Method for producing a three-dimensional human multiple-myeloma model

Abstract

The present invention relates to the method for producing a three-dimensional (3D) model of multiple myeloma (MM), in the form of spheroids, by co-culturing stem cells / mesenchymal stromal cells, endothelial progenitors and primary plasma cells of one or more MM patients. The present invention also relates to the spheroids obtained by said method, and uses thereof.

Description

[0001] TITLE: Method for producing a three-dimensional human multiple myeloma model

[0002] The present invention relates to the method for producing a three-dimensional (3D) model of multiple myeloma (MM), in the form of spheroids, by co-cultivating mesenchymal stem / stromal cells, endothelial progenitors and primary plasma cells from patient(s) suffering from MM. The present invention also relates to the spheroids obtained by said method and their uses.

[0003] Multiple myeloma (MM) is a malignant hematological disease, also known as 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 (BM) that produce antibodies to protect the body against external attacks (bacteria, viruses). During development, genetic abnormalities (deletion, chromosomal translocation) can occur, transforming healthy plasma cells into malignant plasma cells. In the normal state, these plasma cells circulate in the blood, whereas in MM pathology they return to the bone marrow where they cause damage at several levels.

[0004] Multiple myeloma remains an incurable disease despite recent remarkable therapeutic advances. Treatment currently prevents or alleviates symptoms and complications by destroying pathological plasma cells and slowing disease progression.

[0005] To date, multiple myeloma suffers from the lack of relevant preclinical models. Indeed, mouse models are expensive, time-consuming, and not representative of human pathology. Standard two-dimensional (2D) models do not allow 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.

[0006] Human adult ex vivo bone marrow reproduction is increasingly described in the literature to overcome animal models that are costly, time-consuming, and species-dependent. Studies have begun to demonstrate 3D ex vivo human bone marrow models combining the mesenchymal and vascular compartments, generally from lineage-derived cells. For example, the vascular compartment, playing an active role in hematopoietic stem cell (HSC) proliferation, is often incorporated via HlIVEC endothelial cell lines. Other models require a step in mice to allow vascularization or functional approaches.Furthermore, the short half-life of plasma cells does not allow their autologous incorporation into current 3D models and requires ignoring them or adding tumor plasma cell lines, which does not allow a relevant response of the model compared to the patient.

[0007] The inventors have created a novel human 3D tissue model of multiple myeloma. It includes the mesenchymal compartment, the vascular compartment, and plasma cells, obtained from sample(s) of multiple myeloma patient(s), without the use of a cell line. In particular, the maintenance of plasma cell viability in coculture for more than 14 days has been resolved. This allows the generation of a fully human preclinical ex vivo model of multiple myeloma, genetically relevant to said patient, and comprising the mesenchymal, vascular, and plasma cell compartments in spheroid form.

[0008] This model allows us to envisage a breakthrough in personalized medicine thanks to the rapid creation of a representative model of the tumor tissues of the bone marrow of each patient.

[0009] Detailed description of the invention

[0010] Process for producing human multiple myeloma spheroids

[0011] The invention provides a method for producing human multiple myeloma (MM) spheroids, comprising: a. Cultivating 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 CD138+ primary plasma cells from a patient with MM under conditions allowing for spheroid formation.

[0012] By "mesenchymal stem cells", also called "mesenchymal stromal cells", we mean stem cells of mesodermal origin. They are phenotypically characterized by the co-expression of a certain number of markers such as for example CD73, CD90, CD105, CD146, and the absence of expression of other markers, in particular CD45, CD31 and CD34. They can 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 suffering from MM or a healthy subject. In a preferred mode, the mesenchymal stem / stromal cells cultured in step a. are primary cells.

[0013] Endothelial progenitors are cells that are engaged in endothelial differentiation but are not yet recognizable as endothelial cells under the microscope. They are characterized phenotypically by the expression of a number of markers such as CD133, CD34, CD31, VEGFR2.

[0014] Endothelial cells are cells that are fully differentiated in the endothelial pathway and therefore recognizable as endothelial cells under the microscope. They are phenotypically characterized by the expression of a number of markers such as CD31, VE-Cadherin, von Willebrand factor, VEGFR2.

[0015] Endothelial progenitors and endothelial cells have the ability to organize themselves into endothelial cell networks, or vascular networks, and therefore to organize themselves into vessels.

[0016] In a preferred embodiment, 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 bone marrow mononuclear cells.

[0017] In one embodiment, the MSCs, endothelial cells and endothelial progenitors were obtained from the same subject, i.e., from the same healthy subject or from the same patient with MM. Preferably, the MSCs, endothelial cells and endothelial progenitors were obtained from a single or a single sample, in particular a bone marrow sample, from said healthy subject or patient with MM.

[0018] By "primary cell" we mean a cell directly derived from a tissue and / or from a cell sample from an individual.

[0019] The term "culture" refers to the selection and multiplication of cultured cells.

[0020] In one embodiment of the method, in step a), the mesenchymal stem / stromal cells (MSCs), endothelial progenitors and endothelial cells are co-cultured in the same culture medium and, preferably, in the same culture container. After extraction of the raw marrow, the cells are seeded for example at a density of 50,000 cells / cm 2 in flasks. All three cell types coexist and proliferate in this culture.

[0021] In one embodiment, the culture takes place in 2 dimensions (2D), in the form of an at least partially adherent monolayer. The culture preferably takes place until the cells reach confluence. The culture typically lasts for 3 to 30 days, preferably for 5 to 25 days, or alternatively for 10 to 20 days, 12 to 16 days, 13 to 15 days, or approximately 2 weeks.

[0022] Preferably, cells are not cultured in the presence of a hydrogel or solid support (ossified tissue or other scaffold).

[0023] A "hydrogel" is a gel in which the swelling agent is water. The matrix of a hydrogel is generally a network of polymers. Examples of hydrogels include Matrigel, which can be made from fibrin, collagen, agarose, gelatin, synthetic polymer, or a mixture of these.

[0024] Preferably, cells are not cultured with an exogenous supply of complex biomolecules (e.g., cytokine, growth factors, hormones).

[0025] “Culture medium” means a medium suitable for the culture of mesenchymal stem / stromal cells, endothelial progenitors and endothelial cells. The culture medium is, for example, RPMI medium supplemented with 10% fetal calf serum (FCS), minimal essential medium a (MEM a), or Endothelial cell Growth Medium 2 (EGM2, from Promocell) supplemented with 2% FCS or platelet lysate (PL). It can be in different forms but is preferably liquid and allows the culture of eukaryotic cells, in particular mammalian cells and more particularly human cells.

[0026] According to the invention, the healthy subject or patient suffering from MM is a human being. According to certain embodiments, the patient has just been diagnosed as suffering from MM. According to certain embodiments, the patient suffering from MM is diagnosed as having relapsed.

[0027] At the end of culture step a), the cultured MSCs, endothelial cells, and endothelial progenitors are harvested. Harvesting is typically done by trypsinization and subsequent washing. Other agents that allow the detachment of adherent cells without damage can replace trypsin.

[0028] Harvested MSCs, endothelial cells, and endothelial progenitors are then co-cultured with CD138+ primary plasma cells from an MM patient under conditions that allow spheroid formation. “Plasma cells” are bone marrow (BM)-derived immune system cells expressing the CD138+ marker. In MM, plasma cells express both CD38+ and CD138+ markers.

[0029] By "spheroids" is meant a grouping of cells linked together in the three dimensions of space. Preferably a spheroid comprises from 500 to 750,000 cells, or 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.

[0030] Preferably, cells are not cultured in the presence of a hydrogel or solid support (ossified tissue or other scaffold).

[0031] Preferably, cells are not cultured with an exogenous supply of complex biomolecules (e.g. cytokine, growth factors, hormones, etc.).

[0032] Indeed, the spheroids according to the invention are formed by self-organization of MSCs, endothelial cells and endothelial progenitors with plasma cells. The spheroids according to the invention are therefore formed without hydrogel, support or exogenous supply of complex biomolecule. This approach makes it possible to limit bias and to get closer to what is observed in vivo. The use of hydrogel, support or exogenous supply of complex biomolecule can alter the behavior of certain products and thus lead to underestimation or overestimation of the action potential of these products in vivo.

[0033] According to one embodiment, the CD138+ primary plasma cells originate from a patient with MM different from the patient with MM, or the healthy subject, from which the cultured MSCs, endothelial cells and endothelial progenitors were obtained. The production method then allows the production of heterologous human multiple myeloma (MM) spheroids. The resulting heterologous human MM spheroid model allows the combination of a stroma from a patient or healthy subject with the tumor plasma cells from another patient with MM in order to study the impact of MM plasma cells on a healthy stroma and reveal therapeutic targets. Conversely, healthy plasma cells can be associated with an MM stroma in order to study the impact of an MM stroma on healthy plasma cells and reveal therapeutic targets.

[0034] According to another embodiment, the MSCs, endothelial cells, endothelial progenitors and CD138+ primary plasma cells were obtained from the same patient suffering from MM. The production method then allows the production of autologous human multiple myeloma (MM) spheroids. Since bone marrow collection is an invasive procedure, preferably, the MSCs, endothelial cells, endothelial progenitors and CD138+ primary plasma cells were obtained from the same bone marrow collection of said patient suffering from MM.

[0035] This embodiment presents the additional difficulty of successfully preserving the primary CD138+ plasma cells, and preserving their viability, for the duration of the culture of the MSCs, endothelial cells, and endothelial progenitors, i.e. for 3 to 30 days of culture and generally around two weeks. Indeed, to be able to constitute autologous spheroids without resorting to a new bone marrow harvest from the patient suffering from MM, it is necessary to freeze the primary CD138+ plasma cells while ensuring optimal viability of these plasma cells during the subsequent culture of the spheroids.

[0036] Thus, preferably, the primary CD138+ plasma cells from the same patient sample as the MSCs, endothelial cells, and endothelial progenitors were preserved, before co-culture, by freezing at a temperature lower than or equal to -70°C, preferably lower than or equal to -75°C or even -80°C, in a cryopreservation medium. This cryopreservation medium may be a medium consisting of 90% FCS + 10% DMSO (v / v), or 90% 4% human albumin solution + 10% DMSO (v / v), or commercial cryopreservation solutions such as, for example, Cryostor® CS10 (Sigma-Aldrich C2874). Preferably, freezing of the CD138+ primary plasma cells is carried out within a time interval not exceeding 2 hours after isolation of the CD138+ primary plasma cells from the bone marrow sample.

[0037] Co-culture of MSCs, endothelial cells and endothelial progenitors with CD138+ primary plasma cells is carried out in ULA (Ultra-Low Adherence) plates to promote spheroid formation. The CD138+ primary plasma cells and MSCs are co-cultured with a ratio, in number, of 1:1 to 4:1, preferably a ratio of approximately 2:1. The co-culture is carried out for 4 to 14 days, for example 4 to 10 days, preferably for 6 to 8 days or approximately 7 days. Preferably, the co-culture step is carried out with shaking, preferably gentle shaking.

[0038] The culture medium is, for example, RPMI medium supplemented with 10% fetal calf serum (FCS), minimal essential medium a (MEM a), or preferably, Endothelial Growth Medium 2 (EGM2, from Promocell) supplemented with 2% FCS or platelet lysate (PL). Preferably, the cells are not cultured with an exogenous supply of complex biomolecules (e.g., cytokine, growth factors, hormones, etc.).

[0039] The invention also relates to spheroids obtained or capable of being obtained by the above spheroid production method. These human multiple myeloma (MM) spheroids comprise a stroma, a vascular compartment and CD138+ plasma cells from a patient suffering from MM.

[0040] Spheroids are preferably autologous, obtained by co-culture of MSCs, endothelial cells and endothelial progenitors with CD138+ primary plasma cells from the same patient with MM, preferably from the same sample.

[0041] Spheroids can also be heterologous in the case where the primary CD138+ plasma cells are derived from an MM patient different from the MM patient or healthy subject from which the cultured MSCs, endothelial cells, and endothelial progenitors were obtained.

[0042] Use of spheroids

[0043] The subject of the invention is the use of autologous multiple myeloma spheroids for the selection of a therapeutic treatment adapted to the patient suffering from MM, that is to say for the selection of a therapeutic treatment to which a patient suffering from multiple myeloma (MM) is likely to respond.

[0044] Indeed, the construction of a 3D model of 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 patient suffering from MM (the one from which the cells used to constitute the spheroids come) 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.

[0045] By "treatment" or "treat" is meant herein the achievement, partially or substantially, of one or more of the following results: partially or totally reducing the extent of the disease, improving a clinical symptom or 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.

[0046] By “subject”, “patient” or “patient” we mean a human being suffering from multiple myeloma.

[0047] Process for selecting a therapeutic treatment

[0048] 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 from said patient. This selection method includes:

[0049] - the culture of autologous spheroids of 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,

[0050] - the harvesting of autologous spheroids and their dissociation in order to collect their myeloma plasma cells,

[0051] - analysis of the viability of the collected myeloma plasma cells; and

[0052] - the selection of said at least one drug candidate as a therapeutic treatment to which the patient suffering from MM is likely to respond, on the basis of the measured viability of the collected myeloma plasma cells.

[0053] According to one embodiment, said at least one drug candidate is selected as a therapeutic treatment to which the patient suffering from MM is likely to respond if the measured viability of the myeloma plasma cells is reduced compared to the viability of myeloma plasma cells obtained from autologous spheroids cultured under control conditions (i.e. without the addition of a drug candidate or with the addition of a control buffer), or cultured in the presence of at least one other drug candidate.

[0054] The spheroids are cultured under the same conditions as previously defined in the method for producing autologous spheroids, except for the addition of said at least one drug candidate. According to one embodiment, the selection method includes the preparation of autologous spheroids according to the method of the invention.

[0055] 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.

[0056] This screening method is used 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 approximately 7 days.

[0057] In parallel, a control culture is carried out, without the presence of drug candidate or in the presence of buffer, under the same culture conditions as in the presence of said at least one drug candidate. 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,

[0058] [Chem 1]

[0059] The spheroids are then harvested and mechanically dissociated, for example in a thermomixer and / or by repeated suction and discharge using a micropipette, with or without the aid of one or more chemical agents such as trypsin, collagenase, or AccuMax dissociation solution (Capricorn Scientific GmbH). In particular, the dissociation of the 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 the AccuMax solution) with stirring (for example in a thermomixer at 37°C and at 1200-1500 rpm or approximately 1400 rpm, for approximately 10 min), then dissociating the spheroids by suction / discharge using a micropipette, and harvesting the dissociated cells (for example by centrifugation).

[0060] 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 in particular, and CD138 to identify CD38+ CD138+ myeloma plasma cells. The cells are then resuspended and filtered before being analyzed by flow cytometry (FACS). The fluorochrome markers associated with specific antibodies can 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 70pm. The viability of the myeloma plasma cells is compared between the different culture conditions (control condition or with at least one drug candidate), a reduced viability of the myeloma plasma cells compared to the control being the sign of a promising treatment.

[0061] This process is faster than using mouse models and provides a more relevant response due to the genetic proximity between the model and the patient, especially when the spheroidal model is autologous. In fact, spheroids can be generated in about 2 weeks, on average, and the selection of a treatment adapted to the patient can be carried out in about 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.

[0062] Description of figures

[0063] [Fig 1 ] Figure 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 10pM, lenalidomide 10pM, C34 5pM, combination melphalan 10pM + C34 5pM, combination lenalidomide 10pM + C34 5pM) 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 plasma cell component of the spheroid. Each number corresponds to a given MM patient.

[0064] [Fig 2] Figure 2 shows the rate of live MM plasma cells within spheroids following their culture for 7 days with one or more drug candidates (Carfilzomib 5-50nM, Pomalidomide 1 -1 OpM, Dexamethasone 1 pM, Isatuximab 1 pg / ml, Daratumumab 1 pg / ml) as well as a control condition (NT). On the abscissa, the different treatments tested:

[0065] DCD: Daratumumab / Carfilzomib / Dexamethasone,

[0066] DPD: Daratumumab / Pomalidomide / Dexamethasone,

[0067] ICD: Isatuximab / Carfilzomib / Dexamethasone,

[0068] IPD: Isatuximab / Pomalidomide / Dexamethasone,

[0069] CD: Carfilzomib / Dexamethasone,

[0070] PD: Pomalidomide / Dexamethasone).

[0071] Examples

[0072] Example 1: Studies of different plasma cell freezing protocols and cell viability studies

[0073] In this example, the inventors sought to select a plasma cell freezing / thawing protocol that was most effective for preserving plasma cell viability. They studied this impact by quantifying their viability after thawing using trypan blue counting on Malassez cells.

[0074] Following a total bone marrow sample at diagnosis in patients affected by multiple myeloma (MM), MM plasma cells are separated and then counted as Malassez cells. They were then subjected to freezing according to one of the following protocols:

[0075] A: 90% fetal calf serum (FCS) buffer: 10% DMSO; freezing -80°C immediately

[0076] B: Buffer 90% human serum albumin (HSA): 10% DMSO; freezing -80°C immediately

[0077] - C: CryoStor® CS10 buffer (Sigma-Aldrich, product reference C2874); immediate freezing at -80°C

[0078] D: CryoStor® CS 10 buffer; freezing at -20°C for 2 hours then -80°C

[0079] E: CryoStor® CS10 buffer; freeze at -20°C overnight then -80°C

[0080] The freezing time was 31 days on average.

[0081] The plasma cells were then thawed and counted using trypan blue in Malassez cells.

[0082] [Table 1]

[0083] The results in the table above show that common freezing conditions, namely 90% FCS or HSA + 10% DMSO, appear less effective compared to immediate freezing conditions with CryoStor®.

[0084] Example 2: Culture of heterologous and autologous spheroids and study of cell viability by flow cytometry

[0085] In this example, the inventors sought to demonstrate the low impact on plasma cell viability of the freezing and thawing steps, culture within spheroids, and dissociation of the spheroids before labeling. They studied this impact by quantifying their viability using flow cytometry. To do this, they sought to label the cells with anti-CD38 and anti-CD138 antibodies coupled with fluorochromes to specifically select MM plasma cells.

[0086] Following a total bone marrow collection at diagnosis in patients affected by multiple myeloma (MM), MM plasma cells are separated and then counted in Malassez cells. They are then frozen in CRYOSTOR or used directly fresh for heterologous cocultures. From another patient, MM total marrow cells are seeded according to their initial number and the cells are incubated at a temperature of 37°C and under an atmosphere with 5% carbon dioxide for approximately 2 weeks. Once confluence is reached, mesenchymal stem / stromal cells (MSCs), endothelial cells and endothelial progenitors are treated with trypsin and counted.

[0087] On ULA (Ultra-Low Adherence) plates, trypsin-treated CSM, endothelial cells and endothelial progenitors and fresh or thawed plasma cells are suspended in 50 μL of RPMI medium (10% FCS, 1% PS) in a ratio of 1 CSM to 2 plasma cells. The cells are then incubated at a temperature of 37°C and an atmosphere with 5% carbon dioxide with shaking. 150 μL of complete RPMI medium is added after 24 hours of incubation and the medium is renewed twice a week by removing 100 μL of culture supernatant and adding 100 μL of complete RPMI medium.

[0088] Spheroid cells are mechanically dissociated at 1400rpm with AccuMax® and then transferred to a flow cytometry tube and 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 and resuspended in MACS buffer medium and filtered at 70pm, labeled with DAPI before undergoing flow cytometry.

[0089] The inventors observed that quantification of MM plasma cell viability was possible with this protocol and that MM plasma cell viability remains high after the manipulations of this protocol, even after 14 days of co-culture.

[0090] Example 3: 3D spheroids with plasma cells from a patient with multiple myeloma and response to treatment with melphalan

[0091] In this example, the inventors sought to demonstrate the viability of plasma cells within the spheroids as well as their accessibility for the therapeutic molecules tested. Following a total bone marrow sample at diagnosis in patients affected by multiple myeloma (MM), the MM plasma cell cells are separated and then counted in Malassez cells. They are then used freshly for cocultures or frozen in CRYOSTOR. The MM total marrow cells are seeded according to their initial number and the cells are incubated at a temperature of 37°C and under an atmosphere with 5% carbon dioxide for approximately 2 weeks. Once confluence is reached, the mesenchymal stem / stromal cells (MSCs), endothelial cells and endothelial progenitors are treated with trypsin and counted.

[0092] On ULA (Ultra-Low Adherence) plates, trypsin-treated MSCs, endothelial cells and endothelial progenitors, and fresh or thawed plasma cells are suspended in 50 μL of RPMI medium (10% FCS, 1% PS) in a ratio of 1 MSC to 2 plasma cells. The cells are then incubated at a temperature of 37°C and an atmosphere with 5% carbon dioxide with shaking. 150 μL of RPMI medium is added after 24 hours of incubation and the medium is renewed twice a week by removing 100 μL of supernatant.

[0093] A 10 pm melphalan treatment is added to the spheroid culture medium 48 hours after their formation and the culture is continued for 14 days. This selection process is completed by a control situation, without the presence of drug candidate. Part of the cultures is stopped after 7 (D+7) and 11 (D+11) days to analyze the viability of the plasma cells while the rest of the cultures is stopped after 14 days of culture (D+14).

[0094] Spheroid cells are mechanically dissociated at 1400rpm with AccuMax® and then transferred into a flow cytometry tube and 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 medium and filtered at 70pm, and finally labeled with the viability marker DAPI, before undergoing flow cytometry.

[0095] The viability of plasma cells in untreated MM spheroids increased from 50% at D+7 and D+1 to 60% at D+14, whereas plasma cells in MM spheroids treated with 10 pM melphalan had a viability of 5% at D+7, less than 5% at D+1 and less than 10% at D+14.

[0096] Firstly, the inventors show that the viability of plasma cells in untreated spheroids is greater (D7, D11, D14) than in the case of 2D cultures where the primary plasma cells do not survive beyond a few days.

[0097] Then, the inventors show that the localization of plasma cells within the spheroids does not prevent a strong response to treatment (here melphalan 10 pM). Example 4: Response of heterologous MM spheroids to different drug candidates

[0098] Following a total bone marrow collection at diagnosis in patients affected by multiple myeloma (MM), MM plasma cells are separated and then counted in Malassez cells. They are then used fresh or frozen at -80°C in CRYOSTOR. From the same or a different patient, the MM total marrow cells are seeded according to their initial number and the cells are incubated at a temperature of 37°C and under an atmosphere with 5% carbon dioxide for approximately 2 weeks. Once confluence is reached, mesenchymal stem / stromal cells (MSCs), endothelial cells and endothelial progenitors are treated with trypsin and counted.

[0099] On ULA (Ultra-Low Adherence) plates, trypsin-treated MSCs, endothelial cells and endothelial progenitors, and fresh or thawed plasma cells are suspended in 50 μL of RPMI medium (10% FCS, 1% PS) in a ratio of 1 MSC to 2 plasma cells. The cells are then incubated at a temperature of 37°C and an atmosphere with 5% carbon dioxide with shaking. 150 μL of RPMI medium is added after 24 hours of incubation and the medium is renewed twice a week by removing 1 OopL of culture supernatant and adding 1 OopL of RPMI medium.

[0100] The drug candidate, the candidate combination or the combination of candidates are added to the culture medium of the spheroids between the time of their formation and 48 hours after their formation and the culture is continued for 7 days. This selection process is completed by a control situation, without the presence of the drug candidate, of the same culture duration as in the presence of the drug candidate.

[0101] The treatments tested are as follows:

[0102] - melphalan 10pM, lenalidomide 10pM,

[0103] - C34 5pM,

[0104] - combination melphalan 10pM + C34 5pM, combination lenalidomide 10pM + C34 5pM

[0105] The spheroid cells were mechanically dissociated at 1400rpm with AccuMax® and then transferred into a flow cytometry-adapted tube, washed with PBS, and labeled with the specific antibodies CD38 FITC and CD138 AF700 in MACS buffer. The cells were then incubated at 4°C for 30 minutes, washed and resuspended in MACS buffer medium and filtered at 70pm, and finally labeled with DAPI before undergoing flow cytometry. Figure 1 shows the viability rate of MM plasma cells within heterologous spheroids following their culture for 7 days with the different potential treatments tested as well as a control condition. On the x-axis, the MM MSCs (MM91, MM97, MM100) represent the stroma component of the spheroid. MM plasma cells (P64 / 65, P66) are the plasma cell component of the spheroid. Each number corresponds to a given MM patient.

[0106] Figure 1 clearly shows the difference in plasma cell behavior under different conditions and the effectiveness of the combined treatments melphalan + C34 and lenalidomide + C34 in reducing the viability of MM plasma cells.

[0107] Example 5: Response of spheroids to different drug candidates and combination of candidates

[0108] Whole marrow cells from patients with multiple myeloma (MM) are first seeded into flasks in EGM2 medium according to their starting number in the tube. The cells are incubated at 37°C and 5% CO2 for approximately 2 weeks.

[0109] Plasma cells from patients with multiple myeloma (MM) are first enumerated using Malassez cells and Trypan blue. The cells are either frozen in CryoStor (autologous culture) or used directly for heterologous coculture in spheroids.

[0110] The MSCs to be used for co-culture are trypsinized and then counted.

[0111] On a 96-well ULA plate, and in RPMI medium (10% FCS):

[0112] - For each sample of recovered plasma cells (fresh or thawed), a control (in triplicate) with cells alone is produced (50,000 plasma cells / well).

[0113] - Each plasma cell sample is co-cultured with each sample of trypsinized MSCs (ratio 1:2 = 50,000 MSCs per 100,000 plasma cells per well, 50 pL / well)

[0114] - The plate is incubated at 37°C and 5% CO2 with orbital shaking for 24 hours at 73 rpm.

[0115] - 150 pL of medium (with or without drug) is added to the medium before incubating the ULA plate again. The medium is changed once a week by removing 100 pL / well and adding 100 pL of fresh medium (with or without drug). - The culture stops at day 7 and the spheroids are dissociated following the protocol described previously.

[0116] Carfilzomib is used at a concentration between 5-50nM, Pomalidomide is used at a concentration between 1 -1 OpM, Dexamethasone is used at a concentration of 1 pM, Isatuximab is used at a concentration of 1 pg / ml, Daratumumab is used at a concentration between 1 pg / ml.

[0117] Figure 2 shows the viability rate of MM plasma cells within heterologous spheroids following their culture for 7 days with the different treatments tested as well as a control condition (NT). The different treatments tested are indicated on the abscissa. DCD: Daratumumab / Carfilzomib / Dexamethasone,

[0118] DPD: Daratumumab / Pomalidomide / Dexamethasone,

[0119] ICD: Isatuximab / Carfilzomib / Dexamethasone,

[0120] IPD: Isatuximab / Pomalidomide / Dexamethasone,

[0121] CD: Carfilzomib / Dexamethasone,

[0122] PD: Pomalidomide / Dexamethasone.

[0123] On the left of the graph, frozen plasma cells were used, each modality was repeated 4 times (n=4). On the right of the graph, fresh plasma cells were used, i.e., they were not frozen before being cocultured to form the spheroids; here each modality was repeated 3 times (n=3).

Claims

CLAIMS 1. A method for producing human multiple myeloma (MM) spheroids, comprising: a. Cultivating 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 CD138+ primary plasma cells from a patient with MM under conditions allowing spheroid formation.

2. Production method according to claim 1, wherein the CSM, endothelial cells, endothelial progenitors and CD138+ primary plasma cells were obtained from the same patient suffering from MM.

3. Production method according to claim 1 or 2, wherein the CSM, endothelial cells, endothelial progenitors and CD138+ primary plasma cells were obtained from the same bone marrow sample from said patient suffering from MM.

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

5. Production method according to any one of claims 1 to 4, in which the primary CD138+ plasma cells have been preserved, before co-culture, by freezing at a temperature less than or equal to -70°C in a cryopreservation medium.

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

1.

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

8. Human multiple myeloma (MM) spheroids obtained by a production method according to any one of claims 1 to 7, comprising a stroma, a vascular compartment and CD138+ plasma cells from patient(s) suffering from 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. Cultivating 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 patient suffering from MM 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. 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 the autologous spheroids between the time of formation of the spheroids and up to 48 h after their formation, and the culture is continued for said period of at least 3 days.

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