comprises cancerous and noncancerous cells, such as stromal cells. The tissue and cells that are isolated from a solid tumor are primary tissue and cells. In some embodiments, the solid tissue comprises tissue isolated from a subject having a disease.
(24) In some embodiments, the solid tissue comprises tissue isolated from a tumor. The tissue isolated from a tumor is not limiting. In some embodiments, the isolated tumor tissue is selected from the group consisting of lung cancer tissue, stomach cancer tissue, colon cancer tissue, lymph node metastatic cancer tissue, melanoma tissue, renal cell carcinoma tissue, squamous non-small cell lung (NSCLC) tissue, non-squamous NSCLC tissue, rectal cancer tissue, prostate cancer tissue, ovarian cancer tissue, hepatocellular carcinoma tissue, pancreatic carcinoma tissue, squamous cell carcinoma tissue of the head and neck, esophageal cancer tissue, gastrointestinal tract cancer tissue, breast cancer tissue, bone cancer tissue, brain cancer tissue, thyroid cancer tissue, testicular cancer tissue and vaginal cancer tissue.
(25) In some embodiments, the tissue is isolated directly from a non-removed tumor, from a tumor biopsy, or from a tumor that is surgically removed. In some embodiments, the isolated tissue is grown in another animal, such as a mouse, (tumorgrafts, PDX), isolated from the animal and then analyzed.
(26) In some embodiments, the solid tissue is isolated by mechanical means, such as dissection, from the subject. In some embodiments, the solid tissue is not subjected to enzymatic digestion prior to culturing. In some embodiments, the solid tissue is surgically removed and cut into small pieces for use in the culturing method of the invention. In some embodiments, the solid tissue is cut into pieces having a diameter of less than about 1 mm, less than about 0.75 mm, less than about 0.5 mm or less than about 0.25 mm.
(27) The method for obtaining serum from the subject for use in the invention can be by conventional means and is not limiting. In some embodiments, 15-ml of fresh blood without anti-coagulant is withdrawn from each patient before and is stored at room temperature until coagulation is complete. In some embodiments, about 5 ml, 10 ml, 15 ml, 20 ml or 25 ml of fresh blood without anti-coagulant is withdrawn from each patient before and is stored at room temperature until coagulation is complete. The blood can be centrifuged and the serum isolated. In some embodiments, one or more antibiotics can be added to the serum. The antibiotic that can be added is not limiting. In some embodiments, the antibiotic is one that is commonly used in cell culture techniques, such as, for example, penicillin or streptomycin. In some embodiments cefoperazone is added to the serum (Pfizer Dalian Pharmaceutical Plant) to a final concentration of 20 μg/ml.
(28) In some embodiments, the tissue is cultured in a media comprising 50-100% serum (v/v) from the subject. In some embodiments, the tissue is cultured in a media comprising about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% serum (v/v) from the subject. In some embodiments, a base media is combined with the subject's serum. The base media is not limiting and can include, for example, saline, phosphate buffered saline, Dulbecco's Modification of Eagle's Medium (DMEM), Ham's F-12 (F12), Ham's F-10 (F10), RPMI 1640, Iscove's Modified Dulbecco's Medium (IMDM) or a combination thereof. In some embodiments, the media comprises a 1:1 mixture of the subject's serum and RPMI 1640. In some embodiments, the media comprises about a 1:1, 2:1, 3:1 or 4:1 (v/v) mixture of the subject's serum and RPMI 1640. In some embodiments, exogenous factors, such as antibodies, growth factors, cytokines, or FBS are not added to media and the tissue is nourished primarily by the natural body fluid without artificial modification.
(29) In accordance with the invention, the subject's solid tissue is cultured in a three-dimensional biocompatible matrix in the presence of the subject's serum. The three-dimensional biocompatible matrix is not limiting. In some embodiments, the three-dimensional biocompatible matrix replicates or mimics the extracellular matrix (ECM). In some embodiments, the three-dimensional biocompatible matrix can comprise a scaffold-free platform for spheroid growth, scaffold, gel, bioreactor, or microchip.
(30) In some embodiments, the three-dimensional biocompatible matrix comprises one or more cell matrix proteins. In some embodiments, the cell matrix proteins comprise one or more of laminin, entactin, collagen, nidogen, or heparan sulfate proteoglycans.
(31) In some embodiments, the three-dimensional biocompatible matrix comprises a gel. In general, gels have a soft tissue-like stiffness and aim to mimic the ECM. Gels made from ECM mixtures of natural origin, such as collagen, and alginate, have been used as substrates for 3D cell culture. In some embodiments, the gel is MATRIGEL. MATRIGEL is a reconstituted basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in ECM proteins, such as laminin and collagen, plus growth factors and enzymes. MATRIGEL is produced and marketed by Corning Life Sciences. MATRIGEL contains approximately 60% laminin, 30% collagen IV, and 8% entactin. In some embodiments, the three-dimensional biocompatible matrix comprises MATRIGEL.
(32) Culturing times and conditions are not limiting. In some embodiments, the cells are cultured for 3-4 days and then the media is refreshed. In some embodiments, the cells can be cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or 30 days.
(33) In some embodiments, the method comprises coating a culture surface with a first composition comprising one or more types of cell matrix proteins; placing the solid tissue onto the coated surface; adding a second composition comprising one or more types of cell matrix proteins to the solid tissue, so that the solid tissue is immersed in the second composition and polymerizing the second composition to form the three-dimensional biocompatible matrix; and adding the serum to the immersed solid tissue and culturing the tissue.
(34) In some embodiments, the first composition comprises MATRIGEL or a mixture of MATRIGEL and serum from the subject. In some embodiments, the first composition comprises a 1:1 (v/v) mixture of the subject's serum and MATRIGEL. In some embodiments, the first composition comprises about a 1:1, 1:2, 1:3 or 1:4 (v/v) mixture of the subject's serum and MATRIGEL. The first composition can comprise the three-dimensional biocompatible matrix described herein.
(35) The thickness of the coating of the first composition is not limiting. In some embodiments, the coating has a thickness less than or equal to 0.5 mm. In some embodiments, the coating has a thickness of about 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm or 0.1 mm. In some embodiments, the coating has a thickness of about 0.1 to 0.4 mm.
(36) In some embodiments, the second composition comprises MATRIGEL, or a mixture of MATRIGEL and serum from the subject. In some embodiments, the second composition comprises a 1:1 (v/v) mixture of the subject's serum and MATRIGEL. In some embodiments, the second composition comprises about a 1:1, 1:2, 1:3 or 1:4 (v/v) mixture of the subject's serum and MATRIGEL. The second composition can comprise the three-dimensional biocompatible matrix described herein.
(37) Culturing Primary Cells from Ascites Fluid in Autologous Ascites Fluid
(38) In one embodiment, the invention provides a method of culturing primary cells from ascites fluid from a subject comprising i) obtaining the cells from ascites fluid from the subject; ii) seeding the cells on a surface of a biocompatible matrix; and iii) culturing the cells in a media comprising ascites fluid from the subject.
(39) Ascites fluid can be collected from the patient in any suitable amount. In some embodiments, 100-2000 ml of ascites fluid is collected from the patient. In other embodiments, 500-800 ml ascites fluid is collected from the patient. Cells can be obtained from the ascites fluid by any suitable means including centrifugation, density gradient centrifugation and filtration. In some embodiments, cells present in the collected ascites fluid are pelleted by centrifugation, re-suspended in PBS, subjected to density gradient centrifugation in FICOLL, washed in PBS and re-suspended in media comprising autologous ascites fluid. In some embodiments, other types of density gradient centrifugation media can be used, like PERCOLL. In some embodiments, centrifugation is performed at 2000 rpm.
(40) Any suitable amount of cells from ascites fluid can be seeded on the surface of the bio-compatible matrix. In some embodiments, about 1×10.sup.5, 2×10.sup.5, 3×10.sup.5, 4×10.sup.5, 5×10.sup.5, 6×10.sup.5, 7×10.sup.5, 8×10.sup.5, 9×10.sup.5, or 1×10.sup.6 cells are seeded on the surface of a biocompatible matrix coating a 100 mm culture dish or other type of culture plate or dish.
(41) The cells can comprise noncancerous or cancerous cells and is not limiting. In some embodiments, the cells comprise cancer cells. The cancer cells can comprise stomach cancer cells, endometrium cancer cells, ovarian cancer cells, breast cancer cells, pancreatic cancer cells, uterine cancer, colon cancer cells, melanoma cells, leukemia cells, or lymphoma cells. The cells can comprise a mixed population of cells present in the ascites fluid. The mixed population can comprise cancer cells, blood cells and mesothelial cells at various proportions.
(42) The biocompatible matrix is not limited. In some embodiments, the biocompatible matrix comprises one or more cell matrix proteins. In some embodiments, the cell matrix proteins comprise one or more of laminin, entactin, collagen, nidogen, or heparan sulfate proteoglycans.
(43) In some embodiments, the biocompatible matrix comprises a gel. In some embodiments, the biocompatible matrix comprises a gel and autologous ascites fluid. In some embodiments, the gel is MATRIGEL. In some embodiments, the biocompatible matrix comprises a mixture of MATRIGEL and autologous ascites fluid. In some embodiments, the biocompatible matrix comprises a 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, or 4:1 (v/v) mixture of autologous ascites fluid and gel. In some embodiments, the biocompatible matrix comprises a 1:1 mixture of MATRIGEL and autologous ascites fluid.
(44) The biocompatible matrix can be on a suitable culture surface. The thickness of the coating is not limiting. In some embodiments, the coating has a thickness of at least 0.5 mm. In some embodiments, the coating has a thickness of about 1 mm, 2 mm, 3 mm, or 4 mm. In some embodiments, the coating has a thickness of about 1 to 4 mm.
(45) Media comprising ascites fluid from the subject can comprise 100% autologous ascites fluid with or without one or more suitable antibiotics. The antibiotic that can be added is not limiting. In some embodiments, the antibiotic is one that is commonly used in cell culture techniques, such as, for example, penicillin or streptomycin. In some embodiments cefoperazone is added to the media (Pfizer Dalian Pharmaceutical Plant) to a final concentration of 20 μg/ml. In some embodiments, the media comprises about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% (v/v) autologous ascites fluid. In some embodiments, a base media is combined with the autologous ascites fluid. The base media is not limiting, and can include, for example, saline, phosphate buffered saline, Dulbecco's Modification of Eagle's Medium (DMEM), Ham's F-12 (F12), Ham's F-10 (F10), RPMI 1640, Iscove's Modified Dulbecco's Medium (IMDM), or a combination thereof. In some embodiments, exogenous factors, such as antibodies, growth factors, cytokines, or FBS are not added to media and the cells are nourished primarily by the autologous ascites fluid without artificial modification.
(46) Autologous ascites fluid can be obtained by any suitable means. In some embodiments, the autologous ascites fluid is obtained by filtration and/or centrifugation of ascites fluid. In some embodiments, autologous ascites fluid is obtained by centrifuging ascites fluid obtained from the patient to pellet cells contained in the ascites fluid at 2000 rpm followed by removing the ascites fluid supernatant and filtering the supernatant through a grade GF/F glass microfiber filter (Sigma-Aldridge Cat #WHA 1825025), followed by a 250-ml filter unit with 0.45μ filter (Fisher Scientific, Cat #09-740-24B) after adding cefoperazone to a final concentration of 20 μg/ml.
(47) Culturing times and conditions are not limiting. In some embodiments, the cells are cultured for 3-4 days and then the media is refreshed. In some embodiments, the cells are passaged after digestion with a cell dissociation enzyme to release the cells from the biocompatible matrix. In some embodiments, the cells can be cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or 30 days.
(48) In some embodiments, the method comprises: coating a culture surface with a composition comprising one or more types of cell matrix proteins; adding cells onto the coated surface, which cells are suspended in the ascites fluid from the subject; and optionally adding a composition comprising more ascites fluid to continue the culture.
(49) The composition comprising one or more cell matrix proteins is not limiting. The composition comprising one or more cell matrix proteins can comprise the biocompatible matrix described herein. In some embodiments, the composition comprising one or more cell matrix proteins is a 1:1 (v/v) mixture of MATRIGEL and autologous ascites fluid. In some embodiments, the composition comprising one or more cell matrix proteins is coated at a thickness of at least 0.5 mm on the culture surface. In some embodiments, the composition has a thickness of about 1 to 4 mm.
(50) The cells can be cultured for any suitable period and is not limiting. In some embodiments, the cells are cultured for any period as described herein. The ascites fluid can be refreshed at any suitable time and is not limited. In some embodiments, a composition comprising ascites fluid is refreshed every 3-4 days.
(51) Culturing Primary Cells from Pleural Effusion in Autologous Pleural Effusion Fluid
(52) In one embodiment, the invention provides a method of culturing primary cells from pleural effusion fluid from a subject comprising i) obtaining the cells from pleural effusion fluid from the subject; ii) seeding the cells on a surface of a biocompatible matrix; and iii) culturing the cells in a media comprising pleural effusion fluid from the subject.
(53) Pleural effusion fluid can be collected from the patient in any suitable amount. In some embodiments, 100-2000 ml of pleural effusion is collected from the patient. In other embodiments, 500-800 ml pleural effusion fluid is collected from the patient. Cells can be obtained from the pleural effusion fluid by any suitable means including centrifugation, density gradient centrifugation and filtration. In some embodiments, cells present in the collected pleural effusion fluid are pelleted by centrifugation, re-suspended in PBS, subjected to density gradient centrifugation in FICOLL, washed in PBS and re-suspended in autologous media comprising autologous pleural effusion fluid. In some embodiments, other types of density gradient centrifugation media can be used, like PERCOLL. In some embodiments, centrifugation is performed at 2000 rpm.
(54) Any suitable amount of cells from the pleural effusion fluid can be seeded on the surface of the bio-compatible matrix. In some embodiments, about 1×10.sup.5, 2×10.sup.5, 3×10.sup.5, 4×10.sup.5, 5×10.sup.5, 6×10.sup.5, 7×10.sup.5, 8×10.sup.5, 9×10.sup.5, or 1×10.sup.6 cells are seeded on the surface of a biocompatible matrix coating a 100 mm culture dish or other type of culture plate or dish.
(55) The cells can comprise noncancerous or cancerous cells and is not limiting. In some embodiments, the cells comprise cancer cells. The cancer cells can comprise stomach cancer cells, endometrium cancer cells, ovarian cancer cells, breast cancer cells, pancreatic cancer cells, uterine cancer, colon cancer cells, melanoma cells, leukemia cells, or lymphoma cells. The cells can comprise a mixed population of cells present in the ascites fluid. The mixed population can comprise cancer cells, blood cells and mesothelial cells at various proportions.
(56) The biocompatible matrix can be any biocompatible matrix described herein. In some embodiments, the biocompatible matrix comprises one or more cell matrix proteins. In some embodiments, the cell matrix proteins comprise one or more of laminin, entactin, collagen, nidogen, or heparan sulfate proteoglycans.
(57) In some embodiments, the biocompatible matrix comprises a gel. In some embodiments, the biocompatible matrix comprises a gel and autologous pleural effusion fluid. In some embodiments, the gel is MATRIGEL. In some embodiments, the biocompatible matrix comprises a mixture of MATRIGEL and autologous pleural effusion fluid. In some embodiments, the biocompatible matrix comprises a 1:1, 1:2, 1:3, 1:4, 2:1, 3:1, or 4:1 (v/v) mixture of a gel and autologous pleural effusion fluid. In some embodiments, the biocompatible matrix comprises a 1:1 (v/v) mixture of MATRIGEL and autologous pleural effusion fluid.
(58) The biocompatible matrix can be a coating on a suitable culture surface. The thickness of the coating is not limiting. In some embodiments, the coating has a thickness of at least 0.5 mm. In some embodiments, the coating has a thickness of about 1 mm, 2 mm, 3 mm, or 4 mm. In some embodiments, the coating has a thickness of about 1 to 4 mm.
(59) Media comprising pleural effusion from the subject can comprise 100% autologous pleural effusion fluid with or without one or more suitable antibiotics. The antibiotic that can be added is not limiting. In some embodiments, the antibiotic is one that is commonly used in cell culture techniques, such as, for example, penicillin or streptomycin. In some embodiments, cefoperazone is added to the media (Pfizer Dalian Pharmaceutical Plant) to a final concentration of 20 μg/ml. In some embodiments, the media comprises about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% (v/v) autologous pleural effusion fluid.
(60) Autologous pleural effusion fluid can be obtained by any suitable means. In some embodiments, the autologous pleural effusion fluid is obtained by filtration and/or centrifugation of pleural effusion fluid obtained from a patient to remove cells present in the pleural effusion fluid. In some embodiments, autologous pleural effusion fluid is obtained by centrifuging pleural effusion fluid obtained from the patient to pellet cells contained in the pleural effusion fluid at 2000 rpm followed by removing the pleural effusion fluid supernatant and filtering the supernatant through a grade GF/F glass microfiber filter (Sigma-Aldridge Cat #WHA 1825025), followed by a 250-ml filter unit with 0.45μ filter (Fisher Scientific, Cat #09-740-24B) after adding cefoperazone to a final concentration of 20 μg/ml.
(61) Culturing times and conditions are not limiting. In some embodiments, the cells are cultured for 3-4 days and then the media is refreshed. In some embodiments, the cells are passaged after digestion with a cell dissociation enzyme to release the cells from the biocompatible matrix. The cells can be cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or 30 days.
(62) In some embodiments, the method comprises: coating a culture surface with a composition comprising one or more cell matrix proteins; adding cells onto the coated surface, which cells are suspended in the pleural effusion fluid from the subject; and optionally adding a composition comprising more pleural effusion fluid to continue the culture.
(63) The composition comprising one or more cell matrix proteins can comprise a biocompatible matrix described herein. In some embodiments, the composition comprising one or more cell matrix proteins is a 1:1 mixture of MATRIGEL and autologous pleural effusion fluid. In some embodiments, the composition comprising one or more cell matrix proteins is coated at a thickness of at least 0.5 mm on the culture surface. In some embodiments, the coating has a thickness of about 1 mm, 2 mm, 3 mm, or 4 mm. In some embodiments, the coating has a thickness of about 1 to 4 mm.
(64) The cells can be cultured for any suitable period and is not limiting. In some embodiments, the cells are cultured for any period as described herein. The pleural effusion can be refreshed at any suitable time and is not limited. In some embodiments, pleural effusion is refreshed every 3-4 days.
(65) Culturing Cells from Blood in Autologous Serum
(66) In one embodiment, the invention provides a method of culturing cells from blood from a subject comprising i) obtaining the cells from the blood of the subject; ii) obtaining serum from the subject; and iii) culturing the cells in the presence of a media comprising at least 50% (v/v) of the serum.
(67) Obtaining cells from blood of the subject can be by any conventional means and is not limited. In some embodiments, cells are obtained by centrifuging an anti-coagulate treated blood sample to pellet the cells. In some embodiments, the cell pellet is further purified by density gradient centrifugation. In some embodiments, FICOLL can be used for density gradient centrifugation. In some embodiments, other types of density gradient centrifugation media can be used, like PERCOLL.
(68) In some embodiments, the cells from the blood of the subject comprise a mixture of cell populations present in blood. In some embodiments, the cells from the blood of the subject comprise cancerous and noncancerous cells. In some embodiments, the cells from the blood of the subject comprise PBMC. In some embodiments, the cells from the blood of the subject comprise CTC. In some embodiments, the cells from the blood of the subject comprise CTC and PBMC. In some embodiments, the CTC comprise cancer cells selected from the group consisting of lung cancer cells, stomach cancer cells, colon cancer cells, lymph node metastatic cancer cells, melanoma cells, renal cell carcinoma cells, squamous non-small cell lung (NSCLC) cells, non-squamous NSCLC cells, rectal cancer cells, prostate cancer cells, ovarian cancer cells, hepatocellular carcinoma cells, pancreatic carcinoma cells, squamous cell carcinoma cells of the head and neck, esophageal cancer cells, gastrointestinal tract cancer cells, breast cancer cells, bone cancer cells, brain cancer cells, thyroid cancer cells, testicular cancer cells and vaginal cancer cells.
(69) The method of obtaining the serum from the subject for use in the invention can be by conventional means and is not limiting. In some embodiments, 10-15 ml of fresh blood with anti-coagulant is withdrawn from each patient and is centrifuged to precipitate blood cells and collect the serum. In some embodiments, about 5 ml, 10 ml, 15 ml, 20 ml or 25 ml of fresh blood with anti-coagulant is withdrawn from each patient and is centrifuged to precipitate blood cells and collect the serum. One or more antibiotics can be added to the serum. The antibiotic that can be added is not limiting. In some embodiments, the antibiotic is one that is commonly used in cell culture techniques, such as, for example, penicillin or streptomycin. In some embodiments, cefoperazone is added to the serum (Pfizer Dalian Pharmaceutical Plant) to a final concentration of 20 μg/ml.
(70) In some embodiments, the cells are cultured in a media comprising 50-100% (v/v) serum from the subject. In some embodiments, the cells are cultured in a media comprising about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% (v/v) serum from the subject. In some embodiments, a base media is combined with the subject's serum. The base media is not limiting, and can include, for example, saline, phosphate buffered saline, Dulbecco's Modification of Eagle's Medium (DMEM); Ham's F-12 (F12); Ham's F-10 (F10); RPMI 1640; Iscove's Modified Dulbecco's Medium (IMDM); or a combination thereof. In some embodiments, the media comprises a 1:1 mixture of the subject's serum and RPMI 1640. In some embodiments, the media comprises about a 1:1, 1:2, 1:3 or 1:4 (v/v) mixture of RPMI 1640 and the subject's serum. In some embodiments, exogenous factors, such as antibodies, growth factors, cytokines, or FBS are not added to media and the cells are nourished by the natural body fluid without artificial modification.
(71) In some embodiments, about 1×10.sup.5, 2×10.sup.5, 3×10.sup.5, 4×10.sup.5, 5×10.sup.5, 6×10.sup.5, 7×10.sup.5, 8×10.sup.5, 9×10.sup.5, or 1×10.sup.6 cells per well of cells obtained from blood are cultured in a 24 well culture plate in media comprising autologous serum. In some embodiments, the cells are cultured in media comprising autologous serum for at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more days. In some embodiments, PMBCs are cultured in media comprising autologous serum for 4-5 days.
(72) In some embodiments, blood is collected from a cancer patient and a cell population comprising PBMCs and CTCs are cultured in media comprising at least 50% autologous serum. In some embodiments, blood is collected from a cancer patient and a cell population comprising PBMCs and CTCs are cultured in media comprising 100% autologous serum.
(73) In some embodiments, the invention provides a method of enriching circulating tumor cells (CTCs) in a population of blood cells, comprising obtaining a cell fraction comprising PBMCs from the blood of the subject; obtaining serum from the subject; and culturing the cells in the presence of serum from the subject for a period of time whereby the CTCs are enriched in the cell population.
(74) In some embodiments, culturing cells in the presence of serum comprises culturing the cells in media comprising autologous serum as described herein. In other embodiments, the cells are cultured in 100% serum with or without added antibiotics as described herein.
(75) The cells can be cultured for any suitable amount of time and is not limiting. In some embodiments, the cells are cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days or more. In some embodiments, the CTCs are enriched by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold or 1000-fold or more.
(76) Methods of Testing the Toxicity or Effectiveness of a Therapeutic Agent and Methods of Treating Cancer
(77) In one embodiment, the invention provides a method for testing the effectiveness or toxicity of a therapeutic agent, comprising: i) culturing cells or solid tissue from a subject according to any of the methods taught herein, ii) contacting the cultured cells or solid tissue with the therapeutic agent, and iii) assessing the effectiveness or toxicity of the therapeutic agent as a treatment for the subject.
(78) In some embodiments, the methods further comprise administering an effective amount of the therapeutic agent to the subject to treat a disease or condition if the therapeutic agent shows efficacy.
(79) In one embodiment, the invention provides a method of treating cancer in a subject, comprising i) culturing cancer cells or solid cancer tissue from a subject according to any of the methods described herein; ii) contacting the cultured cells or solid tissue with a therapeutic agent; iii) assessing the effectiveness or toxicity of the therapeutic agent as a treatment for the subject; and iv) administering to the subject an effective amount of the therapeutic agent.
(80) Culturing cells or solid tissue can be by any method taught herein using autologous fluid or serum. The cells or solid tissue can be any taught herein and can comprise cancerous or noncancerous cells or solid tissue. The cells or solid tissue can be contacted with the therapeutic agent at any suitable time and is not limiting. In some embodiments, the therapeutic agent can be contacted with the cells or solid tissue immediately or after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of culturing of the cells or solid tissue by the methods taught herein. The concentration of therapeutic agent contacted with the cells or solid tissue is not limiting. In some embodiments, a therapeutically effective amount of the therapeutic agent is contacted with the cells or solid tumor.
(81) The therapeutic agent is not limiting. The therapeutic agent can be used to treat a disease or condition in a subject. The disease or condition is not limiting. In some embodiments, the therapeutic agent could be chemotherapy (as listed below), immunotherapy, gene therapy or other types of targeted therapies.
(82) In some embodiments, the therapeutic agent is a cancer therapeutic selected from the group consisting of Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Avastin (Bevacizumab), Axitinib, Azacitidine, BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Bevacizumab, Bexarotene, Bexxar (Tositumomab and I 131 Iodine Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabozantinib-S-Malate, CAF, Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPDX, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CeeNU (Lomustine) Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), CMF, Cometriq (Cabozantinib-S-Malate), COPP, COPP-ABV, Cosmegen (Dactinomycin), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine, Liposomal, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Liposomal Cytarabine), DepoFoam (Liposomal Cytarabine), Dexrazoxane Hydrochloride, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Efudex (Fluorouracil), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston (Toremifene), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), Imatinib Mesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Intron A (Recombinant Interferon Alfa-2b), Iodine 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax (Romidepsin), Ixabepilone, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Liposomal Cytarabine, Lomustine, Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lupron Depot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megace (Megestrol Acetate), Megestrol Acetate, Mekinist (Trametinib), Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilotinib, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, OEPA, Ofatumumab, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Pamidronate Di sodium, Panitumumab, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Di sodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), Rituximab, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Ruxolitinib Phosphate, Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synovir (Thalidomide), Synribo (Omacetaxine Mepesuccinate), TAC, Tafinlar (Dabrafenib), Talc, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thiotepa, Toposar (Etoposide), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and I 131 Iodine Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Vandetanib, VAMP, Vectibix (Panitumumab), VeIP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, VePesid (Etoposide), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), and Zytiga (Abiraterone Acetate). In some embodiments, the drug is selected from the group consisting of Paclitaxel, Curcumin, Docetaxel, Ixabepilone, Vinblastine, Colchicine, Y-27632 Fasudil, SU6656 Dasatinib, HDAC inhibitors, ROCK inhibitors, Parthenolide, Costunolide and ML-7 Jazplakinolide.
(83) The effectiveness or toxicity of the therapeutic agent as a treatment for the subject can be assessed by any suitable means and is not limiting. In some embodiments, the effectiveness or toxicity of the therapeutic agent as a treatment for the subject is assessed by microscopy, special staining (histochemistry), fluorescent labeling, radiation material labeling, and/or various cell apoptosis and viability assays.
(84) In some embodiments, the cells or solid tissue comprises cancer cells from solid tumor and the effectiveness of the therapeutic agent as a cancer treatment for the subject is assessed. In some embodiments, cells comprise PBMC and the toxicity of the therapeutic agent for the subject is assessed. In some embodiments, the cells comprise CTC and the effectiveness of the therapeutic agent as a cancer treatment for the subject is assessed. In some embodiments, the cells comprise cancer cells obtained from ascites fluid or pleural effusion fluid and the effectiveness of the therapeutic agent as a cancer treatment for the subject is assessed.
(85) A Method of Screening for Anti-Cancer Agents
(86) In one embodiment, the invention provides a method of screening for anti-cancer agents, comprising i) culturing cells or solid tissue from a subject according to according to any of the methods taught herein; ii) contacting the cells or solid tissue with one or more candidate anti-cancer agents; and iii) assessing the effectiveness or toxicity of the candidate anti-cancer agent as a cancer treatment.
(87) Culturing cells or solid tissue can be by any method taught herein using autologous fluid or serum. The cells or solid tissue can be any taught herein. The cells or solid tissue can comprise cancerous or noncancerous cells. The cells or solid tissue can be contacted with the one or more candidate anti-cancer agents at any suitable time and is not limiting. In some embodiments, the one or more candidate anti-cancer agents can be contacted with the cells or solid tissue immediately or after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of culturing of the cells or solid tissue by the methods taught herein.
(88) In some embodiments, the effectiveness or toxicity of the candidate anti-cancer agent as a cancer treatment is assessed by any suitable means and is not limiting. In some embodiments, the effectiveness or toxicity of the candidate anti-cancer agent as a cancer treatment is assessed by microscopy, special staining (histochemistry), fluorescent labeling, radiation material labeling, and/or various cell apoptotic and viability assays.
(89) In some embodiments, the cells or solid tissue comprises cancer cells from solid tumor and the effectiveness of the candidate anti-cancer agent as a cancer treatment is assessed. In some embodiments, cells comprise PBMC and the toxicity of the candidate anti-cancer agent is assessed. In some embodiments, the cells comprise CTC and the effectiveness of the candidate anti-cancer agent as a cancer treatment is assessed. In some embodiments, the cells comprise cancer cells obtained from ascites fluid or pleural effusion fluid and the effectiveness of the candidate anti-cancer agent as a cancer treatment is assessed.
(90) Methods of Testing the Toxicity and Effectiveness of a Therapeutic Agent
(91) In one embodiment, the invention provides a method for simultaneously testing the toxicity and effectiveness of a therapy in order to select the treatment that is suitable for a subject, comprising i) culturing cells or tissue according to any of the methods taught herein; ii) contacting the cells or solid tissue with one or more candidate therapeutic agents; and iii) assessing the effectiveness of the candidate therapeutic agents of ii); iv) culturing PBMC according to any of the methods taught herein; v) contacting the PBMC with the one or more candidate therapeutic agents of ii); vi) assessing the toxicity of the candidate therapeutic agents on the PBMC; and vii) administering to the subject the therapeutic agent that exhibits therapeutic effectiveness against the cancer and low toxicity to the PBMC.
(92) In some embodiments, the therapeutic agent is an anti-cancer agent. The anti-cancer agent is not limiting and can include any of the anti-cancer agents described herein.
(93) The cells or solid tissue can be cultured with autologous serum or fluid by any method taught herein. The cells or solid tissue can be any taught herein and can comprise cancerous or noncancerous cells or solid tissue. The cells or solid tissue can be contacted with the one or more candidate therapeutic agents or anti-cancer agents at any suitable time and is not limiting. In some embodiments, the one or more candidate therapeutic agents or anti-cancer agents can be contacted with the cells or solid tissue immediately or after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of culturing of the cells or solid tissues by the methods taught herein.
(94) In some embodiments, the effectiveness of the one or more candidate therapeutic agents or anti-cancer agents is assessed by any suitable means and is not limiting. In some embodiments, the effectiveness of the one or more candidate therapeutic agents or anti-cancer agents is assessed by microscopy, special staining (histochemistry), fluorescent labeling, radiation material labeling, and/or various cell apoptotic and viability assays.
(95) The PBMC can be cultured with autologous serum or fluid by any method taught herein. The PBMC can be contacted with the one or more candidate therapeutic agents or anti-cancer agents at any suitable time and is not limiting. In some embodiments, the one or more candidate therapeutic agents or anti-cancer agents can be contacted with the PBMC immediately or after about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days of culturing of the PBMC by the methods taught herein.
(96) In some embodiments, the toxicity of the one or more candidate therapeutic agents or anti-cancer agents is assessed by any suitable means and is not limiting. In some embodiments, the effectiveness of the one or more candidate therapeutic agents or anti-cancer agents is assessed by microscopy, special staining (histochemistry), fluorescent labeling, radiation material labeling, and/or various cell apoptotic and viability assays.
(97) The present invention is further illustrated by the following Examples. These Examples are provided to aid in the understanding of the invention and are not to be construed as a limitation thereof.
EXAMPLES
Example 1
(98) Materials and Methods
(99) Clinical Samples Information:
(100) All clinical materials were collected from Dalian Central Hospital, Dalian, China, during 2014 and 2015. This study was approved and monitored under the hospital Humane Society and Research Committee (YN2014-023-01). Primary cultures were performed for fourteen solid tumors and thirteen serous tumors (malignant ascites and pleural effusions). The solid tumors were from seven male and seven female patients whose ages ranged from 40 to 80, with an average of 61. These patients were diagnosed clinically as stomach cancers (n=6), lung cancers (n=7), and lymph node metastatic cancer from a stomach cancer patient (n=1). For the lymph node metastatic cancer from a stomach cancer patient an enlarged lymph node (LN; 2.5×1×1 cm.sup.3) was used. Pathology confirmed that the enlargement of this LN was caused by cancer metastasis. All these cases were confirmed as cancers by histopathology (Table 1). For serous tumors, samples were collected from seven male and six female patients (n=13) and specimens included ascites (n=8) or pleural effusions (n=5) (see Table 2). Most of these body liquids were hemorrhagic (500-800 ml) and were diagnosed clinically as cancer-generated body fluids (Table 2). Patients' ages in this group ranged from 25 to 87, with an average of 59. In addition, PBMC from seven cancer patients were isolated. The tumor types are listed in Table 3.
(101) TABLE-US-00001 TABLE 1 Source of solid tumors Case# Sex Age specimen Pathology Dx 1 M 68 Stomach Gastric poorly differentiated adenocarcinoma 2 F 75 Lung Pulmonary adenocarcinoma 3 M 62 Lung Pulmonary moderately/poorly differentiated adenocarcinoma 4 F 80 Stomach Gastric mixed mucinous carcinoma and poorly differentiated adenocarcinoma 5 M 51 Stomach Gastric mixed mucinous carcinoma and poorly differentiated adenocarcinoma 6 M 40 Stomach Gastric mucinous carcinoma 7 M 54 Lung Pulmonary well-differentiated adenocarcinoma 8 F 69 Stomach Gastric mixed adenocarcinoma and mucinous carcinoma 9 F 70 Lymph node Metastatic adenocarcinoma (stomach) 10 M 62 Stomach Gastric poorly differentiated adenocarcinoma 11 F 58 Lung Pulmonary well differentiated adenocarcinoma 12 F 62 Lung Pulmonary well differentiated adenocarcinoma 13 M 58 Lung Pulmonary well/modertely differentiated adenocarcinoma 14 F 47 Stomach Gastric poorly differentiated adenocarcinoma
(102) TABLE-US-00002 TABLE 2 Samples from ascites and pleural effusions Case# Sex Age Specimen/ml Clinical Dx Pathology Dx 1 M 42 Ascites/800 Stomach cancer; two years post Some cells proliferate actively (biopsy gastrostomy. Two months post chemo of endoscope) 2 F 74 Ascites/800 Ovary carcinoma; two years post Ovary serous papillary cystic adenoma ovariectomy, two weeks post chemo (biopsy of ovariosalpingectomy) 3 F 60 Ascites/800 Endometrium carcinoma; one year Poorly differentiated adenocarcinoma post uteroectomy. (biopsy of endometrium) 4 M 46 Ascites/300 Stomach cancer; two years post Adenocarcinoma (Cytology) gastrostomy. 5 F 69 Pleural effusion/200 Lung cancer Adenocarcinoma (Cytology) 6 M 58 Pleural effusion/500 Pleural Mesothelioma Pleural Mesothelioma (Cytology) 7 F 87 Pleural effusion/650 Lung cancer Adenocarcinoma (Cytology) 8 M 60 Ascites/750 Stomach cancer Consider adenocarcinoma (Cytology) 9 F 61 Pleural effusion/600 Unknown reason No tumor cell was found (Cytology) 10 F 25 Ascites/500 Stomach cancer Adenocarcinoma (Cytology) 11 M 39 Ascites/300 Pancreatic cancer Adenocarcinoma (Cytology) 12 M 77 Ascites/800 Pancreatic cancer Contains cancerous cells (Cytology) 13 M 76 Pleural effusion/600 Lung metastatic cancer ND. Consider stomach cancer (Clinic)
(103) TABLE-US-00003 TABLE 3 PBMC samples PBMC source (patient *Days in Case # with cancer) Serum type culture 1 lung HS/FBS 8 2 stomach HS/FBS 7 3 breast HS/FBS 8 4 lung HS/FBS 7 5 breast HS/FBS 8 6 stomach HS/FBS 8 7 stomach HS only 10 HS: autologous human serum; FBS: fetal bovine serum
Autologous Culture Medium Preparations:
(104) Two types of autologous culture media (ACM) were prepared for tissue/cell cultures. For the cultures of solid tumors, 10-15 ml of fresh blood without anti-coagulant was withdrawn from each patient before anesthesia and stored at room temperature until coagulation was complete and tumors were removed surgically. The blood was then centrifuged at 2,000 rpm for 10 minutes. The resultant serum was then immediately transferred to a clean polypropylene tube (usually 8-10 ml), supplemented with cefoperazone (Pfizer Dalian Pharmaceutical Plant) to a final concentration of 40 μg/ml, and then stored at 4° C. until use. Most of the autologous plasma was 1:1 diluted in RPMI-1640 medium (with 50% human plasma) to serve as an autologous culture medium for individual solid tumor growth (s-ACM). The second type of ACM is from ascites or pleural effusions. After collection (
(105) Primary Cultures for Solid Tumors:
(106) Wells in a pre-cooled 12- or 24-well plate were first evenly coated (≤0.5 mm in thickness) with MATRIGEL (basement membrane matrix) (BD Biosciences, Cat #356243) that was well-mixed with autologous plasma at a 1:1 ratio and polymerized in a 37° C. incubator for 30 min. Surgically removed tumor tissues (
(107) To compare the culturing effects between 3D-tissue culture and previous, commonly used 3D-cell culture, part of the tumor pieces were digested in Dispase (BD Biosciences, Cat #354235) solution for 30-60 min while shaken, to obtain single cells suspension (
(108) Primary Cultures of Cancer Cells from Ascites/Pleural Effusions:
(109) A pre-cooled culture dish (100 mm) was first evenly coated (1-3 mm in thickness) with 1:1 diluted MATRIGEL (basement membrane matrix) in f-ACM and then placed in a 37° C. incubator for gel polymerization. Cell pellets from body fluids (
(110) Cultures of PBMCs from Cancer Patients:
(111) Anticoagulants-treated 8-10 ml peripheral blood was centrifuged at 2,000 rpm for 10 mins. PBMC was isolated with routine FICOLL centrifugation protocol (TBD, Cat #LTS0770125). Cells were then seeded into a 24-well culture plate at 5×10.sup.5/well. The growth conditions of cells were monitored and imaged with microscopy every day for 7-10 days. Autologous serum and commercial FBS were used individually as culture media; each condition was duplicated in both media for all time points, for comparison purposes. The cellular viability and total cells numbers were recorded daily.
(112) Experimental Results and Discussion
(113) Survival Rate of Primary Cultures:
(114) A total of 27 clinical specimens were processed. After tissues or cells were seeded into culture wells, cell growth was monitored under microscope daily, and morphology images were taken at least every 2-3 days. Among all clinical specimens, the success rate for culturing cells isolated from ascites or pleural effusions (n=13) was nearly 100%, and for solid tumors (n=14) was 86% (Table 4 and Table 5).
(115) TABLE-US-00004 TABLE 4 Summary of solid tumor cultures 3D cell 3D tissue *Days in autologous *Days in FBS Total days in Case # culture culture medium medium culture 1 + + 8 8 2 + + 16 10 26 3 + + 15 10 25 4 + + 15 10 25 5 + + 14 14 6 + + 13 13 7 + + 8 8 8 + + 8 8 9 + + 8 8 10 + + 4 4* 11 + 8 8 12 + 6 6* 13 + 15 14 29 14 + 20 6 26 *Culture was contaminated.
(116) TABLE-US-00005 TABLE 5 Summary of body fluid sample cultures Days for Case # confluent Passages 1 21 0 2 41 0 3 7 5 4 7 1 5 11 1 6 12 2 7 8 5 8 7 4 9 17 0 10 4 3 11 8 1 12 8 2 13 9 2
(117) All 3D-tissue cultures (n=14) survived well for at least 8 days, except for two cases of contamination. The overall survival rate was 86%. Among the 14 samples, seven cultures were stopped before day-14 because of a shortage of s-ACM (Table 4). It is noteworthy that the duration of these cultures was mostly limited by either a shortage of s-ACM or by research requirements, not by cell growth failure.
(118) Cell growth conditions of the present 3D-tissue culture were compared to previously established 3D-cell culture. Among the 14 solid tumors processed, these two types of cultures were performed side by side in nine cases. Cells in 3D-cell cultures did not survive more than seven days. A gradual reduction in cell numbers was observed after day 3. At day 7, most of the cells had degenerated or were dead (
(119) This primary culture technique for solid tumors provides many benefits that traditional and other previous tissue/cell cultures do not provide. When solid tumors grow in patients, they have special 3D tissue structures to allow tumor cells to communicate with each other and with other body cells. Sufficient blood supply provides not only nutrients but also other bio-chemicals that are required for tumor cell growth. The present culture technique maximally mimicked these physiological conditions. First, the culture is a multicellular environment composed of both tumor and their autologous surrounding tissue cells (
(120) The survival rates for cells from ascites and pleural effusions were 100% (Table 5). There are also mixed populations in these body fluids, which contain tumor cells, blood cells, mesothelial cells, and possibly other type of cells in different proportions. Therefore, the time for cells to become confluent in a dish ranged from 4-41 days (Table 5), even though all cases were started with a similar amount of cells. Cells isolated from cases #1, #2, and #9 took a much longer time to become confluent in culture dishes than cells from other cases (Table 4). By reviewing treatment histories, it was apparent that patients #1 (21 days) and #2 (41 days) had received chemotherapy intraperitoneally not long before samples were taken. Additionally, it was suspected that patient #9 had tuberculosis, and no tumor cells were subsequently found in this patient by cytology. This explains the slow growth of these samples. Morphologically, most of the cells in these three cultures were more uniform in size and shape, with a large amount of fibers produced by cells in the dishes. This suggests that there were fewer or no tumor cells in the original samples, just different types of “normal” cells (
(121) The physiological conditions of cells isolated from ascites and pleural effusions are different as compared to those from solid tumors; therefore, the culture condition was modified accordingly. Generally, to survive, tumor cells floating in the ascites or pleural effusions need to penetrate the surface of the plasma membrane of a peritoneal or pleural cavity (transcoelomic metastasis). To mimic this physiological condition, we used f-ACM-MATRIGEL (basement membrane matrix) (1:1) coated plates and seeded the cells on top of the gel without embedding them in gel (
(122) Histopathology Comparison Before and after Cultures:
(123) Unlike in cultures previously reported by others, tumor cells grown in the present novel culture conditions preserved the ability to form tissue structures similar to what they were in human bodies. As shown in Table 1, all the solid tumors in the present studies were adenocarcinomas with different differentiation levels from either stomach or lung. Tumor cells in the culture wells showed a strong tendency to form adenoid structures (
(124) Immunophenotype Comparison Before and after Cultures:
(125) Furthermore, cells in the present culture conditions were also highly consistent in their immunophenotypes before and after cultures. Immunohistochemistry (IHC) was performed in 10 samples (six solid tumors and four serous tumors) and the expressions of markers tested were compared in samples before and after cultures. Antibody against CK, Napsin-A, and TTF-1 were used in lung cancers (
(126) PBMC and CTC Growing in Autologous Serum:
(127) Two morphological differences between HS (autologous human serum) and FBS were observed in all seven samples tested. First, the red blood cells (RBC) in FBS showed damage within 48 hours and became aggregated and clumpy (
(128) To summarize, the high success rate of primary culture with this novel technique is due to three major factors: use of autologous body fluid, retention of multicellular communications and imitation of growth conditions in the body. With similar components and levels of various nutrients, growth factors, chemokines/cytokines, and hormones in autologous body fluid, both tumor and stroma cells easily adapted to the new but similar growth environment in vitro. In the present culture system, with the support of autologous tumor-surrounding cells, tumor cells grow not alone but as a tissue in which the physiological cellular communications in vivo are preserved. Because the CTCs' living condition remains the same, they also proliferate well. Since the present culture system maintains nearly the same conditions as tumors growing in the patients, drug-sensitivity testing can more accurately predict the tumors' responses in patients. Furthermore, the use of ACM is more cost-effective for cancer patients because no additional commercial cells, growth factors, or stimuli are needed in this culture system. Additionally, this culture system only requires a small amount of cells and body fluid, and so causes no harm to the patient.
(129) While there have been shown and described what are presently believed to be the preferred embodiments of the present invention, those skilled in the art will realize that other and further embodiments can be made without departing from the spirit and scope of the invention described in this application, and this application includes all such modifications that are within the intended scope of the claims set forth herein. All patents and publications mentioned and/or cited herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated as having been incorporated by reference in its entirety.