Methods for producing a three-dimensional human multiple myeloma model

Abstract

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 stem / mesenchymal stromal cells, endothelial progenitor cells and primary plasma cells of one or more MM patients. The present invention also relates to the spheroids obtained by said method and their uses.

Description

[Technical Field]

[0001] 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 patients suffering from MM. The present invention further relates to the spheroids obtained by said method and their uses. [Background technology]

[0002] Multiple myeloma (MM), also known as bone marrow cancer, is a hematological malignancy. MM is characterized by the overgrowth of abnormal plasma cells, a type of white blood cell, in the bone marrow. 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 (deletions, chromosomal translocations) can occur, transforming healthy plasma cells into malignant plasma cells. Under normal conditions, plasma cells circulate in the blood, but in the pathology of MM, plasma cells home to the bone marrow, where they cause damage at several levels.

[0003] Multiple myeloma remains an incurable disease despite significant therapeutic advances in recent years. Care currently focuses on preventing or alleviating symptoms and complications, destroying diseased plasma cells, and slowing disease progression.

[0004] To date, multiple myeloma has suffered from a 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 provide viability of primary patient plasma cells and favor the use of immortalized cell lines, which distances the models from the pathophysiology of MDM disease.

[0005] Reproducing adult human ex vivo bone marrow has been gradually described in the literature, overcoming animal models that are expensive, time-consuming, and dependent on species barriers. Research has begun to develop 3D models of human ex vivo bone marrow that combine mesenchymal and vascular compartments, typically derived from cells derived from immortalized cell lines. For example, the vascular compartment, which plays an active role in hematopoietic stem cell (HSC) proliferation, is often incorporated via HUVEC endothelial immortalized cell lines. Other models require mouse procedures for angiogenesis or functional approaches. Furthermore, the short half-life of plasma cells does not allow for their autologous integration into current 3D models, leading to either their neglect or the addition of tumor-immortalized plasma cell lines, which would lead to significant patient responses in the model. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 115476 A1 Summary of the Invention [Means for solving the problem]

[0007] The present inventors have created a novel human 3D tissue model of multiple myeloma. This model does not involve the use of any immortalized cell lines and contains mesenchymal and vasculature compartments and plasma cells obtained from samples derived from patients with multiple myeloma. In particular, the maintenance of plasma cell viability in co-culture for more than 14 days was resolved. As a result, a fully human preclinical ex vivo model of multiple myeloma was generated that is genetically related to the patient and contains mesenchymal, vasculature, and plasma cell compartments in a spheroid morphology.

[0008] Such models make it possible to envision advances in personalized medicine using the rapid construction of models representative of each patient's bone marrow tumor tissue.

[0009] Detailed Description of the Invention Methods for producing human multiple myeloma spheroids The present invention provides a method for producing human multiple myeloma (MM) spheroids, comprising: a. culturing mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells in a culture medium; b. harvesting the cultured MSCs, endothelial cells, and endothelial progenitor cells; and c. Co-culturing the collected MSCs, endothelial cells, and endothelial progenitor cells with CD138+ primary plasma cells from a patient with MM under conditions conducive to spheroid formation. The present invention relates to a method for manufacturing the same, comprising:

[0010] "Mesenchymal stem cells", also referred to as "mesenchymal stromal cells", refer to stem cells of mesodermal origin. Mesenchymal stem cells are phenotypically characterized by the co-expression of a number of markers, such as CD73, CD90, CD105, CD146, and the absence of other markers, more particularly CD45, CD31, and CD34. Mesenchymal stem cells can be derived from bone marrow, adipose tissue, and umbilical cord blood. The mesenchymal stem or stromal cells are of human origin and are derived from patients with MM or healthy subjects. In a preferential manner, the mesenchymal stem / stromal cells cultured in step a. are primary cells.

[0011] "Endothelial progenitor cells" refer to cells that are involved in endothelial differentiation but are not yet recognizable as endothelial cells under microscopic observation. Endothelial progenitor cells are phenotypically characterized by the expression of a number of markers, such as CD133, CD34, CD31, and VEGFR2.

[0012] By "endothelial cell" is meant a cell that is fully differentiated in the endothelial pathway and is therefore recognizable as an endothelial cell under microscopic observation. Endothelial cells are phenotypically characterized by the expression of a number of markers, such as CD31, VE-cadherin, von Willebrand factor, and VEGFR2.

[0013] Endothelial progenitor cells and endothelial cells have the potential to organize into networks of endothelial cells, or vascular networks, and therefore into blood vessels.

[0014] 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 bone marrow mononuclear cells.

[0015] In one embodiment, the MSCs, endothelial cells and endothelial progenitor cells are obtained from the same subject, i.e., from the same healthy subject or from the same patient suffering from MM. Preferably, the MSCs, endothelial cells and endothelial progenitor cells are obtained from only one or the same sample, more particularly a bone marrow sample, from said healthy subject or patient suffering from MM.

[0016] By "primary cells" is meant cells derived directly from an individual's tissue and / or from a sample of cells.

[0017] "Culturing" refers to the selection and propagation of cells to be cultured.

[0018] In one embodiment of the method, in step a), mesenchymal stem / stromal cells (MSCs), endothelial progenitor cells and endothelial cells are cultured together in the same culture medium and preferably in the same culture vessel. After extraction of raw bone marrow, the cells are cultured, for example, at 50,000 cells / cm in a flask. 2 In such cultures, the three cell types coexist and proliferate.

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

[0020] Preferably, the cells are not cultured in the presence of a hydrogel or solid support (ossified tissue or other scaffold).

[0021] "Hydrogel" refers to a gel in which the swelling agent is water. The matrix of a hydrogel is generally an array of polymers. Hydrogels include, in particular, Matrigel, and can be based on fibrin, collagen, agarose, gelatin, synthetic polymers, or mixtures thereof.

[0022] Preferably, the cells are not cultured with an exogenous supply of complex biomolecules (eg, cytokines, growth factors, hormones).

[0023] By "culture medium" is meant 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 calf serum (FCS), minimum essential medium alpha (MEMα), or endothelial cell growth medium 2 (EGM2, from Promocell) supplemented with 2% FCS or platelet lysate (PL). The culture medium can be in different forms but is preferably liquid and is used for culturing eukaryotic cells, more particularly mammalian cells and more particularly human cells.

[0024] According to the present invention, the healthy subject or patient suffering from MM is a human. According to certain embodiments, the patient has just been diagnosed with MM. In certain embodiments, the patient with MM is diagnosed with relapse.

[0025] At the end of the culturing step a), the cultured MSCs, endothelial cells and endothelial progenitor cells are harvested. Harvesting is typically performed by trypsinization followed by washing. Other agents used for the detachment of adherent cells without damage can replace trypsin.

[0026] The harvested MSCs, endothelial cells and endothelial progenitor cells are then co-cultured with CD138+ primary plasma cells from patients with MM under conditions that result in the formation of spheroids.

[0027] By "plasma cell" is meant a cell of the immune system derived from the bone marrow (BM) that expresses the marker CD138+. In the case of MM, plasma cells express the markers CD38+ and CD138+.

[0028] "Spheroid" refers to a grouping of cells connected to one another in three-dimensional space. Preferably, the spheroid contains 500 to 750,000 cells, or alternatively 1,000 to 500,000 cells. The spheroids of the composition according to the invention have an average diameter comprised between 50 μm and 750 μm, preferably between 100 μm and 500 μm.

[0029] Preferably, the cells are not cultured in the presence of a hydrogel or solid support (ossified tissue or other scaffold).

[0030] Preferably, the cells are not cultured with an exogenous supply of complex biomolecules (eg, cytokines, growth factors, hormones, etc.).

[0031] In fact, the spheroids according to the present invention are formed by the self-assembly of MSCs, endothelial cells, and endothelial progenitor cells with plasma cells. Therefore, the spheroids according to the present invention are formed without the external provision of any hydrogel, support, or complex biomolecules. Such an approach limits bias and allows for closer observations to those observed in vivo. The use of external provision of hydrogel, support, or complex biomolecules may alter the behavior of some products, thus resulting in an underestimation or overestimation of their potential in vivo.

[0032] In one embodiment, the CD138+ primary plasma cells are derived from a patient with MM different from the patient with MM, or from a healthy subject from which the cultured MSCs, endothelial cells, and endothelial progenitor cells were obtained. The manufacturing method is then used to obtain heterogeneous human multiple myeloma (MM) spheroids. The obtained heterogeneous human MM spheroid model allows for the combination of stroma from a patient or healthy subject with tumor plasma cells from another patient with MM to study the effects of MM plasma cells on healthy stroma and elucidate therapeutic targets. Alternatively, healthy plasma cells can be combined with MM stroma to study the effects of MM stroma on healthy plasma cells and elucidate therapeutic targets.

[0033] According to another embodiment, MSCs, endothelial cells, endothelial progenitor cells, and CD138+ primary plasma cells are obtained from the same patient suffering from MM. Subsequently, the production method allows for obtaining 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 from the patient with MM.

[0034] Such embodiments present the additional challenge of successfully preserving and maintaining the viability of CD138+ primary plasma cells over the culture time of MSCs, endothelial cells, and endothelial progenitor cells, i.e., 3-30 days of culture, and generally about 2 weeks. Indeed, to be able to construct autologous spheroids without relying on fresh 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 culture of the spheroids.

[0035] Thus, primary CD138+ plasma cells derived from the same patient samples as MSCs, endothelial cells, and endothelial progenitor cells are preferably preserved before co-culture by freezing them in a cryopreservation medium at temperatures below -70°C, preferably below -75°C, or even below -80°C. The cryopreservation medium can be a medium consisting of 90% FCS + 10% DMSO (v / v), or 90% 4% + 10% DMSO (v / v) human albumin solution, or a commercially available cryopreservation solution such as CryoStor® CS10 (Sigma-Aldrich, C2874). Preferably, the freezing of CD138+ primary plasma cells is performed within 2 hours after isolation of CD138+ primary plasma cells from the bone marrow sample.

[0036] Co-culture of MSCs, endothelial cells, and endothelial progenitor cells with CD138+ primary plasma cells is performed in an ultra-low attachment (ULA) plate to promote spheroid formation. The CD138+ primary plasma cells and MSCs are co-cultured at a ratio of 1:1 to 4:1, preferably about 2:1, in terms of number. The co-culture is performed for 4 to 14 days, for example, 4 to 10 days, preferably 6 to 8 days, or otherwise about 7 days. Preferably, the co-culture step is performed with agitation, preferably low agitation.

[0037] 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) preferably supplemented with 2% FCS or platelet lysate (PL). Preferably, the cells are not cultured with an exogenous supply of complex biomolecules (e.g., cytokines, growth factors, hormones, etc.).

[0038] The present invention further relates to spheroids obtained or obtainable by the above-described method for producing spheroids. Human multiple myeloma (MM) spheroids comprise stroma, vascular compartments, and CD138+ plasma cells derived from patients with MM.

[0039] The spheroids are preferably autologous and are obtained by co-culturing MSCs, endothelial cells and endothelial progenitor cells with CD138+ primary plasma cells from the same patient with MM, preferably from the same sample.

[0040] Spheroids can also be heterogeneous if the primary CD138+ plasma cells are derived from a patient with MM that is different from the patient with MM or healthy subject from which the cultured MSCs, endothelial cells, and endothelial progenitor cells were obtained.

[0041] Use of spheroids The subject of the present invention is the use of autologous multiple myeloma spheroids for the selection of a suitable therapeutic treatment for a patient suffering from MM, i.e. for the selection of a therapeutic treatment to which a patient suffering from multiple myeloma (MM) is likely to respond.

[0042] In fact, the construction of a 3D model of MM that represents the patient's tumor as closely as possible allows for clinical follow-up and personalized medicine. The spheroid model according to the present invention, which is preferably autologous, can be used to study the response of tumor tissue of a patient with MM (the patient from whom the cells used to produce the spheroids are derived) to different treatments or combinations of treatments. The aim is thereby to select the therapeutic treatment to which the patient is most likely to respond.

[0043] "Treatment" or "treating," as used herein, means partially or substantially achieving one or more of the following results: partially or completely reducing the extent of the disease; ameliorating clinical symptoms or indicators associated with the disease; slowing, inhibiting, or preventing the progression of the disease; or partially or completely slowing, inhibiting, or preventing the occurrence of recurrence of the disease.

[0044] By "subject," "patient," or "disease" herein is meant a human suffering from multiple myeloma.

[0045] How to Select a Therapeutic Treatment The present invention further includes a method of using patient-derived autologous spheroids to select a therapeutic treatment to which said patient with multiple myeloma (MM) is likely to respond, comprising: - culturing the patient's autologous spheroids 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; - harvesting the autologous spheroids and dispersion thereof to collect the myeloma plasma cells; - Analysis of the viability of collected myeloma plasma cells; and - Selection of at least one candidate pharmaceutical product as a therapeutic treatment to which patients with MM are likely to respond based on the measured viability of the collected myeloma plasma cells. Includes:

[0046] According to one embodiment, at least one drug candidate is selected as a therapeutic treatment to which patients with MM are likely to respond if the measured viability of 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 drug candidate or with the addition of a control buffer) or in the presence of at least one other drug candidate.

[0047] The spheroids are cultured under the same conditions as previously defined in the method for producing autologous spheroids, except for the addition of at least one drug candidate. According to one embodiment, the selection method comprises the preparation of autologous spheroids according to the method of the present invention.

[0048] 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.

[0049] Such a selection method functions to test the effectiveness of a drug candidate or drug combination, which is added to the spheroid culture medium between the time of spheroid formation and up to 48 hours after formation, and the culture is continued for a period of at least 3 days, e.g., 4-10 days, preferably 6-8 days, or otherwise about 7 days.

[0050] In parallel, control cultures are performed under the same culture conditions as in the presence of said at least one drug candidate, but without the presence of a drug candidate or in the presence of a buffer.

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

[0052] [ka]

[0053] Examples include:

[0054] The spheroids are then recovered and mechanically dispersed, for example, with or without one or more chemical agents, such as trypsin, collagenase, or AccuMax Dispersion Solution (Capricorn Scientific GmbH), in a thermomixer and / or by repeated aspiration and ejection with a micropipette. More specifically, the spheroids can be dispersed by incubating the spheroids in a dispersion solution (e.g., AccuMax solution, containing protease and / or collagenase, and preferably containing an association of protease, collagenase, and DNase) with stirring (e.g., in a thermomixer at 37°C, 1200-1500 rpm, or again at about 1400 rpm, for about 10 minutes), followed by dispersing the spheroids by aspiration and ejection with a micropipette and recovering the dispersed cells (e.g., by centrifugation).

[0055] The cells are then labeled with a marker to identify myeloma plasma cells, for example, using a fluorescent dye associated with a specific antibody, such as CD38 to specifically label plasma cells, and CD138 to identify myeloma plasma cells CD38+CD138+. The cells are then resuspended, filtered, and then analyzed by flow cytometry (FACS). The fluorescent dye marker associated with the specific antibody can be, for example, CD38 FITC and CD138 AF700. The suspension and labeling solution is preferably MACS solution, and the cells are preferably filtered through a 70 μm filter. The viability of the myeloma plasma cells is compared between different culture conditions (control condition or containing at least one drug candidate), and reduced viability of the myeloma plasma cells compared to the control is an indication of a promising treatment.

[0056] Such a method is faster than the use of mouse models and, especially when the spheroid model is autologous, is a more significant response due to the genetic proximity between the model and the patient. In fact, spheroids can be generated in an average of about two weeks, and the selection of an appropriate treatment for the patient can be performed in about one week starting from the acquisition of the spheroids, which in principle represents a total of three weeks to be able to define an individualized treatment for a patient with MM. [Brief explanation of the drawings]

[0057] [Figure 1] Figure 1 shows the viability levels of MM plasma cells in heterogeneous spheroids after 7 days of culture with one or more of the drug candidates (melphalan 10 μM, lenalidomide 10 μM, C34 5 μM, a combination of melphalan 10 μM + C34 5 μM, a combination of lenalidomide 10 μM + C34 5 μM) and control conditions. On the horizontal axis, MM MSC (MM91, MM97, MM100) represents the stromal component of the spheroids. Plasma cell MM (P64 / 65, P66) is the plasma cell component of the spheroids. Each number corresponds to a given MM patient. [Figure 2] 1 shows the levels of viable MM plasma cells in spheroids after 7 days of culture with one or more drug candidates (carfilzomib 5-50 nM, pomalidomide 1-10 μM, dexamethasone 1 μM, isatuximab 1 μg / mL, daratumumab 1 μg / mL) and control conditions. On the horizontal axis are the various treatments tested: - DCD: daratumumab / carfilzomib / dexamethasone, - DPD: daratumumab / pomalidomide / dexamethasone, - ICD: isatuximab / carfilzomib / dexamethasone, - IPD: isatuximab / pomalidomide / dexamethasone, - CD: carfilzomib / dexamethasone, - PD: pomalidomide / dexamethasone). DETAILED DESCRIPTION OF THE INVENTION [Example]

[0058] Example 1: Investigation of various plasma cell freezing protocols and cell viability studies In this example, we attempted to select the plasma cell freezing / thawing protocol that is most effective for preserving plasma cell viability. We studied the effect by quantifying their viability after thawing by counting with trypan blue in a Malassez cell.

[0059] Following total bone marrow sampling at the time of diagnosis in patients with multiple myeloma (MM), MM plasma cells are isolated and then enumerated in a Malassez cell. The cells are then frozen according to one of the following protocols: - A: Buffer 90% fetal calf serum (FCS): 10% DMSO; Instant freezing -80℃ - B: Buffer 90% Human Serum Albumin (HSA): 10% DMSO; Instant freezing -80℃ - C: CryoStor® CS10 buffer (Sigma-Aldrich, product reference C2874); Instant freezing -80℃ - D: CryoStor® CS10 buffer; Freeze at -20°C for 2 hours, then at -80°C. - E: CryoStor® CS10 buffer; Freeze at -20°C overnight, then at -80°C.

[0060] The average freezing time was 31 days.

[0061] Subsequently, the plasma cells were thawed and then counted using trypan blue in a Malassez cell.

[0062] [Table 1]

[0063] The results in the table herein above show that the current freezing conditions, i.e., 90% FCS or HSA + 10% DMSO, appear to be ineffective compared to the immediate freezing conditions using CryoStor®.

[0064] Example 2: Culture of heterologous and autologous spheroids and study of cell viability by flow cytometry In this example, we attempted to demonstrate the minimal impact of the freeze-thaw, spheroid culture, and spheroid dispersion processes prior to labeling on plasma cell viability. We investigated this impact by quantifying their viability by flow cytometry. To this end, we attempted to label cells with fluorescent dye-conjugated anti-CD38 and anti-CD138 antibodies to specifically select MM plasma cells.

[0065] Following total bone marrow sampling at the time of diagnosis in patients with multiple myeloma (MM), MM plasma cells are isolated and then counted in a Malassez cell. The cells are then frozen in a CRYOSTOR or used fresh directly for heterogeneous co-culture. MM total bone marrow cells from another patient were seeded according to their original number, and the cells were incubated for approximately two weeks at 37°C in an atmosphere containing 5% carbon dioxide. Upon reaching confluence, mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells were treated with trypsin and counted.

[0066] Trypsinized MSC cells, endothelial cells, and endothelial progenitor cells, as well as fresh or thawed plasma cells, were suspended in 50 μL of RPMI medium (10% FCS, 1% PS) at a ratio of 1 MSC:2 plasma cells on an ultra-low attachment (ULA) plate. The cells were then incubated with stirring at 37°C in an atmosphere containing 5% carbon dioxide. After 24 hours of incubation, 150 μL of complete RPMI medium was added, 100 μL of culture supernatant was removed, and 100 μL of complete RPMI medium was added to replenish the medium twice a week.

[0067] Spheroid cells were mechanically dispersed using an AccuMax® at 1400 rpm, 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. Cells were then incubated for 30 minutes at 4°C, washed, resuspended in MACS buffer, filtered through a 70 μm filter, and labeled with DAPI before flow cytometry.

[0068] We observed that quantification of MM plasma cell viability was possible using such a protocol, and that MM plasma cell viability remained high following protocol manipulation, even after 14 days of co-culture.

[0069] Example 3: 3D spheroids of plasma cells from patients with multiple myeloma and response to treatment with melphalan In this example, we sought to demonstrate the viability of plasma cells within the spheroids as well as their accessibility to the therapeutic molecules tested.

[0070] Following total bone marrow sampling at the time of diagnosis in patients with multiple myeloma (MM), MM plasma cells were isolated and subsequently counted in a Malassez cell. The cells were then used fresh for co-culture or frozen in a CRYOSTOR. MM total bone marrow cells from another patient were seeded according to their original cell numbers, and the cells were incubated for approximately two weeks at 37°C in an atmosphere containing 5% carbon dioxide. Upon reaching confluence, mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells were treated with trypsin and counted.

[0071] Trypsinized MSC cells, endothelial cells, and endothelial progenitor cells, as well as fresh or thawed plasma cells, were suspended in 50 μL of RPMI medium (10% FCS, 1% PS) at a ratio of 1 MSC:2 plasma cells on an ultra-low attachment (ULA) plate. The cells were then incubated with stirring at 37°C in an atmosphere containing 5% carbon dioxide. After 24 hours of incubation, 150 μL of RPMI medium was added, 100 μL of culture supernatant was removed, and the medium was replenished twice a week.

[0072] 48 hours after their formation, the spheroid culture medium was treated with 10 μM melphalan, and the culture was continued for 14 days. A control condition without the presence of the drug candidate was added to the selection method. To analyze the viability of plasma cells, part of the culture was stopped after 7 (D+7) and 11 (D+11) days, and the rest of the culture was stopped after 14 days of culture (D+14).

[0073] Spheroid cells were mechanically dispersed using an AccuMax® at 1400 rpm, then transferred to a tube suitable for flow cytometry, washed with PBS, and labeled with anti-CD38 FITC and anti-CD138 AF700 specific antibodies in MACS buffer. Cells were then incubated for 30 minutes at 4°C, washed, resuspended in MACS buffer, filtered through a 70 μm filter, and finally labeled with the viability marker DAPI before flow cytometry.

[0074] The viability of plasma cells in untreated MM spheroids increased from 50% at D+7 and D+11 to 60% at D+14, whereas plasma cells in MM spheroids treated with 10 μM melphalan had a viability of 5% at D+7, less than 5% at D+11, and less than 10% at D+14.

[0075] First, we therefore showed that plasma cell viability in untreated spheroids was higher than in 2D cultures, where primary plasma cells did not survive beyond a few days (D7, D11, D14).

[0076] Subsequently, we showed that the localization of plasma cells within the spheroids did not prevent a robust response to treatment (here melphalan 10 μM).

[0077] Example 4: Response of heterogeneous MM spheroids to various drug candidates Following total bone marrow sampling at the time of diagnosis in patients with multiple myeloma (MM), MM plasma cells were isolated and subsequently counted in a Malassez cell. Cells were then used fresh or frozen at -80°C in a CRYOSTOR. MM total bone marrow cells from the same or different patients were seeded according to their initial cell numbers, and the cells were incubated for approximately 2 weeks at 37°C in an atmosphere containing 5% carbon dioxide. Upon reaching confluence, mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells were treated with trypsin and counted.

[0078] Trypsinized MSC cells, endothelial cells, and endothelial progenitor cells, as well as fresh or thawed plasma cells, were suspended in 50 μL of RPMI medium (10% FCS, 1% PS) at a ratio of 1 MSC:2 plasma cells on an ultra-low attachment (ULA) plate. The cells were then incubated with stirring at 37°C in an atmosphere containing 5% carbon dioxide. After 24 hours of incubation, 150 μL of RPMI medium was added, 100 μL of culture supernatant was removed, and 100 μL of RPMI medium was added to replenish the medium twice a week.

[0079] Drug candidates, candidate combinations, or candidate combinations were added to the culture medium of the spheroids between the time of their formation and 48 hours after their formation, and the culture was continued for 7 days. This selection method was complemented by a control situation without the presence of any drug candidates, with the same culture duration as in the presence of the drug candidates.

[0080] The treatments tested were as follows: - melphalan 10 μM, - lenalidomide 10 μM, - C34 5 μM, - a combination of melphalan 10 μM + C34 5 μM, - Lenalidomide 10μM + C34 5μM combination

[0081] Spheroid cells were mechanically dispersed using an AccuMax® at 1400 rpm, then transferred to a tube suitable for flow cytometry, washed with PBS, and labeled with anti-CD38 FITC and anti-CD138 AF700 specific antibodies in MACS buffer. Cells were then incubated for 30 minutes at 4°C, washed, resuspended in MACS buffer, filtered through a 70 μm filter, and finally labeled with DAPI before flow cytometry.

[0082] Figure 1 shows the viability levels of MM plasma cells in heterogeneous spheroids after 7 days of culture using various potential treatments tested, as well as control conditions. On the horizontal axis, MM MSC (MM91, MM97, MM100) represent the stromal component of the spheroids. Plasma cell MM (P64 / 65, P66) are the plasma cell component of the spheroids. Each number corresponds to a given MM patient.

[0083] FIG. 1 clearly shows the differences in plasma cell behavior under various conditions and the effectiveness of combined melphalan+C34 and lenalidomide+C34 treatment in reducing MM plasma cell viability.

[0084] Example 5: Spheroid response to various drug candidates and combinations of candidates Total bone marrow cells from patients with multiple myeloma (MM) were first seeded into flasks in EGM2 medium according to the initial cell count in the test tube. The cells were incubated at 37°C and 5% CO for approximately 2 weeks.

[0085] Plasma cells from patients with multiple myeloma (MM) were first counted in Malassez cells and trypan blue. Cells were either frozen in CryoStor (autologous cultures) or used directly for heterologous spheroid cocultures.

[0086] MSCs to be used for co-culture were trypsinized and then counted.

[0087] On a 96-well ULA plate and in RPMI medium (10% FBS): - For each plasma cell sample collected (fresh or thawed), a control (in triplicate) containing cells only was generated (50,000 plasma cells / well). - Each plasma cell sample was co-cultured with each trypsinized MSC sample (ratio 1:2 = 50,000 MSCs:100,000 plasma cells per well, 50 μL / well) - Plates were incubated at 37°C and 5% CO2 with orbital shaking at 73 rpm for 24 hours. - 150 μL of medium (with or without drugs) was added to the culture medium, after which the ULA plates were re-incubated. Medium was changed once a week by removing 100 μL / well and adding 100 μL of fresh medium (with or without drugs). - The culture was stopped at D7 and the spheroids were dispersed according to the protocol described herein above.

[0088] Carfilzomib was used at concentrations of 5 to 50 nM, pomalidomide was used at concentrations of 1 to 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.

[0089] Figure 2 shows the viability levels of MM plasma cells in heterogeneous spheroids after 7 days of culture using the various treatments and control conditions tested. The various treatments tested are indicated on the horizontal axis: DCD: daratumumab / carfilzomib / dexamethasone; DPD: daratumumab / pomalidomide / dexamethasone; ICD: Isatuximab / Carfilzomib / Dexamethasone, IPD: isatuximab / pomalidomide / dexamethasone; CD: carfilzomib / dexamethasone; PD: pomalidomide / dexamethasone).

[0090] For the left side of the graph, frozen plasma cells were used, and each mode was repeated four times (n=4). For the right side of the graph, fresh plasma cells were used, i.e., plasma cells were not frozen before co-culturing to form spheroids; here, each mode was repeated three times (n=3).

Claims

1. 1. A method for producing human multiple myeloma (MM) spheroids, comprising: a. culturing mesenchymal stem / stromal cells (MSCs), endothelial cells, and endothelial progenitor cells in a culture medium; b. harvesting the cultured MSCs, endothelial cells, and endothelial progenitor cells; and c. Co-culturing the collected MSCs, endothelial cells, and endothelial progenitor cells with CD138+ primary plasma cells from a patient with MM under conditions conducive to spheroid formation. A method comprising:

2. The method of claim 1, wherein the MSCs, endothelial cells, endothelial progenitor cells and CD138+ primary plasma cells are obtained from the same patient suffering from MM.

3. 3. The method of claim 1, wherein the MSCs, endothelial cells, endothelial progenitor cells and CD138+ primary plasma cells are obtained from the same bone marrow sample from the patient with MM.

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

5. 5. The method of any one of claims 1 to 4, wherein the CD138+ primary plasma cells are preserved by freezing at a temperature of -70°C or below in a cryopreservation medium prior to the co-culture step.

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

1.

7. 7. The method according to claim 1, wherein the step of co-culturing MSCs, endothelial cells and endothelial progenitor cells with primary plasma cells is carried out for 4 to 14 days.

8. A human multiple myeloma (MM) spheroid obtained by the manufacturing method described in any one of claims 1 to 7, comprising stroma, vascular compartment and CD138+ plasma cells of a patient suffering from MM.

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

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

11. 1. A method for selecting a therapeutic treatment to which a patient with multiple myeloma (MM) is likely to respond, comprising: a. Cultivating the autologous spheroids of 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 dispersion thereof to collect the myeloma plasma cells present in the autologous spheroids; c. Analysis of the viability of collected myeloma plasma cells; and d. Selection of at least one candidate pharmaceutical product as a therapeutic treatment to which patients with MM are likely to respond based on the measured viability of the collected myeloma plasma cells. A method of selecting a therapeutic treatment, comprising:

12. 12. The method of claim 11, wherein at least one drug candidate is selected as a therapeutic treatment to which a patient with MM is likely to respond if the measured viability of 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 drug candidate.

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

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