Method for generating three-dimensional human multiple myeloma model

Abstract

The present application relates to a method for producing a three-dimensional (3D) model of multiple myeloma (MM) in spheroid form by co-culturing mesenchymal stem / stromal cells, endothelial progenitor cells and primary plasma cells of an MM patient. The present application also relates to spheroids obtained by said method and uses thereof.

Description

Technical Field

[0001] The present application relates to a method for generating a spheroid-based three-dimensional (3D) model of multiple myeloma (MM) by co-culturing mesenchymal stem cells / stromal cells, endothelial progenitor cells, and primary plasma cells from MM patients. The present application also relates to the spheroids obtained by the method and their uses. Background Art

[0002] Multiple myeloma (MM) is a hematologic malignancy, also known as bone marrow cancer. MM is characterized by the excessive proliferation of a type of white blood cell (plasma cell) in the bone marrow that has become abnormal. Plasma cells are immune system cells originating from the bone marrow (BM) and produce antibodies to protect the body from external attacks (bacteria, viruses). During development, genetic abnormalities (deletions, chromosomal translocations) can occur, transforming healthy plasma cells into malignant ones. Normally, plasma cells circulate in the blood, but in MM pathology, they return to the bone marrow, causing damage on multiple levels.

[0003] Despite recent significant therapeutic advances, multiple myeloma remains an incurable disease. Current treatments can prevent or alleviate symptoms and complications, destroy pathological plasma cells, and slow the progression of the disease.

[0004] To date, there has been a lack of relevant preclinical models for multiple myeloma. Indeed, mouse models are expensive, time-consuming, and do not represent human pathology. Standard two-dimensional (2D) models do not provide the viability of primary patient plasma cells and tend to use immortalized cell lines, which distances the models from the pathophysiology of MM disease.

[0005] The propagation of adult ex vivo bone marrow is increasingly described in the literature to overcome animal models that are expensive, time-consuming and dependent on species barriers. Studies have begun to reveal 3D models of human ex vivo bone marrow that integrate mesenchymal and vascular compartments (usually cells derived from immortalized cell lines). For example, the vascular compartment, which plays an active role in the proliferation of hematopoietic stem cells (HSCs), is often incorporated through the HUVEC endothelial immortalized cell line. Other models require a step in mice to conduct angiogenesis or functional pathways. In addition, the short half-life of plasma cells does not allow their autologous incorporation into current 3D models and forces the omission of plasma cells or the addition of tumor-immortalized cell lines of plasma cells, which does result in model-relevant responses to patients.

[0006] The inventors have created a new 3D tissue model of human multiple myeloma. It comprises a mesenchymal compartment, a vascular compartment, and plasma cells obtained from samples from patients with multiple myeloma, without the use of any immortalized cells. Specifically, the problem of maintaining plasma cell viability for more than 14 days in co-culture has been solved. This results in a fully humanized preclinical in vitro model of multiple myeloma that is genetically linked to the patient and includes mesenchymal, vascular, and plasma cell compartments in the form of spheroids.

[0007] This model enables advances in personalized medicine by rapidly constructing a model representative of each patient's bone marrow tumor tissue. Detailed Description of the Invention

[0009] Methods for generating human multiple myeloma spheroids

[0010] The present application relates to a method for generating human multiple myeloma (MM) spheroids, comprising:

[0011] a. Culturing mesenchymal stem cells / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells in culture medium;

[0012] b. harvesting cultured MSCs, endothelial cells, and endothelial progenitor cells; and

[0013] c. Harvested MSCs, endothelial cells, and endothelial progenitor cells were co-cultured with CD138+ primary plasma cells from MM patients under conditions that resulted in spheroid formation.

[0014] "Mesenchymal stem cells," also known as "mesenchymal stromal cells," refer to stem cells derived from the mesoderm. Their phenotypic characteristics are the co-expression of a certain number of markers, such as CD73, CD90, CD105, and CD146, and the absence of other markers, more specifically CD45, CD31, and CD34. They can be derived from bone marrow, adipose tissue, or umbilical cord blood. Mesenchymal stem cells or stromal cells are derived from humans and are from MM patients or healthy subjects. In a preferred embodiment, the mesenchymal stem cells / stromal cells cultured in step a are primary cells.

[0015] "Endothelial progenitor cells" are cells that participate in endothelial differentiation but are not yet recognizable as endothelial cells under a microscope. Their phenotype is characterized by the expression of a certain number of markers, such as CD133, CD34, CD31, and VEGFR2.

[0016] "Endothelial cells" refer to cells that have fully differentiated through the endothelial pathway and are therefore identifiable as such under a microscope. Their phenotype is characterized by the expression of a number of markers, such as CD31, VE-cadherin, Willebrand factor, and VEGFR2.

[0017] Endothelial progenitor cells and endothelial cells have the ability to organize into endothelial cell networks or vascular networks and are therefore able to organize into blood vessels.

[0018] In a preferred embodiment, the endothelial progenitor cells and endothelial cells cultured in step a) are primary cells. The endothelial progenitor cells and endothelial cells can be obtained, for example, from mononuclear cells of bone marrow.

[0019] In one embodiment, MSCs, endothelial cells and endothelial progenitor cells are obtained from the same subject, i.e., from the same healthy subject or from the same MM patient. Preferably, MSCs, endothelial cells and endothelial progenitor cells are obtained from only one or the same sample (more specifically a bone marrow sample) from the healthy subject or MM patient.

[0020] "Primary cells" refer to cells that are directly derived from an individual's tissue and / or cell sample.

[0021] "Cultivation" refers to the selection and proliferation of cultured cells.

[0022] In one embodiment of the method, in step a), mesenchymal stem cells / stromal cells (MSCs), endothelial progenitor cells and endothelial cells are co-cultured in the same culture medium and preferably in the same culture container. After extracting the original bone marrow, the cells are cultured at a density of, for example, 50,000 cells / cm 2 The three cell types coexist and proliferate in this culture.

[0023] In one embodiment, the culture is carried out in two dimensions (2D) in the form of an at least partially adherent monolayer. Preferably, the culture is carried out until the cells are confluent. The culture typically lasts for 3 to 30 days, preferably for 5 to 25 days, or for 10 to 20 days, 12 to 16 days, 13 to 15 days, or about 2 weeks.

[0024] Preferably, the cells are not cultured in the presence of a hydrogel or solid support (ossified tissue or other scaffold).

[0025] "Hydrogel" refers to a gel in which the swelling agent is water. The matrix of a hydrogel is typically a polymer matrix. Specifically, hydrogels include Matrigel and can be formed based on fibrin, collagen, agarose, gelatin, synthetic polymers, or mixtures thereof.

[0026] Preferably, the cells are not cultured with exogenously supplied complex biomolecules (eg, cytokines, growth factors, hormones).

[0027] "Culture medium" refers to a culture medium suitable for culturing mesenchymal stem cells / stromal cells, endothelial progenitor cells, and endothelial cells. Examples of the culture medium include RPMI medium supplemented with 10% fetal calf serum (FCS), Minimum Essential Medium α (MEMα), or Endothelial Cell Growth Medium 2 (EGM2, from Promocell) supplemented with 2% FCS or platelet lysate (PL). It can be in various forms, but is preferably liquid and is used for culturing eukaryotic cells, more specifically mammalian cells, and more specifically human cells.

[0028] According to the present application, the healthy subject or MM patient is a human. According to certain embodiments, the patient has just been diagnosed with MM. In certain embodiments, the MM patient is diagnosed with relapse.

[0029] At the end of the culturing step a), the cultured MSCs, endothelial cells, and endothelial progenitor cells are harvested. Harvesting is typically accomplished by treatment with trypsin followed by washing. Other agents for separating adherent cells without damage may be substituted for trypsin.

[0030] Harvested MSCs, endothelial cells, and endothelial progenitor cells were then co-cultured with CD138+ primary plasma cells from MM patients under conditions that resulted in spheroid formation.

[0031] "Plasma cells" refer to immune system cells derived from the bone marrow (BM) that express the marker CD138+. In the case of MM, plasma cells express the markers CD38+ and CD138+.

[0032] "Spheroids" refer to aggregates of cells connected to each other in three dimensions. Preferably, the spheroids contain 500 to 750,000 cells, or 1,000 to 500,000 cells. The average diameter of the spheroids in the composition according to the present application is between 50 μm and 750 μm, preferably between 100 μm and 500 μm.

[0033] Preferably, the cells are not cultured in the presence of a hydrogel or solid support (ossified tissue or other scaffold).

[0034] Preferably, the cells are not cultured with exogenously supplied complex biomolecules (eg, cytokines, growth factors, hormones, etc.).

[0035] In fact, the spheroids according to the present application are formed by the self-organization of MSCs, endothelial cells and endothelial progenitor cells with plasma cells. Therefore, the spheroids according to the present application are formed without any hydrogels, supports or exogenously supplied complex biomolecules. This method can limit deviations and more closely resemble what is observed in vivo. The use of hydrogels, supports or exogenously supplied complex biomolecules can change the behavior of certain products, leading to underestimation or overestimation of the product's potential for action in vivo.

[0036] In one embodiment, CD138+ primary plasma cells are derived from a MM patient that is different from the MM patient or healthy subject from which cultured MSCs, endothelial cells, and endothelial progenitor cells are obtained. The production method is then used to obtain heterologous human multiple myeloma (MM) spheroids. The heterologous human MM spheroid model obtained makes it possible to combine the matrix of a patient or healthy subject with the tumor plasma cells of another MM patient to study the effects of MM plasma cells on healthy matrix and reveal therapeutic targets. On the other hand, healthy plasma cells can be associated with the MM matrix to study the effects of the MM matrix on healthy plasma cells and reveal therapeutic targets.

[0037] According to another embodiment, MSCs, endothelial cells, endothelial progenitor cells, and CD138+ primary plasma cells are obtained from the same MM patient. Then, the production method can obtain autologous human multiple myeloma (MM) spheroids. Since bone marrow sampling is an invasive procedure, preferably, MSCs, endothelial cells, endothelial progenitor cells, and CD138+ primary plasma cells are obtained from the same bone marrow sample of the MM patient.

[0038] This embodiment has the additional difficulty of successfully preserving CD138+ primary plasma cells and maintaining their viability during the culture period of MSCs, endothelial cells and endothelial progenitor cells (i.e., 3 to 30 days, usually about two weeks). In fact, in order to be able to construct autologous spheroids without using new bone marrow samples from patients with MM, it is necessary to freeze primary CD138+ plasma cells while ensuring optimal viability of the plasma cells during subsequent spheroid culture.

[0039] Therefore, preferably, before co-culture, primary CD138+ plasma cells derived from the same patient sample as MSCs, endothelial cells and endothelial progenitor cells are stored by freezing in a freezing medium at a temperature lower than or equal to -70°C (preferably lower than or equal to -75°C, or even -80°C). The freezing medium can be 90% FCS + 10% DMSO (v / v), or 90% 4% human albumin solution + 10% DMSO (v / v), or a commercial freezing medium such as Preferably, the CD138+ primary plasma cells are frozen within 2 hours after being isolated from the bone marrow sample.

[0040] MSCs, endothelial cells and endothelial progenitor cells are co-cultured with CD138+ primary plasma cells in ULA (ultra-low adsorption) plates to promote spheroid formation. CD138+ primary plasma cells are co-cultured with MSCs in a quantitative ratio of 1:1 to 4:1, preferably about 2:1. Co-culture is performed for 4 to 14 days, for example 4 to 10 days, preferably 6 to 8 days or about 7 days. Preferably, the co-culture step is performed under stirring, more preferably with low-speed stirring.

[0041] The culture medium is, for example, RPMI medium supplemented with 10% fetal calf serum (FCS), minimum essential medium alpha (MEMα), or endothelial growth medium 2 (EGM2, from Promocell) supplemented with 2% FCS or platelet lysate (PL). Preferably, the cells are not cultured with exogenously supplied complex biomolecules (e.g., cytokines, growth factors, hormones, etc.).

[0042] The present application also relates to spheroids obtained or obtainable by the above-mentioned method for producing spheroids.Human multiple myeloma (MM) spheroids include stroma, vascular compartments and CD138+ plasma cells from MM patients.

[0043] Spheroids are preferably autologous, obtained by co-culturing MSCs, endothelial cells and endothelial progenitor cells with CD138+ primary plasma cells from the same MM patient, preferably from the same sample.

[0044] Spheroids can also be allogeneic if the CD138+ primary plasma cells are derived from an MM patient rather than from an MM patient or healthy subject from whom the cultured MSCs, endothelial cells, and endothelial progenitor cells were derived.

[0045] Uses of spheroids

[0046] The subject of this application is the use of autologous multiple myeloma spheroids for selecting therapeutic treatment suitable for MM patients, ie for selecting treatment to which multiple myeloma (MM) patients are likely to respond.

[0047] Indeed, constructing MM 3D models that are as representative as possible of the patient's tumor enables clinical follow-up and personalized medicine. The spheroid models according to the present application, preferably autologous spheroid models, can be used to study how tumor tissue from MM patients (from whom the cells used to prepare the spheroids come) responds to different treatments or treatment combinations. Thus, the goal is to select the treatment to which the patient is most likely to respond.

[0048] "Treatment" or "treat" as used herein refers to partially or substantially achieving one or more of the following results: partially or completely alleviating the extent of the disease; improving clinical symptoms or indicators associated with the disease; delaying, inhibiting or preventing the progression of the disease; or partially or completely delaying, inhibiting or preventing the occurrence of recurrence of the disease.

[0049] A "subject," "patient," or "patient" as used herein refers to a human suffering from multiple myeloma.

[0050] Choosing a treatment method

[0051] The present application also includes a method for selecting a treatment that a multiple myeloma (MM) patient may respond to using autologous spheroids derived from the patient. The selection method comprises:

[0052] - culturing the patient's autologous spheroids in culture for at least 3 days in the presence of at least one candidate drug for the treatment of MM,

[0053] - Harvesting autologous spheroids and dissociating them to collect their myeloma plasma cells,

[0054] - Analyzing the viability of collected myeloma plasma cells, and

[0055] - Selecting at least one candidate drug as a treatment to which the MM patient is likely to respond based on the measured viability of the collected myeloma plasma cells.

[0056] According to one embodiment, at least one candidate drug is selected as a treatment to which the MM patient 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 the candidate drug or with the addition of a control buffer) or in the presence of at least another candidate drug.

[0057] The cultivation of the spheroids is performed under the same conditions as previously defined in the method for generating autologous spheroids, except for the addition of said at least one candidate drug. According to one embodiment, the selection method comprises preparing autologous spheroids according to the method of the present application.

[0058] The MSCs, endothelial cells, endothelial progenitor cells and plasma cells that form the autologous spheroids are derived from the same patient, preferably from the same bone marrow sample.

[0059] This selection method is used to test the effectiveness of a candidate drug or drug combination. The candidate drug or drug combination is added to the spheroid culture medium between the time of spheroid formation and 48 hours after formation, and culture is continued for at least 3 days, such as 4 to 10 days, preferably 6 to 8 days or about 7 days.

[0060] At the same time, a control culture is performed in the absence of the candidate drug or in the presence of a buffer under the same culture conditions as in the presence of the at least one candidate drug.

[0061] Examples of drugs or drug candidates that can be used to treat MM include melphalan, lenalidomide, bortezomib, dexamethasone, C34 (a compound of formula (I) described in application WO 2018 / 115476 A1,

[0062] Compound 1

[0063]

[0064] The spheroids are then harvested and mechanically dissociated, for example, in a thermomixer and / or by repeated aspiration / discharge using a micropipette, with or without the use of one or more chemical reagents, such as trypsin, collagenase, or AccuMax dissociation solution (Capricorn Scientific GmbH). More specifically, the spheroids can be dissociated by incubating the spheroids with stirring (e.g., in a thermomixer at 37° C., 1200-1500 rpm, or again at about 1400 rpm, for about 10 minutes) in a dissociation solution (comprising a protease and / or collagenase, and preferably a combination of a protease, collagenase, and DNase, such as an AccuMax solution), followed by dissociation of the spheroids by aspiration / discharge using a micropipette, and harvesting the dissociated cells (e.g., by centrifugation).

[0065] The cells are then labeled with markers to identify myeloma plasma cells, for example, using fluorescent dyes associated with specific antibodies, such as CD38 (particularly for labeling plasma cells) and CD138 (for identifying CD38+CD138+ myeloma plasma cells). The cells are then resuspended and filtered and then analyzed by flow cytometry (FACS). The fluorescent dye markers associated with specific antibodies can be, for example, CD38FITC and CD138 AF700. The suspension solution and the labeling solution are preferably MACS solutions, and the cells are preferably filtered through 70 μm. The viability of myeloma plasma cells is compared between different culture conditions (control conditions or at least one drug candidate). Compared to the control, the reduction in the viability of myeloma plasma cells is a sign of promising treatment.

[0066] This approach is faster than using mouse models, and responses are more relevant due to the genetic proximity between the model and the patient, especially when the spheroid model is autologous. In fact, spheroids can be generated in an average of approximately 2 weeks, and from the time the spheroids are obtained, the appropriate treatment for the patient can be selected in approximately 1 week, generally taking a total of 3 weeks, enabling the development of personalized treatments for MM patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] [ Figure 1 ] Figure 1 Figure 2 shows the viability of MM plasma cells in allogeneic spheroids after 7 days of culture with one or more candidate drugs (melphalan 10 μM, lenalidomide 10 μM, C34 5 μM, melphalan 10 μM + C34 5 μM combination, lenalidomide 10 μM + C34 5 μM combination) and control conditions. On the abscissa, MM MSCs (MM91, MM97, MM100) represent the stromal component of the spheroids. MM plasma cells (P64 / 65, P66) are the plasma cell component of the spheroids. Each number corresponds to a specific MM patient.

[0068] [ Figure 2 ] Figure 2 The levels of viable MM plasma cells in spheroids after 7 days of culture with one or more candidate drugs (carfilzomib 5-50 nM, pomalidomide 1-10 μM, dexamethasone 1 μM, isatuximab 1 μg / ml, daratumumab 1 μg / ml) and control conditions (NT) are shown. On the abscissa, different treatments were tested:

[0069] -DCD: daratumumab / carfilzomib / dexamethasone,

[0070] -DPD: daratumumab / pomalidomide / dexamethasone,

[0071] -ICD: isatuximab / carfilzomib / dexamethasone,

[0072] -IPD: isatuximab / pomalidomide / dexamethasone,

[0073] -CD: Carfilzomib / dexamethasone,

[0074] -PD: pomalidomide / dexamethasone. Example

[0075] Example 1: Study of different plasma cell freezing protocols and cell viability studies

[0076] In this example, the inventors attempted to select the plasma cell freezing / thawing protocol that was most effective in maintaining plasma cell viability. They investigated this effect by quantifying post-thaw viability using trypan blue counting in a Malassez cell.

[0077] Whole bone marrow samples are collected from patients with multiple myeloma (MM) at diagnosis, and MM plasma cells are isolated and counted in a Malassez cell counting chamber. The cells are then frozen according to one of the following protocols:

[0078] -A: 90% fetal calf serum (FCS) buffer: 10% DMSO;

[0079] Freeze immediately at -80°C

[0080] -B: 90% human serum albumin (HSA) buffer: 10% DMSO;

[0081] Freeze immediately at -80°C

[0082] -C: CS10 buffer (Sigma-Aldrich, product number C2874);

[0083] Freeze immediately at -80°C

[0084] -D: CS10 buffer;

[0085] Freeze at -20℃ for 2 hours and transfer to -80℃

[0086] -E: CS10 buffer;

[0087] Freeze at -20℃ overnight and transfer to -80℃

[0088] The average freezing time is 31 days.

[0089] The plasma cells were then thawed and counted using trypan blue in a Malassez-type cell counting chamber.

[0090] [Table 1]

[0091]

[0092] The results in the above table show that The current freezing conditions (ie 90% FCS or HSA + 10% DMSO) appeared to be less effective compared to the immediate freezing conditions of the cells.

[0093] Example 2: Culture of allogeneic and autologous spheroids and study of cell viability by flow cytometry

[0094] In this example, the inventors sought to demonstrate that the steps of freezing and thawing, culturing within spheroids, and dissociating the spheroids before labeling had a low impact on plasma cell viability. They investigated this effect by quantifying their viability by flow cytometry. To this end, they attempted to label the cells with anti-CD38 and anti-CD138 antibodies conjugated to fluorescent dyes to specifically select for MM plasma cells.

[0095] Whole bone marrow samples were collected from multiple myeloma (MM) patients at diagnosis, and MM plasma cells were isolated and counted in a Malassez cell counting chamber. The cells were then frozen in a CRYOSTOR or used fresh for allogeneic co-culture. MM whole bone marrow cells from another patient were seeded according to their initial number and incubated at 37°C in an atmosphere containing 5% carbon dioxide for approximately 2 weeks. Once confluence was achieved, mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells were trypsinized and counted.

[0096] On ULA (ultra-low attachment) plates, MSC cells, endothelial cells, and endothelial progenitor cells treated with trypsin were suspended in 50 μL RPMI culture medium (10% FCS, 1% PS) with fresh or thawed plasma cells at a ratio of 1 MSC per 2 plasma cells. The cells were then incubated with stirring at a temperature of 37°C and an atmosphere containing 5% carbon dioxide. After incubation for 24 hours, 150 μL complete RPMI culture medium was added, and the culture medium was replaced twice a week, with 100 μL of culture supernatant removed and 100 μL of complete RPMI culture medium added each time.

[0097] use Spheroids were mechanically dissociated at 1400 rpm and then transferred to a tube suitable for flow cytometry, washed with PBS, and labeled with anti-CD38 FITC and anti-CD138 AF700 antibodies in MACS buffer. The cells were then incubated at 4°C for 30 minutes, washed, and resuspended in MACS buffer medium, filtered through 70 μm, and labeled with DAPI before flow cytometry.

[0098] The inventors observed that the viability of MM plasma cells could be quantified using this protocol and that the viability of MM plasma cells remained high even after 14 days of co-culture following operation of this protocol.

[0099] Example 3: 3D spheroids containing plasma cells from multiple myeloma patients and response to melphalan treatment

[0100] In this example, the inventors sought to demonstrate the viability of plasma cells within spheroids and the accessibility of spheroids to tested therapeutic molecules.

[0101] Whole bone marrow samples were collected from multiple myeloma (MM) patients at diagnosis, and MM plasma cells were isolated and counted in a Malassez cell counting chamber. The cells were then freshly used for co-culture or frozen in a CRYOSTOR. MM whole bone marrow cells from another patient were seeded based on their initial number and incubated at 37°C in an atmosphere containing 5% carbon dioxide for approximately 2 weeks. Once confluence was achieved, mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells were trypsinized and counted.

[0102] On ULA (ultra-low attachment) plates, trypsinized MSCs, endothelial cells, and endothelial progenitor cells were suspended in 50 μL of RPMI medium (10% FCS, 1% PS) with fresh or thawed plasma cells at a ratio of one MSC per two plasma cells. The cells were then incubated with agitation at 37°C in an atmosphere containing 5% carbon dioxide. After 24 hours of incubation, 150 μL of RPMI medium was added, and the medium was replaced twice a week, with 100 μL of supernatant removed each time.

[0103] After 48 hours of spheroid formation, 10 μM melphalan was added to the spheroid culture medium and cultured for an additional 14 days. This selection was supplemented by a control without the candidate drug. Some cultures were stopped after 7 (D+7) and 11 days (D+11) to analyze plasma cell viability, while others were stopped after 14 days (D+14).

[0104] use Spheroids were mechanically dissociated at 1400 rpm and then transferred to a tube suitable for flow cytometry, washed with PBS, and labeled with CD38 FITC and CD138 AF700-specific antibodies in MACS buffer. The cells were then incubated at 4°C for 30 minutes, washed and resuspended in MACS buffer medium, filtered through 70 μm, and finally labeled with the viability marker DAPI before flow cytometry.

[0105] The plasma cell viability in untreated MM spheroids increased from 50% on D+7 and D+11 to 60% on D+14; whereas the plasma cell viability in MM spheroids treated with 10 μM melphalan was 5% on D+7, less than 5% on D+11, and less than 10% on D+14.

[0106] Firstly, the inventors thus showed that the viability of plasma cells in untreated spheroids was higher (D7, D11, D14) than in the case of 2D culture, where primary plasma cells could not survive for more than a few days.

[0107] The inventors then showed that the localization of plasma cells within spheroids did not prevent a strong response to treatment (here melphalan 10 μM).

[0108] Example 4: Response of heterogeneous MM spheroids to different drug candidates

[0109] Whole bone marrow samples were collected from multiple myeloma (MM) patients at diagnosis, and MM plasma cells were isolated and counted in a Malassez cell counting chamber. The cells were then used fresh or frozen at -80°C in a CRYOSTOR. MM whole bone marrow cells from the same or different patients were seeded according to their initial number and incubated at 37°C in an atmosphere containing 5% carbon dioxide for approximately 2 weeks. Once confluence was achieved, mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells were trypsinized and counted.

[0110] On ULA (ultra-low attachment) plates, trypsinized MSCs, endothelial cells, and endothelial progenitor cells were suspended in 50 μL RPMI medium (10% FCS, 1% PS) with fresh or thawed plasma cells at a ratio of 1 MSC per 2 plasma cells. The cells were then incubated with stirring at 37°C in an atmosphere containing 5% carbon dioxide. After 24 hours of incubation, 150 μL RPMI medium was added, and the medium was replaced twice a week, with 100 μL of culture supernatant removed and 100 μL of RPMI medium added each time.

[0111] Between the time of spheroid formation and 48 hours after formation, the candidate drug or drug combination is added to the spheroid culture medium and cultured for an additional 7 days. This selection is completed by a control in the absence of any candidate drug, which is cultured for the same period of time as in the presence of the candidate drug.

[0112] The treatments tested were as follows:

[0113] - Melphalan 10 μM,

[0114] - Lenalidomide 10 μM,

[0115] -C34 5μM,

[0116] -Melphalan 10μM + C34 5μM combination,

[0117] -Lenalidomide 10μM + C34 5μM combination

[0118] use Spheroids were mechanically dissociated at 1400 rpm and then transferred to flow cytometry-compatible tubes, washed with PBS, and labeled with CD38 FITC and CD138 AF700-specific antibodies in MACS buffer. The cells were then incubated at 4°C for 30 minutes, washed, and resuspended in MACS buffer medium, filtered through 70 μm, and finally labeled with DAPI before flow cytometry.

[0119] Figure 1 Figure 2 shows the viability of MM plasma cells in allogeneic spheroids after 7 days of culture under various potential treatments tested, as well as control conditions. On the abscissa, MM MSCs (MM91, MM97, MM100) represent the stromal component of the spheroids. MM plasma cells (P64 / 65, P66) represent the plasma cell component of the spheroids. Each number corresponds to a specific MM patient.

[0120] Figure 1 The differences in plasma cell behavior under different conditions and the effectiveness of combined treatment with melphalan+C34 and lenalidomide+C34 in reducing MM plasma cell viability were clearly demonstrated.

[0121] Example 5: Response of spheroids to different drug candidates and drug candidate combinations

[0122] Whole bone marrow cells from multiple myeloma (MM) patients were first seeded into culture flasks containing EGM2 medium according to the initial number in the tube. The cells were incubated at 37° C. and 5% CO 2 for approximately 2 weeks.

[0123] Plasma cells from multiple myeloma (MM) patients were first counted in a Malassez cell counting chamber using trypan blue. Cells were either frozen in Cryostor (autologous culture) or used directly in allogeneic spheroid co-cultures.

[0124] MSCs used for co-culture were trypsinized and then counted.

[0125] In 96-well ULA plates, in RPMI medium (10% FBS):

[0126] - For each recovered plasma cell sample (fresh or thawed), generate a cell-only control (50,000 plasma cells / well) in triplicate.

[0127] - Each plasma cell sample was co-cultured with each trypsinized MSC sample (ratio 1:2 = 50,000 MSCs per 100,000 plasma cells, 50 μL / well).

[0128] - Incubate the plate at 37°C and 5% CO2 with orbital agitation at 73 rpm for 24 hours.

[0129] - Before re-incubating the ULA plate, add 150 μL of medium (with or without drug) to the culture medium. Change the medium once a week by removing 100 μL / well and adding 100 μL of new medium (with or without drug).

[0130] - The culture was stopped at D7 and spheroids were dissociated according to the protocol above.

[0131] Carfilzomib was used at a concentration of 5-50 nM, pomalidomide was used at a concentration of 1-10 μM, dexamethasone was used at a concentration of 1 μM, isatuximab was used at a concentration of 1 μg / mL, and daratumumab was used at a concentration of 1 μg / mL.

[0132] Figure 2 Shown are the viability levels of MM plasma cells in allogeneic spheroids after 7 days of culture under the different treatments tested and control conditions (NT).On the abscissa, the different treatments tested are shown.

[0133] DCD: daratumumab / carfilzomib / dexamethasone,

[0134] DPD: daratumumab / pomalidomide / dexamethasone,

[0135] ICD: isatuximab / carfilzomib / dexamethasone,

[0136] IPD: isatuximab / pomalidomide / dexamethasone,

[0137] CD: Carfilzomib / dexamethasone,

[0138] PD: pomalidomide / dexamethasone.

[0139] On the left side of the figure, frozen plasma cells were used and each approach was repeated 4 times (n=4). On the right side of the figure, fresh plasma cells were used, i.e., plasma cells were not frozen before co-culture to form spheres; here, each approach was repeated 3 times (n=3).

Claims

1. A method for generating human multiple myeloma (MM) spheroids, comprising: a. Culturing mesenchymal stem cells / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells in culture medium; b. harvesting cultured MSCs, endothelial cells, and endothelial progenitor cells; and c. Harvested MSCs, endothelial cells, and endothelial progenitor cells were co-cultured with CD138+ primary plasma cells from MM patients under conditions that resulted in spheroid formation. 2 . The production method according to claim 1 , wherein the MSCs, endothelial cells, endothelial progenitor cells and the CD138+ primary plasma cells are obtained from the same MM patient. 3 . The production method according to claim 1 , wherein the MSCs, endothelial cells, endothelial progenitor cells and the CD138+ primary plasma cells are obtained from the same bone marrow sample of the MM patient. 4 . The production method according to claim 1 , wherein in step a, the MSCs, endothelial cells and endothelial progenitor cells are cultured for 3 to 30 days. 5 . The production method according to claim 1 , wherein the CD138+ primary plasma cells are preserved in a cryopreservation medium by freezing at a temperature lower than or equal to −70° C. before co-culturing. The production method according to any one of claims 1 to 5, wherein the CD138+ primary plasma cells and the MSCs are co-cultured at a ratio of about 2:

1. 7 . The production method according to claim 1 , wherein the MSCs, endothelial cells and endothelial progenitor cells are co-cultured with the primary plasma cells for 4 to 14 days. 8 . Human multiple myeloma (MM) spheroids obtained by the production method of any one of claims 1 to 7 , comprising stroma, vascular compartments, and CD138+ plasma cells of MM patients.

9. The MM spheroid of claim 8, wherein the spheroid is an autologous spheroid.

10. Use of autologous multiple myeloma (MM) spheroids as defined in claim 9 for selecting MM patients who are likely to respond to treatment.

11. A method for selecting a multiple myeloma (MM) patient likely to respond to a treatment, comprising: a. The autologous spheroids according to claim 9 are cultured in a medium containing at least one candidate drug for treating MM for at least 3 days; b. harvesting the autologous spheroids and dissociating them to collect myeloma plasma cells present in the autologous spheroids; c. analyzing the viability of the collected myeloma plasma cells; as well as d. Based on the measured viability of the collected myeloma plasma cells, selecting at least one candidate drug as a treatment to which the MM patient is likely to respond.

12. The method of selecting a treatment according to claim 11, wherein the at least one candidate drug is selected as a treatment to which the MM patient 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 or in the presence of at least one other candidate drug.

13. The method for selective treatment according to claim 11 or 12, wherein the at least one candidate drug is added to the culture medium of the autologous spheroids between the time of formation and 48 hours after formation of the spheroids, and the culture is continued for at least 3 days.

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