CELLULAR CIS-CO-CULTURE SYSTEMS AND METHODS

Abstract

The invention relates to systems and methods for studying patient cancer samples in cis-co-culture with non-cancer cells from the same patient. For example, the invention provides systems and methods for testing therapeutic agents in vitro in an environment that simulates an in vivo environment to identify agents that are therapeutically effective for the patient.

Claims

1-20. (canceled)

21. A method for cellular analysis comprising: a. placing cancer cells isolated from a patient on a region of a solid support; b. placing non-cancer cells isolated from the same patient in the same region of the solid support; c. co-culturing the cancer and non-cancer cells on the solid support; d. contacting the co-cultured cancer and non-cancer cells with an agent; and e. measuring the effect of the agent on the cancer and/or non-cancer cells, wherein the cancer cells are hematological cancer cells, wherein the cancer cells are placed on the solid support without cryopreservation following isolation, and wherein the non-cancer cells are placed on the solid support without expansion following isolation.

22. The method of claim 21, wherein the patient has been diagnosed with adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, AIDS-related lymphoma, cutaneous T-cell lymphoma, adult Hodgkin lymphoma, childhood Hodgkin lymphoma, Hodgkin lymphoma during pregnancy, mycosis fungoides, adult non-Hodgkin lymphoma, childhood non-Hodgkin lymphoma, non-Hodgkin lymphoma during pregnancy, primary central nervous system lymphoma, Szary syndrome, cutaneous t-cell lymphoma, Waldenstrm macroglobulinemia, chronic myeloproliferative disorders, Langerhans cell histiocytosis, multiple myeloma/plasma cell neoplasm, myelodysplastic syndrome, or myelodysplastic/myeloproliferative neoplasm.

23. The method of claim 21, wherein the patient has been diagnosed with multiple myeloma.

24. The method of claim 21, wherein the patient has been diagnosed with adult acute myeloid leukemia or childhood acute myeloid leukemia.

25. The method of claim 21, wherein the hematological cancer cells are Hodgkin's lymphoma cells, non-Hodgkin's lymphoma cells, acute lymphoblastic leukemia cells, chronic lymphoblastic leukemia cells, or multiple myeloma cells.

26. The method of claim 21, wherein the hematological cancer cells are multiple myeloma cells.

27. The method of claim 21, wherein the cancer cells and the non-cancer cells are collected at the same time.

28. The method of claim 21, wherein the cancer cells and the non-cancer cells are isolated from the same bone marrow aspirate.

29. The method of claim 21, wherein the cancer cells and the non-cancer cells are co-cultured in volume of less than 1 ml.

30. The method of claim 21, wherein the cellular analysis comprises testing the cancer and/or non-cancer cells for sensitivity to the agent.

31. The method of claim 21, wherein the cellular analysis comprises testing the cancer and/or non-cancer cells for resistance to the agent.

32. The method of claim 21, wherein the agent is a small molecule, a proteinaceous molecule, a genetic molecule, or a blood product.

33. The method of claim 21, wherein the agent is a chemotherapeutic agent or a candidate chemotherapeutic agent.

34. A system for patient-specific cellular analysis comprising: a. a device; b. cultured cancer cells from a patient on or in a region of the device; and c. cultured non-cancer cells from the same patient on or in the same region of the device, wherein the cancer cells and the non-cancer cells are co-cultured in volume of less than 1 ml.

35. The system of claim 34, wherein the device is a solid support that is a chip, dish, bead, scaffold, or gel.

Description

DESCRIPTION OF FIGURES

[0027] FIG. 1A and FIG. 1B (together referred to as FIG. 1). Chip design. In some embodiments, the present invention provides a chip for culturing cells and a method using the chip. In FIG. 1A, the chip comprises a central well connected to 2 side chambers, via diffusion channels. The cells are seeded through the inlet ports and fluid is passively flown from the inlet to outlet ports. In some embodiments, the central well has low wall shear stress at the bottom of the well to retain settled non-adherent cells. In this figure, multiple myeloma (MM) cells are used illustratively, but many other types of cells can be used instead. In this illustrative example, the central well comprises CD138+ multiple myeloma cells and the side chambers comprise CD138 mononuclear cells. The diffusion ports (also referred to as channels) allow media and soluble factors to diffuse among the central well and the side chambers, but the diffusion ports are not tall enough for cells to pass through. In some embodiments, the diffusion channels are 100 m wide and 20 m deep. FIG. 1B shows a more three dimensional view of an embodiment of the device of FIG. 1A with a pipette tip shown above the device.

[0028] FIGS. 2a-2r shows primary CD138+ multiple myeloma (MM) and CD138 mononuclear cells from patients that were co-cultured as in Example 1. Graphs show mono-culture response in black and co-culture response in grey. All patients' in vitro responses matched their clinical response. FIG. 2a shows results for patient 314. FIG. 2b shows results for patient 315. FIG. 2c shows results for patient 316. FIG. 2d shows results for patient 317. FIG. 2e shows results for patient 318. FIG. 2f shows results for patient 323. FIG. 2g shows results for patient 329. FIG. 2h shows results for patient 330. FIG. 2i shows results for patient 344. FIG. 2j shows results for patient 345. FIG. 2k shows results for patient 353. FIG. 2l shows results for patient 398. FIG. 2m shows results for patient 419. FIG. 2n shows results for patient 431. FIG. 2o shows results for patient 432. FIG. 2p shows results for patient 435. FIG. 2q shows results for patient 446. FIG. 2r shows results for patient 447. Patient 329 was refractory to bortezomib at the time of the cell harvest. When the patient's MM cells were grown in co-culture with the patient's own mononuclear cells, the MM cells appeared resistant to bortezomib. However, when the MM cells were grown in mono-culture, they exhibited less resistance. In this way, the cis-co-culture method described herein predicted resistance of MM cells to bortezomib more accurately than mono-cultured MM cells. Patient 316 was refractory to bortezomib at the time of the cell harvest. When the patient's MM cells were grown in co-culture with the patient's own mononuclear cells, the MM cells appeared resistant to bortezomib. However, when the MM cells were grown in mono-culture, they exhibited less resistance. In this way, the cis-co-culture method described herein predicted resistance of MM cells to bortezomib more accurately than mono-cultured MM cells. Patient 318 was refractory to bortezomib at the time of the cell harvest. When the patient's MM cells were grown in co-culture with the patient's own mononuclear cells, the MM cells appeared resistant to bortezomib. However, when the MM cells were grown in mono-culture, they exhibited less resistance. In this way, the cis-co-culture method described herein predicted resistance of MM cells to bortezomib more accurately than mono-cultured MM cells. Patient 315 was sensitive to bortezomib at the time of the cell harvest. When the patient's MM cells were grown in co-culture with the patient's own mononuclear cells, the MM cells exhibited a similar response to bortezomib as when the cells were grown in mono-culture. In this way, the cis-co-culture method described herein predicted sensitivity of MM cells to bortezomib as well as mono-cultured MM cells. Patient 344 was sensitive to bortezomib at the time of the cell harvest. When the patient's MM cells were grown in co-culture with the patient's own mononuclear cells, the MM cells exhibited a similar response to bortezomib as when the cells were grown in mono-culture. In this way, the cis-co-culture method described herein predicted sensitivity of MM cells to bortezomib as well as mono-cultured MM cells. Patient 345 was sensitive to bortezomib at the time of the cell harvest. When the patient's MM cells were grown in co-culture with the patient's own mononuclear cells, the MM cells exhibited a similar response to bortezomib as when the cells were grown in mono-culture. In this way, the cis-co-culture method described herein predicted sensitivity of MM cells to bortezomib as well as mono-cultured MM cells.

[0029] FIG. 3. Data from analyzed patient target cells exposed to drug as described in Example 7 were statistically analyzed. FIG. 3 shows a summary graph of the cluster analysis, differentiating responsive cells from non-responsive cells.

[0030] FIG. 4. Patient 442 was tested for sensitivity against a plurality of therapeutic agents (bortezomib, lenalidomide, and pomalidomide). Patient 442's MM cells exhibited resistance to bortezomib in vitro but sensitivity to lenalidomide and pomalidomide. Clinically, Patient 442 was initially treated with a bortezomib-containing regimen but did not respond. Patient 442 then was treated with a lenalidomide-containing regimen and had a partial response. FIG. 4 shows a graph of the data, demonstrating the sensitivity/resistance profile.

DEFINITIONS

[0031] As used herein, the term cell culture or culture refers to the process by and conditions under which cells or tissue is maintained under artificial conditions for a short or long time outside of the organism from which it was originally extracted. Cell culture is a generic term that may also encompass the cultivation of prokaryotes and eukaryotes. The term mono-culture refers to a type of cell culture in which all cultured cells are the same type. Culturing, as used herein, can include, but does not require, cell division of cultured cells.

[0032] As used herein, the term cellular co-culture or co-culture refers to the process by which a mixture of two or more different cell types are grown together or the mixture itself. The term trans-co-culture refers to the process or system of growing together different cell types from different patients. For example, trans-co-culture describes a system in which Patient A's cancer cells are cultivated with Patient B's stromal cells. The term cis-co-culture refers to the process or system of growing or maintaining together different cell types from the same patient. For example, cis-co-culture describes a system in which Patient A's cancer cells are cultivated with Patient A's stromal cells. For either type of co-culture, the different cell types can be harvested at different times and stored for different lengths of time. In some forms of cis-co-culture, cells of one type are frozen or cultured for several days before cells of a different type are collected and the cis-co-culture is assembled.

[0033] As used herein, the term cancer refers to any hyperproliferative disease that includes a malignancy characterized by deregulated or uncontrolled cell growth. Cancers of virtually every tissue are known. Examples of cancers include, but are not limited to carcinoma, lymphoma, blastema, sarcoma, and leukemia or lymphoid malignancies. The term hematological cancer or hematological malignancy refers to any malignancy associated with cells in the bloodstream, bone marrow, or lymphoid system. Hematological cancers include but are not limited to Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic leukemia (acute and chronic), or multiple myeloma. The term multiple myeloma refers to a disseminated malignant neoplasm of plasma cells which is characterized by multiple bone marrow tumor foci and widespread osteolytic lesions. The term Hodgkin's lymphoma refers to cancer originating from the lymphocytes and characterized by the orderly spread of disease from one lymph node group to another and by the development of systemic systems with advanced disease.

[0034] As used herein, the term microfluidic refers to a device or system through which materials, particularly fluid born materials such as liquids, are transported on a microscale, and in some embodiments on a nanoscale. Microfluidic systems are systems arranged to deliver small amounts of fluid, for example, less than 1 ml of fluid.

[0035] The term agent, as used herein, includes a compound that induces a desired pharmacological and/or physiological effect. The term also encompass pharmaceutically acceptable and pharmacologically active ingredients of those compounds including but not limited to their salts, esters, amides, prodrugs, active metabolites and analogs. This term includes the active agent per se, as well as its pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites and analogs. The term agent is not to be construed narrowly but extends to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins as well as compositions comprising them, and genetic molecules, such as RNA, DNA and their mimetics and chemical analogs, as well as cellular agents. The term agent includes a cell which is capable of producing and secreting the polypeptides referred to herein as well as a polynucleotide comprising a nucleotide sequence that encodes this polypeptide. Thus, the term agent extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells. The term agent includes plasma or other blood products.

[0036] As used herein, the term drug refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or function of? the body. The term drug refers to a variety of substances including but in no way limited to afatinib, denosumab, lenalidomide, trametinib, dabrafenib, radium Ra 223 dichloride, erlotinib, ado-trastuzumab emtansine, acetylsalicylic acid, bergamottin, dihydroxybergamottin, paradicin-A, pomalidomide, doxorubicin hydrocholoride, bevacizumab, bortezomib, docetaxel, aziridines, streptozotocin, cytarabine, podophyllotoxin, actinomycin, cyclophosphamide, methotrexate, 5-fluorouracil, vinblastine, dacarbazine, and prednisolone.

[0037] As used herein, the term patient refers to animals, including mammals, preferably humans.

[0038] As used herein, the term in vivo means within a living organism and ex vivo means outside of a living organism. As used herein, the term in vitro refers to operations carried out in an artificial system.

[0039] As used herein, the term fluid connection or fluidically connected or similar means that a system allows fluid to flow between two or more elements. Two reservoirs can be physically isolated by a barrier but fluidically connected by channels that allow fluid to flow between then.

[0040] As used herein, the term channel or diffusion pore refers to pathway in or through a medium that allows for movement of fluids such as liquids or gases, which may contain soluble factors. The term microchannel refers to a channel having cross-sectional dimensions in the range of about 1.0 m to 500 m, preferably between about 15 m and 200 m.

[0041] As used herein, the term target cell refers to a cell which is extracted from a patient, isolated (e.g., to purity or via enrichment in a cell population), and analyzed. Target cells can be isolated using a variety of techniques including but not limited to beads and columns. Sometimes, a cell contains a distinct marker such as CD138 which facilitates cell isolation or enrichment. Target cell can refer to cells of many different types such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic leukemia (acute and chronic), or multiple myeloma cells.

[0042] As used herein, the term stroma or stromal cell refers to connective tissue of an organ such as the endometrium, prostate, or bone marrow. Stromal cells include but are not limited to fibroblasts, macrophages, monocytes, endothelial cells, and pericytes. As an illustrative example, stromal cells in the bone marrow are not directly involved in hematopoiesis. Instead, bone marrow stromal cells provide a proper microenvironment for hematopoiesis by the bone marrow parenchymal cells. For example, bone marrow stromal cells generate colony stimulating factors.

[0043] As used herein, the term solid-support refers to non-gaseous, non-liquid material having a surface. For example, solid-supports can be flat such as glass, silicon, metal, plastic, or composite; or they can be in the form of a bead such as silica gel, controlled pore glass, magnetic or cellulose bead; or they can be pins such as pins suitable for combinatorial synthesis or analysis.

[0044] As used herein, the term chamber or well refers to any structure in which volumes of fluid can be contained.

DETAILED DESCRIPTION

[0045] Hematological malignancies account for a considerable amount of new cancer diagnoses. For example, multiple myeloma (MM) is a universally fatal disease, comprising 15% of hematological malignancies and 1% of all cancers. It has a median survival of 5-7 years from diagnosis. While newer drugs, such as the proteasome inhibitor bortezomib, have increased response to therapy, resistance and relapse still remain a key concern. Therefore, there is a need to understand the mechanism(s) of resistance to cancer-treating agents. Ideally, this is accomplished in a patient-specific manner.

[0046] One approach for patient-specific resistance analysis is to directly examine biological responses of patient target cancer cells in the presence of their own stromal cell components (cis-co-culture or intra-patient analysis, FIG. 1) under different drug conditions. While trans-co-culture (or inter-patient analysis) in which cancer cells and stromal elements are derived from different patients or people has been reported before, the cis-co-culture concept is unique to this application (FIG. 1).

[0047] The invention described herein provides, in some embodiments, microfluidics-based microchannel platforms for analysis of samples in mono- and/or cis-co-culture. Many hematologic cancers can also be treated with the technology described herein. In some embodiments, the present invention is useful in assessment of and treatment of Adult Acute Lymphoblastic Leukemia, Childhood Acute Lymphoblastic Leukemia, Adult Acute Myeloid Leukemia, Childhood Acute Myeloid Leukemia, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Hairy Cell Leukemia, AIDS-Related Lymphoma, Cutaneous T-Cell Lymphoma, Adult Hodgkin Lymphoma, Childhood Hodgkin Lymphoma, Hodgkin Lymphoma During Pregnancy, Mycosis Fungoides, Adult Non-Hodgkin Lymphoma, Childhood Non-Hodgkin Lymphoma, Non-Hodgkin Lymphoma During Pregnancy, Primary Central Nervous System Lymphoma, Szary Syndrome, Cutaneous T-Cell Lymphoma, Waldenstrm Macroglobulinemia, Chronic Myeloproliferative Disorders, Langerhans Cell Histiocytosis, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndromes, and Myelodysplastic/Myeloproliferative Neoplasms. In some embodiments, cells of these cancer types can be analyzed in an accurate in vitro system using the technology described herein.

[0048] In a first step, cells are isolated based on one or more cell-specific markers by affinity binding. For example, in some embodiments, CD138 is used as a marker to sort for patient myeloma cells from bone marrow aspirates. These patient myeloma cells are seeded into a device (e.g., FIGS. 1a and b) comprising a central well comprising a well for retaining a suspension of target patient cells. Next, patient stromal cells are isolated and seeded into one or more side chambers. In some embodiments, CD138 non-cancerous stromal mononuclear cells are seeded into each side chamber. While some embodiments use patient myeloma cells as target cells, other embodiments Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic leukemia (acute and chronic), or other hematological cancer cells as target cells.

[0049] In some embodiments, each side chamber communicates fluidically with the central well via diffusion ports (also referred to as channels) that are large enough for media and soluble factors to diffuse across and small enough to prevent or restrict cells and larger insoluble matter from crossing. In some embodiments, the diffusion ports have dimensions no larger than 300 m wide and 60 m deep. In some embodiments, the diffusion ports have a width of 90-110 m and a depth of 50-70 m. In some embodiments, the central well contains the stromal cells and the side chambers contain the target cells. In further embodiments, the chambers are instead configured such that there are no central or side wells, and instead, the chambers are spread over the device in a symmetrical or asymmetrical pattern. In some embodiments, the chambers are positioned (e.g., with varying altitudes, pressures, or charges) such that fluid from the channels can travel in one direction only. In embodiments where it is desired that cells be physically separated, any mechanism can be used to isolate and separate cells.

[0050] In some embodiments, cultures without the patient's stromal cells (monoculture) and cultures with the patient's stromal cells (cis-coculture) are analyzed for target cell responses to agents in vitro in a patient-specific manner. Some embodiments of the invention relate to analysis of functional responses of primary patient cancer cells.

[0051] In some embodiments, the technology described herein is used to provide patient-specific care in a healthcare setting. In some embodiments, a healthcare provider obtains tissue samples from patients undergoing or beginning treatment with a pharmaceutical agent. In some embodiments, these samples are then separated based on one or more distinctive cell marker, cultured on a solid support, treated with one or more pharmaceutical agents, and observed for effectiveness using the methods, systems, and compositions described herein. The effectiveness results (e.g., the patient's cells are drug resistant or sensitive) are then used to tailor a patient's medical treatment. For example, if the analysis reveals that the patient is resistant to a drug, then the analysis may be repeated with alternative drugs until a drug is found to which that patient is sensitive.

[0052] In some embodiments, the systems and methods assists medical care providers in determining to which pharmaceutical agents a patient is sensitive or resistant. In some embodiments, healthcare providers consider a range of pharmaceutical agents, test the patient for resistance or sensitivity to a variety of these agents using the technology described herein, administer pharmaceutical agents to which the patient is sensitive according to the tests, follow up with the patient to ensure that the selected agent is working effectively, and re-test with the herein described technology if the effectiveness of the agent is not satisfactory.

Example 1Clinical Trials

[0053] A microscale cell culture and analysis device representing one embodiment of the invention was used to analyze CD138+ MM tumor cells' drug responses to the proteasome inhibitor bortezomib/Velcade and whether CD138 mononuclear cells from the same patient provide a protective effect. Cells were routinely cultured at 37 C. with 5% CO.sub.2 in high-glucose DMEM containing 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin (1% P/S), and 10 mM HEPES buffer at 1.0 to 1.5105 cells/mL in tissue culture-treated flasks. A total of 5 L of the concentrated cell suspension was dispensed by passive pumping into each microchamber.

[0054] 7500 CD138+ cells were added in a central well of a device resembling FIG. 1 with or without 8000 CD138 mononuclear cells in each side channel, which were obtained from the same patient bone marrow aspirates. Dose-dependent reductions in cell viability were obtained using calcein AM and ethidium homodimer staining for live and dead cells, respectively. Such MM cell viability was protected by the presence of patients' own CD138 mononuclear cells to varying degrees (FIGS. 2a-r). The blue line represents co-cultured conditions, while the black line represents the mono-cultured conditions. Patients 329, 316, and 318 are currently refractory to treatment and their co-cultured cells appear to show protection from bortezomib to varying degrees in comparison to their mono-cultured cells. Patients 315, 344, and 345 are currently sensitive to treatment and there appears to be no appreciable difference whether their cells are mono- or co-cultured. This analysis is not limited only to bortezomib and can be used to analyze other cancer drugs that are used to treat individual patients at different times in the treatment course (nave, relapse, etc).

Example 2Predictive Assays

[0055] In some embodiments, the invention provides assays that predict patient-specific drug efficacy. Based on data gathered from cis-co-culture experiments and with the knowledge of the next treatment, the technology is used to treat patient cells in vitro with the drugs of the next treatment. This response is then matched to the patients' response in the clinic.

Example 3Cis-Co-Culture Benefits

[0056] Before the development of the present invention, there was no device, whether in macro- or micro-scale, that could culture patient myeloma cells with their own stromal cells (cis-coculture). Others isolate and expand different cell types in the stroma, such as bone marrow stromal cells (BMSCs), derived from different patient sources to co-culture with another patients' myeloma cells. This was avoided in the present technology for two reasons. First, the establishment of BMSCs amenable for conventional co-culture studies takes typically more than two weeks; however, myeloma cells only survive for a maximum of three days ex vivo. Therefore, one needs to cryopreserve myeloma cells while BMSCs are being generated in order to perform cis-co-culture studies. Cryopreservation of myeloma cells is often not efficient and may also alter the biology of the cell. Furthermore, only 20% of the original frozen cells are viable when thawed. Thus, co-culture is classically performed in trans with BMSCs from one patient and myeloma cells from a different patient. Second, artificial enrichment for a specific cell type in the stroma, such as BMSCS, was avoided in the system; rather, all stromal mononuclear cells present in bone marrow aspirates obtained from individual patients were kept in their respective fractions that can vary from patient to patient.

Example 4Microscale Benefits

[0057] The number of myeloma cells obtained from individual patients can vary dramatically and be as low as 10.sup.4 cells per 10 ml aspirate. While certain macroscale cell death assays can work with low cell numbers to provide population-average responses to drugs, in some embodiments, the present invention is able to obtain individual cell responses within a population of cancer cells, as well as population-average responses, because data collection is performed at the single cell level. For example, in some embodiments, cell viability and protein subcellular localization within single cells is analyzed with image analysis consistent with E W Young's methods. Young, E W et al.: Microscale functional cytomics for studying hematologic cancers Blood. 2012 Mar. 8; 119(10).

Example 5Market Need

[0058] Multiple myeloma patients have many options for treatment but also the development of resistance is a common clinical problem. The current method of treatment selection is arbitrary and a more rational method is desperately needed. This invention provides a system for a laboratory assay to predict drug responses in individual patients. In some embodiments, the invention predicts what drugs patients will respond to in the clinic, by mimicking the microenvironment around the patient's malignancy as accurately as possible.

Example 6Device Preparation

[0059] The device was created according to methods described in Young E W et al., Blood. 2012 Mar. 8; 119(10) (hereby incorporated by reference in its entirety). Specifically, embodiments of the device are formed from polydimethylsiloxane (PDMS) that is sequentially bonded via plasma treatment to a glass slide. In some embodiments, the device is made of inert polymers, such as polystyrene to allow for analysis with hydrophobic molecules.

[0060] In some embodiments, the device used soft lithography techniques to fabricate single-use devices consisting of 12 independent microscale cell culture chambers (microchambers) arranged in a 34 array. Xia Y, Soft Lithography Annu Rev Mater Sci. 1998; 28:153-184. A channel layer master mold containing a central well, side chambers, diffusion ports, and an inlet and outlet channel were made. An additional master mold was made for the access port layer. Both layers were made of PDMS. The PDMS was mixed at a 10:1 base-to-curing agent ratio and cured at 80 C. for four hours. The two PDMS layers were sequentially bonded via plasma treatment to a glass slide to produce the final device.

Example 7Rapid Chemosensitivity and Chemoresistance Assay

[0061] The microfluidic platform shown in FIG. 1 was employed in this example. In short, by leveraging pressure differences at differently sized inlet and outlet ports, this platform was operated by passive pumping, requiring only a micropipet. Suspension cells were seeded through the inlet port of the central well and were allowed to settle overnight. Other cell types for coculture were seeded through the inlet ports of the side chambers. This system incorporates the following features: 1) capacity to be performed for all patients (e.g., 7500 cells per endpoint in the current study), 2) both tumor and non-tumor cells incorporated to mimic in vivo environment, and 3) simple and quick to perform without the need to extensively grow the cells in vitro. The entire assay requires only 3 days to complete. Patient MM cell viability within the microchannel during the 3 days was comparable to that of an MM cell line, RPMI8226, for 3 days.

[0062] After being freshly sorted within 24 hours of bone marrow aspiration, CD138.sup.+ tumor cells were cultured in either mono- (MicroMC) or cis-coculture with the patients' own CD138.sup. non-tumor mononuclear cell fractions (MicroC.sup.3). Cryopreservation of MM tumor cells was avoided due to loss of viability of patient cells averaging only 25% of cells being viable after thaw. The following day, the tumor cells were treated with varying doses of bortezomib, ranging from 0 to 300 nM (calculated final concentrations) for 24 hours. The bortezomib concentrations were varied from low to those higher than what is observed in patients (100 nM at peak plasma concentration) because the material used in microchannels, polydimethylsiloxane (PDMS), has the tendency to absorb hydrophobic molecules, and thus lower the effective concentration of drugs that are applied in culture. Due to the limited number of tumor cells obtained, some patient samples could not be analyzed for all bortezomib doses. After bortezomib treatment, the CD138.sup.+ cells were stained with calcein AM and ethidium homodimer to count for live and dead cells, respectively.

[0063] After completion of the assay, live and dead cells were counted using an ImageJ-based in house software program (J experiment). Due to the patient-to-patient variability of the retention of MM cells in the central chambers, live fractions for each dose were calculated and normalized to the 0 dose for both MicroMC and MicroC.sup.3. The percent change in live fraction from the 0 nM to 100 nM dose of bortezomib for MicroMC and MicroC.sup.3 was then calculated in order to classify patients' MM cell ex vivo responses. Patients whose CD138.sup.+ cells did not survive in MicroMC but survived in MicroC.sup.3 were omitted from MicroMC analysis.

[0064] Interestingly, ex vivo responses within MicroC.sup.3 appeared to segregate into two groups, while responses within MicroMC did not (FIG. 3). Therefore, both the AIC (Akaike Information Criterion) and BIC (Bayesian Information Criterion) were calculated for unimodal, bimodal, and trimodal distributions for both MicroMC and MicroC.sup.3. For MicroC.sup.3 and at a clinically relevant treatment dose of 100 nM, a bimodal distribution of the response data was favored by the AIC as well as the BIC over unimodal and trimodal distributions. In contrast, none of these distributions was favored by AIC or BIC for the 100 nM treatment cases in MicroMC.

[0065] As both the AIC and BIC values indicated that a bimodal distribution was favored for MicroC.sup.3, k-means and Gaussian mixture clustering methods were applied to segregate MicroC.sup.3 ex vivo responses. For the 100 nM dose levels, k-means and Gaussian mixture clustering segregated 17 ex vivo responses in MicroC.sup.3 into two clusters: 1non-sensitive (10 cases), 2sensitive (7 cases). The separation of the two resulting clusters (p<10.sup.5) was further in line with the Otsu threshold independently derived for the same data.

[0066] The ex vivo responses within MicroMC and MicroC.sup.3 were then compared with the clinical responses of the same patients. The clinical characteristics of these patients are described in Table 1.

TABLE-US-00001 TABLE 1 MicroMC kM Clinical Ex vivo cluster kM GM cluster Patient response to response (k = 2) Silhouette (k = 2) ID Bortezomib [%] index value index 314 non-responder 26.8 1 0.642 2 316 responder 92.8 2 0.873 2 317 responder 45.2 2 0.299 2 318 responder 83.4 2 0.894 2 323 non-responder n/a n/a n/a n/a 329 non-responder 40.8 2 0.022 2 330 responder 74.2 2 0.892 2 345 responder 97.8 2 0.856 2 353 non-responder 7.0 1 0.954 1 398 non-responder 1.6 1 0.959 1 402 non-responder n/a n/a n/a n/a 419 non-responder 17.7 1 0.927 1 431 non-responder 4.0 1 0.959 1 432 responder 18.8 1 0.837 1 435 responder 5.9 1 0.953 1 442 non-responder n/a n/a n/a n/a 446 non-responder 4.5 1 0.954 1 447 non-responder 19.6 1 0.919 1 AIC unimodal (k = 1) 156.681 AIC bimodal (k = 2) 156.591 AIC trimodal (k = 3) 157.407 BIC unimodal (k = 1) 158.097 BIC bimodal (k = 2) 160.131 BIC trimodal (k = 3) 163.071 Mean Silhouette (k = 2) 0.793 MicroC.sup.3 kM Clinical Ex vivo cluster kM GM cluster Patient response to response (k = 2) Silhouette (k = 2) ID Bortezomib [%] index value index 314 non-responder 5.8 1 0.935 1 316 responder 81.5 2 0.964 2 317 responder 50.5 2 0.736 2 318 responder 86.3 2 0.956 2 323 non-responder 21.0 1 0.923 1 329 non-responder 11.1 1 0.831 1 330 responder 73.6 2 0.965 2 345 responder 97.1 2 0.925 2 353 non-responder 10.3 1 0.841 1 398 non-responder 5.1 1 0.933 1 402 non-responder 62.4 n/a n/a n/a 419 non-responder 13.7 1 0.791 1 431 non-responder 2.3 1 0.909 1 432 responder 65.4 2 0.938 2 435 responder 68.9 2 0.954 2 442 non-responder 11.0 1 0.937 1 446 non-responder 1.0 1 0.924 1 447 non-responder 12.6 1 0.809 1 AIC unimodal (k = 1) 176.397 AIC bimodal (k = 2) 164.746 AIC trimodal (k = 3) 168.091 BIC unimodal (k = 1) 178.064 BIC bimodal (k = 2) 168.079 BIC trimodal (k = 3) 173.091 Mean Silhouette (k = 2) 0.898

[0067] All samples were collected and analyzed ex vivo without the prior knowledge of patients' clinical history to eliminate operator bias. The clinician determining responses was also blinded of the ex vivo response data. Remarkably, the MicroC.sup.3 clusters separated by k-means and Gaussian mixture clustering into non-sensitive and sensitive were correctly identified as either clinically non-responsive or responsive. On the other hand, if the two clustering methods were applied to MicroMC, the results were much less clear. According to k-means, 12/15 (7/8 non-sensitive and 5/7 sensitive) monocultured ex vivo patient responses matched their respective clinical responses. According to Gaussian mixture clustering, only 11/15 (6/8 non-sensitive and 5/7 sensitive) monocultured ex vivo patient responses matched their respective clinical responses.

[0068] These results were further supported when the clustering methods were applied to the changes in live fraction from 0 to 30 nM of bortezomib treatment. MicroC.sup.3 successfully identified 16/16 ex vivo responses, while MicroMC identified 12/15 at this dose of bortezomib. Thus, patients' CD138.sup.+ MM cells in MicroC.sup.3 appear to more uniformly respond to bortezomib (either sensitive or non-sensitive) compared to the same cells analyzed alone without the influence of CD138.sup. cell population.

[0069] One anomalous patient, Pt 402, was removed from the above analyses. Pt 402's CD138.sup.+ cells showed no response to bortezomib ex vivo and the subject's clinical response was non-responsive. However, when included in the clustering analyses, trimodal distributions were favored with Pt 402's ex vivo responses being classified as a third cluster. Peculiarly, Pt 402 was the only patient with a t(14;16) translocation affecting c-Maf oncogene with a very poor prognosis that occurs in 5% of MM patient. It is contemplated that patients with a t(14;16) translocation, or patients with an ex vivo response similar to Pt 402, do comprise a third cluster of patients displaying ex vivo growth response to bortezomib.

[0070] Pt's 317, 318, and 323 were newly diagnosed patients and their tumor cells showed sensitivity to bortezomib in MicroC.sup.3 assay. These patients then went on bortezomib-containing regimens without the clinician's prior knowledge of the ex vivo responses. Clinically, Pt. 317 and 323 had a partial response, and Pt. 318 showed a complete response to bortezomib-containing regimens. Similarly, Pt. 316's tumor cells showed sensitivity to bortezomib in MicroC.sup.3 assay but the patient was classified as refractory to treatment with lenalidomide and dexamethasone at the time of the biopsy. This patient then went on bortezomib-containing therapy and had a partial response. In contrast, Pt. 442 was a newly diagnosed patient whose tumor cells did not respond to bortezomib in MicroC.sup.3. This patient then went on a bortezomib-containing regimen, again without the clinician's prior knowledge of the ex vivo data, and did not respond clinically. Therefore, patient's CD138.sup.+ cell response to bortezomib in the MicroC.sup.3 system predicts patients' clinical response to bortezomib-containing therapy.

[0071] These results demonstrate several important features. First, the MicroC.sup.3 requires only several thousands of CD138.sup.+ cells per condition for functional analysis, thereby enabling the analysis of MM cell responses in virtually all patient bone marrow aspirate samples. This may avoid a potential bias towards samples with larger numbers of tumor cells, which could be unintentionally incorporated when certain assays such as biochemical ones that require a larger starting cell number are used to analyze primary patient samples. Second, both tumor and non-tumor cells are incorporated into the design of MicroC.sup.3 to recapitulate in vivo environment. Given that tumor-associated cell types can have significant impacts on tumor cell drug responses, incorporation of these non-tumor cell populations without artificial enrichment of individual cell components allows tumor cells to behave more similarly to in vivo conditions than when they are cultured alone. Additionally, the MicroC.sup.3 assay is simple and quick to perform taking only three days from the sample acquisition to the analysis of the cell responses, negating the need for culturing and passaging of the patient cells. Incorporation of total CD138.sup. cell population is contemplated to enable ex vivo recapitulation of the tumor cell to non-tumor cell interactions in the cancer microenvironment. Because MicroC.sup.3 is performed with freshly isolated tumor and non-tumor cells, passaging and potential enrichment of the CD138.sup.+ tumor cell subpopulations are also avoided. Furthermore, in some embodiments, MicroC.sup.3 is conducted at the microscale, using fluid volumes of 10 L in the central chamber and 5 L in each of the side chambers. These low fluid volumes are contemplated to concentrate important soluble factors produced by both the tumor and non-tumor companion cells which may otherwise be diluted in conventional culture conditions (e.g., Transwell cultures).

[0072] While all other previous Chemosensitivity-resistance Testing systems (CSRAs) also categorize patients into high and low response groups, MicroC.sup.3 is the only CSRA that has utilized clustering methods to segregate and identify ex vivo patient responses without operator bias. Remarkably, these clustering methods have identified 7 out of 7 patients whose MM cells were sensitive in MicroC.sup.3 were either currently clinically responsive to bortezomib-containing therapy or went on to respond to bortezomib-containing therapy after the treating clinician selected this treatment. Moreover, 10 out of 10 patients with the MicroC.sup.3 non-sensitive designation were either currently relapsed and/or refractory to bortezomib-containing therapy or did not respond to subsequent bortezomib-containing therapy. In comparison, when the same primary MM cells were cultured under the same conditions but without the influence of the CD138.sup. cell fractions, the agreement between ex vivo and clinical responses were much less evident. It should be mentioned that the microchannel-based monoculture assay, MicroMC, was still able to match a large percentage (80%) of ex vivo responses to clinical responses, which is comparable or better than other CSRAs tested for other cancer types that have garnered 30-60% accuracy (see e.g., S. Ugurel et al., Clin. Cancer Res. 12, 5454-63 (2006); T. H. Lippert, H.-J. Ruoff, M. Volm, Int. J. Med. Sci. 8, 245-53 (2011); and M. Suggitt, M. C. Bibby, Clin Cancer Res 11, 971-981 (2005)).

[0073] A predictive CSRA that enables stratification of patients prior to specific therapy allows patients to avoid ineffective therapies as well as unnecessary cost associated with such therapies. Additional therapeutic agents (carfilzomib, lenalidomide, and pomalidomide) were also tested in a small number of cases and MicroC.sup.3 was able to categorize patients into sensitive and non-sensitive groups. For example, FIG. 4 shows data obtained with patient 442 using bortezomib, carfilzomib, lenalidomide, and pomalidomide.

Materials and Methods

Microchannel Fabrication and Preparation

[0074] Single-use devices comprised of 12 or 24 cell culture chambers were fabricated using soft lithography using two master molds established previously (Y. Xia, G. M. Whitesides, Annu. Rev. Mater. Sci. 28, 153-184 (1998); Duffy, J. McDonald, O. Schueller, G. Whitesides, Anal. Chem 70, 4974-4984 (1998)). Polydimethylsiloxane (PDMS) was mixed at a 10:1 base:curing agent ratio, poured on the master molds, and cured at 80 C. for 4 hrs. Two separate PDMS layers were made, one for the channel layers containing the central well, side chambers, diffusion ports, and inlet and outlet channels, and one for the access port layer. The two PDMS layers were then soxhlet extracted and plasma treated to bond to a glass slide. The final device was then baked at 120 C. for 15 min to increase bond strength and release any bubbles.

[0075] Before cell culture, the chambers on the device were filled first with 70% ethanol as a wetting agent as well as a disinfectant. The ethanol was rinsed with 3 volume replacements (VRs) of 1 phosphate-buffered saline (PBS). The 1PBS was then replaced with 3 VRs of the appropriate cell culture medium. The final such prepared device can be stored up to 3 weeks in a 37 C. incubator when encased with appropriate humidifying and sterile conditions. If stored longer than 24 hours, the media is replaced with 1 VR of fresh media prior to cell culture.

Cell Line Culture and Preparation

[0076] RPMI8226 (human MM cell line) was obtained from ATCC. RPMI8226 cells (1.0 to 1.510.sup.5 cells/mL seeding density) were routinely cultured at 37 C. with 5% CO.sub.2 in high-glucose Dulbecco's modified eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 g/mL streptomycin (1% P/S), and 10 mM hydroxyethyl piperazineethanesulfonic acid (HEPES) in tissue culture-treated flasks. Cells were passaged every 2 to 3 days. For experiments conducted in microchambers, RPMI8226 cells were collected and resuspended at 1.010.sup.6 cells/mL of fresh growth media. A total of 54, of the concentrated cell suspension was dispensed by passive pumping into each microchamber.

Primary Patient Cell Culture and Preparation

[0077] Bone marrow aspirates (5-10 ml each) were obtained with informed consent from patients diagnosed with multiple myeloma. Clinical status of the patients was determined by International Myeloma Working Group (IMWG) criteria (N. C. Munshi et al., Blood 117, 4696-4700 (2011)). Sensitive or Responsive is defined as patient achieving at least a partial remission/response. Relapsed is defined as patient having 0.5 g/dl increase in monoclonal protein or >200 mg in 24 hours in urine light chains after obtaining at least a partial remission. Refractory or Resistant is defined as patient having progressed on or within sixty days of treatment. Relapsed and/or refractory patients were defined as non-responsive patients for the purpose of this assay. The researcher assessing clinical statuses of the patients was blinded to assay results and separate from the staff performing the assay. The ex vivo data and the clinical statuses were only compared after the assay was completed.

[0078] Aspirate volumes were doubled with Iscove's Modified Dulbecco's Medium (IMDM)+100 units/mL heparin (Sigma-Aldrich). They were doubled again with IMDM+10 units/mL DNAseI (Roche). After rocking at room temperature for 30 minutes, 2 volumes of cell mixture were put over 1 volume of lymphocyte separation medium (Cellgro) and centrifuged for 35 minutes at 200 g. The interface and 2-3 mLs of the buffy coat layer were taken, and rinsed with PBS+2 mM ethylenediaminetetraacetic acid (EDTA). At this point, the total mononuclear cells were either sorted or cryopreserved with cryopreservation medium (90% FBS, 10% DMSO). CD138.sup.+ magnetic MACS beads (Miltenyi Biotec) were used to positively sort for multiple myeloma cells per the manufacturer's instructions to >95% purity as determined by fluorescence-activated cell sorting (FACS). For sorting with cryopreserved samples, the sample was quickly thawed at 37 C., resuspended in 10 mLs of DMEM and 20% FBS and pelleted to remove DMSO. The sample was then incubated in high glucose DMEM containing 20% FBS and 1% P/S and 10 mM L-glutamine with the addition of DNAseI for 1 hour with agitation approximately every 15 min to break up cell clumps. The sorting protocol was then followed as above with the CD138.sup.+ MACS beads.

[0079] For microchannel experiments, CD138.sup.+ cells were resuspended in high glucose DMEM containing 20% FBS and 1% P/S and 10 mM L-glutamine at a density of 1.510.sup.6/mL and 54, were seeded into the inlet port of each central well for a total of 7500 cells. For coculture experiments, CD138.sup. cells were resuspended at a density of 410.sup.6 cells/mL in the same media as the CD138.sup.+ cells and 2 uL were seeded into the inlet port of each side channel for a total of 8000 cells on each side. After an overnight culture, fresh media containing varying concentrations of drug were added to the input port, resulting in exposures of both MM cells and non-MM cells in the side chambers at the final concentrations needed. If the number of cells permitted, duplicates of drug dose conditions were performed. Cultures were treated with the drug for 24 hrs after which live and dead cells were stained, respectively, as described below.

Reagents

[0080] Bortezomib (PS-341 or Velcade) and carfilzomib (Krypolis) were obtained as a 2.6 mM clinical saline solution and stored at 80 C. in separate aliquots. The drugs were thawed 5 minutes before each treatment, serially diluted to the desired concentrations in media warmed to 37 C., and dispensed into microchambers in 3 sequential VRs followed by aspiration of the outlet port to reach desired final concentrations. Lenalidomide and pomalidomide (Revlimid and Pomalyst, respectively) were purchased from Selleck Chemicals and reconstituted in DMSO to a stock concentration of 100 M and stored at 80 C. These chemicals were thawed, serially diluted, and dispensed into microchambers in the same manner as bortezomib. LIVE/DEAD Viability/Cytotoxicity Assay Kit from Invitrogen was used to detect live and dead cells in microchambers. Both calcein AM (green) and ethidium homodimer (red) were used at a working concentration of 4 M.

Immunofluorescence Image Analysis

[0081] Cells were stained with LIVE/DEAD for 10 min, and washed with one VR of fresh media. All fluorescent images were taken with a Nikon Eclipse Ti inverted fluorescent microscope coupled to a Nikon DS-Qi1Mc CCD camera (Nikon Instruments Inc., Melville, N.Y., USA) at a magnification of 4. Image analysis was performed in ImageJ with custom in-house algorithms and database management to count live and dead cells (J experiment) (E. W. Young et al., Microscale functional cytomics for studying hematologic cancers, Blood 119, e76-85 (2012)).

Statistical Analysis

[0082] Monocultured live fraction responses to bortezomib were normalized to the monoculture 0 dose; cis-cocultured live fraction responses to bortezomib were normalized to the coculture 0 dose. The changes in live fractions as a response to 30 nM and 100 nM doses of bortezomib were calculated. The mean, standard deviation, median, interquartile range (IQR), and Otsu threshold (T. Kurita, N. Otsu, N. Abdelmalek, Pattern Recognit. 25, 1231-1240 (1992)) were calculated for both monoculture and cis-coculture responses in Matlab (MathWorks, Nattick, Mass.) and graphed using Origin (OriginLab, Northampton, Mass.).

Clustering Analysis of Ex Vivo Data

[0083] In order to automatically identify potential distinct subpopulations in an unsupervised fashion and with high discriminative power, ex vivo data were separated using both k-means clustering (J. a Hartigan, M. a Wong, J. R. Stat. Soc. 28, 100-108 (1979)) and Gaussian mixture (GM) modeling (G. McLachlan, D. Peel, Finite Mixture Models (2000; doi (dot) wiley (dot) com/10.1002/0471721182), p. 419.) algorithms. We chose to compare both methods for robustness. In both methods, the data set is iteratively partitioned into k clusters by minimizing the within-cluster variance while maximizing the between-cluster variance. Clustering of the ex vivo response data was carried out using the Statistics Toolbox of Matlab (MathWorks, Nattick, Mass.). The degree of dimensionality (i.e., the numbers of clusters) was determined by a) maximizing the mean Silhouette index of the k-means clusters and by b) minimizing the Akaike and Bayesian information criterion (AIC and BIC, respectively) of the Gaussian mixture model.