Method for producing a three-dimensional human multiple myeloma model

Abstract

Method for producing a three-dimensional human multiple myeloma model. The present invention relates to a method for producing a three-dimensional (3D) model of multiple myeloma (MM), in the form of spheroids, by co-culturing mesenchymal stem / stromal cells, endothelial progenitor cells, and primary plasma cells from MM patient(s). The present invention also relates to the spheroids obtained by said method and their uses. Figure for abstract: none

Description

Title of the invention: Method for producing a three-dimensional model of human multiple myeloma

[0001] The present invention relates to the method of producing a three-dimensional (3D) model of multiple myeloma (MM), in the form of spheroids, by co-culture of mesenchymal stem / stromal cells, endothelial progenitors and primary plasma cells from patient(s) with MM. The present invention also relates to the spheroids obtained by said method and their uses.

[0002] Multiple myeloma (MM) is a malignant hematological disorder, also called bone marrow cancer. It is characterized by the excessive proliferation in the bone marrow of a type of white blood cell, the plasma cell, which has become abnormal. Plasma cells are immune system cells derived from the bone marrow that produce antibodies to protect the body against external attacks (bacteria, viruses). During development, genetic abnormalities (deletion, chromosomal translocation) can occur, thus transforming healthy plasma cells into malignant plasma cells. In a normal state, these plasma cells circulate in the blood, whereas in MM they return to the bone marrow where they cause damage at several levels.

[0003] Multiple myeloma remains an incurable disease despite recent remarkable therapeutic advances. Current treatment makes it possible to prevent or alleviate symptoms and complications by destroying pathological plasma cells and slowing the progression of the disease.

[0004] To date, multiple myeloma suffers from a lack of relevant preclinical models. Indeed, murine models are expensive, time-consuming, and not representative of the human pathology. Standard two-dimensional (2D) models do not allow for the viability of primary plasma cells from patients and favor the use of cell lines, which makes these models too far removed from the pathophysiology of MM disease.

[0005] The reproduction of adult human bone marrow ex vivo is increasingly described in the literature to overcome the limitations of animal models, which are costly, time-consuming, and dependent on the species barrier. Studies have begun to highlight 3D models of human bone marrow ex vivo, incorporating the mesenchymal and vascular compartments, generally from cell lines. For example, the vascular compartment, which plays an active role in the proliferation of hematopoietic stem cells (HSCs), is often incorporated via endothelial cell lines of the HUVEC type. Other models require a mouse step to enable vascularization or functional approaches. Furthermore, the short half-life of plasma cells prevents their autologous incorporation into current 3D models, forcing them to be ignored or the addition of tumor plasma cell lines, which does not allow for a relevant model response compared to the patient.

[0006] The inventors have created a new 3D human tissue model of multiple myeloma. It includes the mesenchymal compartment, the vascular compartment, and plasma cells, obtained from samples of patients with multiple myeloma, without the use of a cell line. In particular, the maintenance of plasma cell viability in co-culture for more than 14 days has been resolved. This makes it possible to generate a fully human preclinical ex vivo model of multiple myeloma, genetically relevant to the patient, and comprising the mesenchymal, vascular, and plasma cell compartments in spheroid form.

[0007] This model makes it possible to envision an advance in personalized medicine thanks to the rapid constitution of a representative model of the tumor tissues of the bone marrow of each patient. Detailed description of the invention

[0008] Process for producing human multiple myeloma spheroids

[0009] The invention relates to a method for producing human multiple myeloma (MM) spheroids, comprising: a. Culture of mesenchymal stem / stromal cells (MSCs), endothelial cells and endothelial progenitors in a culture medium; b. Harvesting of cultured MSCs, endothelial cells, and endothelial progenitors; and c. Co-culture of MSCs, endothelial cells and endothelial progenitors harvested with primary CD138+ plasma cells from a patient with MM under conditions allowing the formation of spheroids.

[0010] The term "mesenchymal stem cells," also called "mesenchymal stromal cells," refers to stem cells of mesodermal origin. They are phenotypically characterized by the co-expression of a number of markers such as, for example, CD73, CD90, CD105, and CD146, and the absence of expression of other markers, in particular CD45, CD31, and CD34. They may be derived from bone marrow, adipose tissue, or umbilical cord blood. The mesenchymal stem or stromal cells are of human origin and come from a patient with MM or a healthy subject. Preferably, the mesenchymal stem / stromal cells cultured in step a are primary cells.

[0011] By "endothelial progenitors" we mean cells engaged in endothelial differentiation but which are not yet recognizable as endothelial cells under the microscope. They are phenotypically characterized by the expression of a number of markers such as, for example, CD133, CD34, CD31, VEGFR2.

[0012] By "endothelial cells" we mean cells that are fully differentiated in the endothelial pathway and therefore recognizable as endothelial cells under a microscope. They are phenotypically characterized by the expression of a number of markers such as, for example, CD31, VE-Cadherin, von Willebrand factor, VEGFR2.

[0013] Endothelial progenitors and endothelial cells have the ability to organize themselves into a network of endothelial cells, or vascular network, and thus to organize themselves into vessels.

[0014] In a preferred mode, the endothelial progenitors and endothelial cells cultured in step a) are primary cells. The endothelial progenitors and endothelial cells can, for example, be obtained from mononuclear cells of the bone marrow.

[0015] In one embodiment, MSCs, endothelial cells and endothelial progenitors were obtained from the same subject, i.e. from the same healthy subject or the same patient with MM. Preferably, MSCs, endothelial cells and endothelial progenitors were obtained from a single sample, in particular a bone marrow sample, from said healthy subject or patient with MM.

[0016] By “primary cell” is meant a cell directly derived from a tissue and / or from a cell sample taken from an individual.

[0017] The term “culture” refers to the selection and multiplication of cultured cells.

[0018] In one embodiment of the process, in step a), mesenchymal stem / stromal cells (MSCs), endothelial progenitors, and endothelial cells are cultured together in the same culture medium and, preferably, in the same culture container. After extraction from the raw bone marrow, the cells are seeded, for example, at a density of 50,000 cells / cm² in flasks. The three cell types coexist and proliferate in this culture.

[0019] In one embodiment, the culture takes place in two dimensions (2D), in the form of a monolayer that is at least partially adherent. The culture preferably proceeds until the cells confluence. The culture typically lasts from 3 to 30 days, preferably from 5 to 25 days, or alternatively from 10 to 20 days, 12 to 16 days, or 13 to 15 days, approximately 2 weeks.

[0020] The term "culture medium" refers to a medium suitable for culturing mesenchymal stem / stromal cells, endothelial progenitor cells, and endothelial cells. The culture medium is, for example, RPMI medium supplemented with 10% fetal bovine serum (FBS), minimal essential medium a (MEM a), or Endothelial Cell Growth Medium 2 (EGM2, from Promocell) supplemented with 2% FBS or platelet lysate (PL). It may be in various forms but is preferably liquid and allows the culture of eukaryotic cells, particularly mammalian cells and, more specifically, human cells.

[0021] According to the invention, the healthy subject or patient with MM is a human being. In some embodiments, the patient has just been diagnosed with MM. In some embodiments, the patient with MM is diagnosed as having a relapse.

[0022] Following culture step a), the cultured MSCs, endothelial cells, and endothelial progenitor cells are harvested. Harvesting is typically performed by trypsinization followed by washing. Other agents that allow the detachment of adherent cells without damage can replace trypsin.

[0023] The harvested MSCs, endothelial cells and endothelial progenitors are then co-cultured with CD 138+ primary plasma cells from an MM patient under conditions allowing the formation of spheroids.

[0024] The term “plasma cells” refers to immune system cells derived from bone marrow (BM) expressing the CD138+ marker. In the case of MM, plasma cells express the CD38+ and CD138+ markers.

[0025] By "spheroids," we mean a grouping of cells linked together in three dimensions. Preferably, a spheroid comprises from 500 to 750,000 cells, or even from 1,000 to 500,000 cells. The spheroids of the composition according to the invention have an average diameter of between 50 µm and 750 µm, preferably between 100 µm and 500 µm.

[0026] According to one embodiment, the primary CD138+ plasma cells are obtained from a patient with multiple myeloma (MM) other than the MM patient or healthy subject from whom the cultured MSCs, endothelial cells, and endothelial progenitor cells were obtained. The production process then allows the production of heterologous human multiple myeloma (MM) spheroids. The resulting heterologous human MM spheroid model allows the stroma of a patient or healthy subject to be combined with tumor plasma cells from another MM patient in order to study the impact of MM plasma cells on a healthy stroma and identify therapeutic targets. Conversely, healthy plasma cells can be combined with an MM stroma in order to study the impact of an MM stroma on healthy plasma cells and identify therapeutic targets.

[0027] According to another embodiment, MSCs, endothelial progenitor cells in CD138+ primary endothelial and plasma cells were obtained from the same patient with multiple myeloma (MM). The production process thus allows for the production of autologous human multiple myeloma (MM) spheroids. Since bone marrow harvesting is an invasive procedure, preferably, mesenchymal stem cells (MSCs), endothelial cells, endothelial progenitors, and CD138+ primary plasma cells were obtained from the same bone marrow sample of the aforementioned MM patient.

[0028] This embodiment presents the additional difficulty of successfully preserving the primary CD138+ plasma cells and maintaining their viability for the duration of the culture of MSCs, endothelial cells, and endothelial progenitors, i.e., for 3 to 30 days of culture and generally about two weeks. Indeed, in order to create autologous spheroids without resorting to a new bone marrow harvest from the MM patient, it is necessary to freeze the primary CD138+ plasma cells while ensuring optimal viability of these plasma cells during the subsequent spheroid culture.

[0029] Preferably, primary CD138+ plasma cells from the same patient sample as MSCs, endothelial cells, and endothelial progenitors were preserved, prior to co-culture, by freezing at a temperature of -70°C or lower, preferably -75°C or lower, or even -80°C, in a cryopreservation medium. This cryopreservation medium may be a medium consisting of 90% FBS + 10% DMSO (v / v), or 90% 4% human albumin solution + 10% DMSO (v / v), or commercial cryopreservation solutions such as Cryostor® CS 10 (Sigma-Aldrich C2874). Preferably, primary CD13 8+ plasma cells are frozen within a time interval not exceeding 2 hours after the isolation of primary CD138+ plasma cells from the bone marrow sample.

[0030] Co-culture of MSCs, endothelial cells, and endothelial progenitors with primary CD138+ plasma cells is performed in an ultra-low adhesion (ULA) plate to promote spheroid formation. Primary CD138+ plasma cells and MSCs are co-cultured in a number ratio of 1:1 to 4:1, preferably approximately 2:1. Co-culture is carried out for 4 to 14 days, for example, 4 to 10 days, preferably 6 to 8 days, or approximately 7 days.

[0031] The culture medium is for example an RPMI medium supplemented with 10% fetal bovine serum (FBS), a minimal essential medium a (MEM a), or preferably, an Endothelial Growth Medium 2 (EGM2, from Promocell) supplemented with 2% FBS or platelet lysate (PL).

[0032] The invention also relates to spheroids obtained or capable of being obtained by the above spheroid production process. These human multiple myeloma (MM) spheroids comprise a stroma, a vascular compartment, and plasma cells CD138+ of a patient with MM.

[0033] The spheroids are preferably autologous, obtained by co-culture of MSCs, endothelial cells and endothelial progenitors with primary CD138+ plasma cells from the same patient with MM, preferably from the same sample.

[0034] Spheroids can also be heterologous in the case where the primary CD 138+ plasma cells come from an MM patient different from the MM patient or healthy subject from whom the cultured MSCs, endothelial cells and endothelial progenitors were obtained.

[0035] Use of spheroids

[0036] The invention relates to the use of autologous multiple myeloma spheroids for the selection of a therapeutic treatment adapted to the patient with MM, that is to say for the selection of a therapeutic treatment to which a patient with multiple myeloma (MM) is likely to respond.

[0037] Indeed, constructing a 3D model of multiple myeloma (MM) that is as representative as possible of the patient's tumors allows for clinical monitoring and personalized medicine. The spheroid models, preferably autologous, according to the invention can be used to study the responses of the tumor tissue of the MM patient (the tissue from which the cells used to construct the spheroids are derived) to different treatments or combinations of treatments. Thus, the objective is to select a therapeutic treatment to which the patient is most likely to respond.

[0038] By "treatment" or "treat" is meant herein the achievement, in part or in part, of one or more of the following results: partially or totally reducing the extent of the disease, improving a clinical symptom or an indicator associated with the disease, delaying, inhibiting or preventing the progression of the disease, or partially or totally delaying, inhibiting or preventing the occurrence of a relapse of the disease.

[0039] The terms “subject”, “patient” or “sick person” here refer to a human being suffering from multiple myeloma.

[0040] Method for selecting a therapeutic treatment

[0041] The invention also includes a method for selecting a therapeutic treatment to which a patient with multiple myeloma (MM) is likely to respond using autologous spheroids derived from said patient. This selection method comprises:

[0042] - the culture of autologous spheroids from said patient in a culture medium in the presence of at least one drug candidate for the treatment of MM, for a period of at least 3 days,

[0043] - the harvesting of autologous spheroids and their dissociation so as to collect their myeloma plasma cells,

[0044] - analysis of the viability of the collected myeloma plasma cells; and

[0045] - the selection of said at least one drug candidate as a therapeutic treatment possible to which the MM patient is likely to respond, based on the measured viability of the collected myeloma plasma cells.

[0046] According to one embodiment, said at least one drug candidate is selected as a therapeutic treatment to which the MM patient is likely to respond if the measured viability of myeloma plasma cells is decreased compared to the viability of myeloma plasma cells obtained from autologous spheroids cultured under control conditions (i.e. without addition of drug candidate or with addition of a control buffer), or cultured in the presence of at least one other drug candidate.

[0047] The spheroid culture is carried out under the same conditions as defined previously in the process for producing autologous spheroids, except for the addition of at least one drug candidate. According to one embodiment, the selection process includes the preparation of autologous spheroids according to the process of the invention.

[0048] The MSCs, endothelial cells, endothelial progenitors and plasma cells constituting the autologous spheroids are derived from the same patient, preferably from the same bone marrow sample.

[0049] This selection method makes it possible to test the efficacy of a drug candidate, or a combination of drugs. The drug candidate, or the combination of drugs, is added to the spheroid culture medium between the time of spheroid formation and up to 48 hours after their formation, and the culture is continued for a period of at least 3 days, for example 4 to 10 days, preferably for 6 to 8 days or even about 7 days.

[0050] In parallel, a control culture is carried out, without the presence of the drug candidate or in the presence of buffer, under the same culture conditions as in the presence of said at least one drug candidate.

[0051] Examples of drugs or drug candidates that can be used for the treatment of MM include melphalan, lenalidomide, bortezomib, dexamethasone, C34 (compound of formula (I) as described in application WO 2018 / 115476 A1,

[0052] [Chem.l]

[0053] The spheroids are then collected and mechanically dissociated, for example in a thermomixer and / or by repeated aspiration and expulsion using a micropipette, with or without the aid of one or more chemical agent(s) such as trypsin, collagenase, or AccuMax dissociation solution (Capricom Scientific GmbH). In particular, the dissociation of spheroids can be carried out by incubating the spheroids in a dissociation solution (comprising a protease and / or collagenase, and preferably comprising a combination of protease, collagenase and DNase, for example AccuMax solution) under agitation (for example in a thermomixer at 37°C and at 1200-1500 rpm or about 1400 rpm, for about 10 min), then dissociating the spheroids by aspiration / discharge using a micropipette, and harvesting the dissociated cells (for example by centrifugation).

[0054] The cells are then labeled with markers to identify myeloma plasma cells, for example, with fluorochromes associated with specific antibodies such as CD38 to label plasma cells, and CD138 to identify CD38+ CD138+ myeloma plasma cells. The cells are then resuspended and filtered before being analyzed by flow cytometry (FACS). Fluorochrome markers associated with specific antibodies may be, for example, CD38 FITC and CD138 AF700. The suspension and labeling solution is preferably a MACS solution, and the cells are preferably filtered at 70 µm. The viability of the myeloma plasma cells is compared between the different culture conditions (control condition or with at least one drug candidate), with decreased viability of the myeloma plasma cells compared to the control being a sign of a promising treatment.

[0055] This method is faster than using murine models and provides a more relevant response due to the genetic similarity between the model and the patient, especially when the spheroidal model is autologous. Indeed, spheroids can be generated in approximately 2 weeks, on average, and the selection of a treatment adapted to the patient can be carried out in approximately 1 week from obtaining the spheroids, which represents a total of 3 weeks, as a general rule, to be able to define a personalized treatment for the patient with MM. Description of the figures

[0056] [Fig. 1] [Fig. 1] shows the viability rate of MM plasma cells within heterologous spheroids following their culture for 7 days with one or more drug candidates (melphalan 1 OpM, lenalidomide 1 OpM, C34 5pM, combination of melphalan 1 OpM + C34 5pM, combination of lenalidomide 1 OpM + C34 5pM) and than a control condition. On the x-axis, MM MSCs (MM91, MM97, MM100) represent the stromal component of the spheroid. MM plasma cells (P64 / 65, P66) are the plasmacytic component of the spheroid. Each number corresponds to a specific MM patient. Examples

[0057] Example 1: Studies of different plasma cell freezing protocols and cell viability study

[0058] In this example, the inventors sought to select the most effective plasma cell freezing / thawing protocol for preserving plasma cell viability. They studied this impact by quantifying their viability after thawing using trypan blue counting with a Malas sez cell.

[0059] Following a whole bone marrow biopsy at diagnosis in patients with multiple myeloma (MM), MM plasma cells are separated and then counted using Malassez counting chambers. They were then frozen according to one of the following protocols: - A: Buffer 90% fetal bovine serum (FBS): 10% DMSO;

[0060] immediate freezing -80°C - B: 90% human serum albumin buffer (HSA): 10% DMSO;

[0061] immediate freezing -80°C - C: CryoStor® CS 10 buffer (Sigma-Aldrich, product reference C2874); immediate freezing at -80°C - D: CryoStor® CS 10 buffer;

[0062] freezing -20°C for 2 hours then -80°C - E: CryoStor® CS 10 buffer;

[0063] freezing -20°C overnight then -80°C

[0064] The freezing time was 31 days on average.

[0065] The plasma cells were then thawed and counted in trypan blue using a Malassez cell.

[0066] [Tables 1] Condition Ratio of live plasma cells after thawing to live plasma cells before freezing (%) Average ratio of viable plasma cells to plasma cells counted after thawing (%) Number of tests A 42.61 60.78 5 B 31.04 61.85 7 C 41.39 71.51 15 D 47.32 69.25 4 E 40.58 66.80 5

[0067] The results in the table above show that the usual freezing conditions, namely 90% SVF or HSA + 10% DMSO, appear to be less effective compared to immediate freezing conditions with CryoStor®.

[0068] Example 2: Culture of heterologous and autologous spheroids and cell viability study by flow cytometry

[0069] In this example, the inventors sought to demonstrate the minimal impact on plasma cell viability of the freezing and thawing, culture within spheroids, and spheroid dissociation steps prior to labeling. They investigated this impact by quantifying viability using flow cytometry. To this end, they labeled cells with anti-CD38 and anti-CD138 antibodies coupled to fluorochromes to specifically select MM plasma cells.

[0070] Following whole bone marrow sampling at diagnosis in patients with multiple myeloma (MM), MM plasma cells are separated and then counted in Malassez counting chambers. They are then frozen in a cryostore or used directly fresh for heterologous co-cultures. From another patient, MM whole bone marrow cells are seeded according to their initial number and incubated at 37°C in a 5% carbon dioxide atmosphere for approximately two weeks. Once confluence is achieved, mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitors are treated with trypsin and counted.

[0071] On ULA (Ultra-Low Adherence) plates, MSCs, endothelial cells and endothelial progenitors treated with trypsin, and fresh or thawed plasma cells are suspended in 50pL of RPMI medium (10% FBS, 1% PS) in a ratio of 1 MSC to 2 plasma cells. The cells are then incubated at 37°C in a 5% carbon dioxide atmosphere with shaking. 150pL of complete RPMI medium are added after 24h of incubation and the medium is renewed twice a week by removing 100pL of culture supernatant and adding 100pL of complete RPMI medium.

[0072] Spheroid cells are mechanically dissociated at 1400 rpm with AccuMax® and then transferred to a tube suitable for flow cytometry. They are washed with PBS and labeled with anti-CD38 FITC and anti-CD138 AF700 antibodies in MACS buffer. The cells are then incubated at 4°C for 30 minutes, washed, resuspended in MACS buffer, filtered at 70 rpm, labeled with DAPI, and then analyzed by flow cytometry.

[0073] The inventors observed that the quantification of the viability of MM plasma cells was possible with this protocol and that the viability of MM plasma cells remains high after the manipulations of this protocol, even after 14 days of co-culture.

[0074] Example 3: 3D spheroids with plasma cells from a patient with multiple myeloma and reaction to treatment with melphalan

[0075] In this example, the inventors sought to demonstrate the viability of plasma cells within spheroids as well as their accessibility for the tested therapeutic molecules.

[0076] Following whole bone marrow sampling at diagnosis in patients with multiple myeloma (MM), MM plasma cells are separated and then counted in Malas sez cells. They are then used fresh for co-cultures or frozen in a cryostore. MM whole marrow cells are seeded according to their initial number and incubated at 37°C in a 5% carbon dioxide atmosphere for approximately 2 weeks. Once confluence is achieved, mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitors are treated with trypsin and counted.

[0077] On ULA (Ultra-Low Adherence) plates, trypsin-treated MSCs, endothelial cells, and endothelial progenitors, and fresh or thawed plasma cells are suspended in 50 pL of RPMI medium (10% FBS, 1% PS). The cells are then incubated at 37°C in a 5% carbon dioxide atmosphere with shaking. 150 pL of RPMI medium is added after 24 hours of incubation, and the medium is renewed twice a week by removing 100 pL of supernatant.

[0078] Melphalan 1Opm is added to the culture medium of the spheroids 48 hours after their formation, and the culture is continued for 14 days. This selection process is complemented by a control condition, without the presence of the drug candidate. Some cultures are stopped after 7 (D+7) and 11 (D+1) days to analyze the viability of the plasma cells, while the remaining cultures are stopped after 14 days of culture (D+14).

[0079] Spheroid cells are mechanically dissociated at 1400 rpm with AccuMax® and then transferred to a tube suitable for flow cytometry. They are washed with PBS and labeled with the specific antibodies CD38 FITC and CD138 AF700 in MACS buffer. The cells are then incubated at 4°C for 30 minutes, washed and resuspended in MACS buffer, filtered at 70 µm, and finally labeled with the viability marker DAPI before being analyzed by flow cytometry.

[0080] The viability of untreated MM spheroid plasma cells increases from 50% at J+7 and J+11 to 60% at J+14, whereas MM spheroid plasma cells treated with melphalan OpM have a viability of 5% at J+7, less than 5% at J+11 and less than 10% at J+14.

[0081] Firstly, the inventors therefore show that the viability of plasma cells in untreated spheroids is greater (J7, J11, J14) than in the case of 2D cultures where primary plasma cells do not survive beyond a few days.

[0082] Next, the inventors show that the localization of plasma cells within spheroids does not prevent a strong response to treatment (here melphalan lOpM).

[0083] Example 4: Response of heterologous MM spheroids to different drug candidates

[0084] Following whole bone marrow sampling at diagnosis in patients with multiple myeloma (MM), MM plasma cells are separated and then counted in Malassez counting chambers. They are then used fresh or frozen at -80°C in a cryostore. From the same or a different patient, MM whole marrow cells are seeded according to their initial number and incubated at 37°C in a 5% carbon dioxide atmosphere for approximately two weeks. Once confluence is achieved, mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitors are treated with trypsin and counted.

[0085] On ULA (Ultra-Low Adherence) plates, trypsin-treated MSCs, endothelial cells, and endothelial progenitors, and fresh or thawed plasma cells are suspended in 50 pL of RPMI medium (10% FBS, 1% PS). The cells are then incubated at 37°C in a 5% carbon dioxide atmosphere with shaking. 150 pL of RPMI medium is added after 24 hours of incubation, and the medium is renewed twice a week by removing 100 pL of culture supernatant and adding 100 pL of RPMI medium.

[0086] The drug candidate, the candidate combination, or the combination of candidates are added to the spheroid culture medium between the time of their formation and 48 hours after their formation, and the culture is continued for 7 days. This process of selection is complemented by a control situation, without the presence of the drug candidate, with the same culture duration as in the presence of the drug candidate.

[0087] The treatments tested are as follows: - melphalan lOpM, - lenalidomide lOpM, C34 5pM, - combination melphalan 10pM + C34 5pM, - combination lenalidomide 1 OpM + C34 5pM

[0088] Spheroid cells are mechanically dissociated at 1400 rpm with AccuMax® and then transferred to a tube suitable for flow cytometry, washed with PBS, and labeled with the specific antibodies CD38 FITC and CD138 AF700 in MACS buffer. The cells are then incubated at 4°C for 30 minutes, washed, resuspended in MACS buffer, filtered at 70 µm, and finally labeled with DAPI before flow cytometry.

[0089] Figure 1 shows the viability rate of MM plasma cells within heterologous spheroids following their culture for 7 days with the various potential treatments tested, as well as a control condition. On the x-axis, MM MSCs (MM91, MM97, MM100) represent the stromal component of the spheroid. MM plasma cells (P64 / 65, P66) are the plasmacytic component of the spheroid. Each number corresponds to a given MM patient.

[0090] Fig. 1 clearly shows the difference in behavior of plasma cells under different conditions and the effectiveness of combined treatments melphalan + C34 and lenalidomide + C34 to decrease the viability of MM plasma cells.

Claims

Demands

1. A method for producing human multiple myeloma (MM) spheroids, comprising: a. Culturing mesenchymal stem / stromal cells (MSCs), endothelial cells and endothelial progenitors in a culture medium; b. Harvesting the cultured MSCs, endothelial cells and endothelial progenitors; and c. Co-culturing the harvested MSCs, endothelial cells and endothelial progenitors with CD13 8+ primary plasma cells from an MM patient under conditions permitting spheroid formation.

2. A production method according to claim 1, wherein MSCs, endothelial cells, endothelial progenitors and CD 138+ primary plasma cells were obtained from the same patient with MM.

3. A production method according to claim 1 or 2, wherein MSCs, endothelial cells, endothelial progenitors and CD 138+ primary plasma cells were obtained from the same bone marrow sample of said patient with MM.

4. A production method according to any one of claims 1 to 3, wherein MSCs, endothelial cells and endothelial progenitors are cultured in step a. for 3 to 30 days.

5. A production method according to any one of claims 1 to 4, wherein primary CD138+ plasma cells were preserved, prior to co-culture, by freezing at a temperature less than or equal to -70°C in a cryopreservation medium.

6. A production method according to any one of claims 1 to 5, wherein primary CD138+ plasma cells and MSCs are co-cultured with a ratio of approximately 2:

1.

7. A production method according to any one of claims 1 to 6, wherein the co-culture of MSCs, endothelial cells and endothelial progenitors with primary plasma cells is carried out for 4 to 14 days.

8. Human multiple myeloma (MM) spheroids obtained by a process production according to any one of claims 1 to 7, comprising a stroma, a vascular compartment and CD138+ plasma cells from patient(s) with MM.

9. MM spheroids according to claim 8, wherein said spheroids are autologous spheroids.

10. Use of autologous multiple myeloma (MM) spheroids as defined in claim 9 for the selection of a therapeutic treatment to which a patient with MM is likely to respond.

11. A method for selecting a therapeutic treatment to which a patient with multiple myeloma (MM) is likely to respond, comprising: a. Culturing autologous spheroids according to claim 9, in a culture medium in the presence of at least one drug candidate for the treatment of MM, for a period of at least 3 days; b. Harvesting the autologous spheroids and dissociating them so as to collect the myeloma plasma cells present in the autologous spheroids; c. Analyzing the viability of the collected myeloma plasma cells; and d. Selecting said at least one drug candidate as a therapeutic treatment to which the patient with MM is likely to respond, based on the measured viability of the collected myeloma plasma cells.

12. A method for selecting a therapeutic treatment according to claim 11, wherein said at least one drug candidate is selected as a therapeutic treatment to which the MM patient is likely to respond if the measured viability of myeloma plasma cells is decreased compared to the viability of myeloma plasma cells obtained from autologous spheroids cultured under control conditions or cultured in the presence of at least one other drug candidate.

13. A method for selecting a therapeutic treatment according to claim 11 or 12, wherein said at least one drug candidate is added to the culture medium of autologous spheroids between the time of spheroid formation and up to 48 hours after their training, and culture is continued during said period of at least 3 days.

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