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US-20260125654-A1 - Method for Producing a Three-Dimensional Human Multiple-Myeloma Model

US20260125654A1US 20260125654 A1US20260125654 A1US 20260125654A1US-20260125654-A1

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 mesenchymal stem/stromal cells, endothelial progenitors and primary plasma cells of patient(s) affected by MM. The present invention further relates to the spheroids obtained by said method and the uses thereof.

Inventors

  • Nicolas Lucien ESPAGNOLLE
  • Mélanie Alexandra GADELORGE
  • Jill CORRE

Assignees

  • ETABLISSEMENT FRANÇAIS DU SANG
  • INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
  • UNIVERSITE TOULOUSE III-PAUL SABATIER
  • CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
  • CENTRE HOSPITALIER UNIVERSITAIRE DE TOULOUSE

Dates

Publication Date
20260507
Application Date
20230929
Priority Date
20220930

Claims (15)

  1. 1 . A method for producing human multiple myeloma (MM) spheroids comprising: a. Culturing mesenchymal stem/stromal cells (MSCs), endothelial cells and endothelial progenitors in a culture medium; b. Harvesting cultured MSCs, endothelial cells and endothelial progenitors; and c. Co-culturing MSCs, endothelial cells and endothelial progenitors harvested with CD138+ primary plasma cells from a patient with MM under conditions that lead to spheroid formation.
  2. 2 . The production method according to claim 1 , wherein the MSCs, endothelial cells, endothelial progenitors and CD138+ primary plasma cells were obtained from the same patient suffering from MM.
  3. 3 . The production method according to claim 1 - or 2 , wherein the MSCs, endothelial cells, endothelial progenitors and CD138+ primary plasma cells were obtained from a same bone marrow sample from said patient with MM.
  4. 4 . The production method according to claim 1 , wherein the MSCs, endothelial cells and endothelial progenitors are cultured in step a. for 3 to 30 days.
  5. 5 . The production method according to claim 1 , wherein the CD138+ primary plasma cells were preserved, prior to co-culturing, by freezing at a temperature less than or equal to −70° C. in a cryopreservation medium.
  6. 6 . The production method according to claim 1 , wherein the CD138+ primary plasma cells and MSCs are co-cultured at a ratio of about 2:1.
  7. 7 . The production method according to claim 1 , wherein the co-culturing of MSCs, endothelial cells and endothelial progenitors with the primary plasma cells is carried out for 4 to 14 days.
  8. 8 . Human multiple myeloma (MM) spheroids obtained by a production method according claim 1 , comprising a stroma, a vascular compartment and CD138+ plasma cells of patient(s) affected by MM.
  9. 9 . MM spheroids according to claim 8 , wherein said spheroids are autologous spheroids.
  10. 10 . A 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. 11 . A method of selecting a therapeutic treatment for which a patient with multiple myeloma (MM) is likely to respond, comprising: i. The culture of 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; ii. The harvesting of autologous spheroids and the dissociation thereof so as to collect the myelomatous plasma cells thereof present in autologous spheroids; iii. The analysis of the viability of collected myelomatous plasma cells; and iv. The selection of the at least one candidate medicinal product as a therapeutic treatment to which the patient with MM is likely to respond, on the basis of the measured viability of the collected myelomatous plasma cells.
  12. 12 . A method of 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 with MM is likely to respond if the measured myelomatous plasma cell viability is decreased compared to the viability of myelomatous plasma cells obtained from autologous spheroids cultured under control conditions or cultured in the presence of at least one other drug candidate.
  13. 13 . A method of selecting a therapeutic treatment according to claim 11 , wherein said 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 h after the formation thereof, and culturing is continued for said period of at least 3 days.
  14. 14 . A method of selecting a therapeutic treatment according to claim 12 , wherein said 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 h after the formation thereof, and culturing is continued for said period of at least 3 days.
  15. 15 . The production method according to claim 2 , wherein the MSCs, endothelial cells, endothelial progenitors and CD138+ primary plasma cells were obtained from a same bone marrow sample from said patient with MM.

Description

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 mesenchymal stem/stromal cells, endothelial progenitors and primary plasma cells from patient(s) affected by MM. The present invention further relates to the spheroids obtained by said method and the uses thereof. Multiple myeloma (MM) is a hematologic malignancy, also known as bone marrow cancer. MM 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 cells of the immune system derived from the bone marrow (BM) that produce antibodies to protect the body against external attacks (bacteria, viruses). During development, genetic anomalies (deletion, chromosomal translocation) may occur, thereby transforming healthy plasma cells into malignant plasma cells. In the normal state, the plasma cells circulate in the blood while in the pathology of MM same return to the bone marrow where same cause damage at several levels. Multiple myeloma remains an incurable disease despite recent remarkable therapeutic advances. Care currently prevents or relieves symptoms and complications, destroying pathological plasma cells and slowing the progression of the disease. To date, multiple myeloma suffers from the absence of relevant preclinical models. Indeed, murine models are expensive, time consuming, and not representative of human pathology. Standard two-dimensional (2D) models do not provide the viability of primary plasma cells of patients and favor the use of immortalized cell lines, which makes the models too far from the pathophysiology of MDM disease The reproduction of adult human ex vivo bone marrow is increasingly described in the literature in order to overcome animal models that are expensive, time consuming and dependent on the species barrier. Studies have begun to reveal 3D models of human ex vivo bone marrow grouping mesenchymal and vascular compartments, generally from cells derived from immortalized cell lines. For example, the vascular compartment, playing an active role in the proliferation of hematopoietic stem cells (HSCs), is often incorporated via HUVEC endothelial immortalized cell lines. Other models require a step in mice for vascularization or functional approaches. Furthermore, the short half-life of the plasma cells does not allow same to be incorporated there in an autologous manner into the current 3D models and forces same to be ignored or tumor immortalized cell lines of plasma cells to be added, which does lead to a relevant response of the model with respect to the patient. The inventors have created a new human 3D tissue model of multiple myeloma. Same comprises the mesenchymal compartment, the vascular compartment and the plasma cells, obtained from samples from patient(s) suffering from multiple myeloma, without the use of any immortalized line cell. In particular, the maintenance of the viability of plasma cells in co-culture over more than 14 days has been resolved. As a result, a fully human preclinical ex vivo model of multiple myeloma is generated, which is genetically relevant to said patient and includes the mesenchymal, vascular and plasma cell compartments in spheroid form. Such model makes it possible to envisage an advance in personalized medicine by means of the rapid construction of a model representative of the tumor tissues of the bone marrow of each patient. DETAILED DESCRIPTION OF THE INVENTION Method for producing human multiple myeloma spheroids The invention relates to a method for producing human multiple myeloma (MM) spheroids, comprising: a. Culturing mesenchymal stem/stromal cells (MSCs), endothelial cells and endothelial progenitors in a culture medium;b. Harvesting cultured MSCs, endothelial cells and endothelial progenitors; andC. Co-culturing MSCs, endothelial cells and endothelial progenitors harvested with CD138+ primary plasma cells from a patient with MM under conditions that lead to spheroid formation. “Mesenchymal stem cells”, also called “mesenchymal stromal cells”, mean stem cells of mesodermal origin. Same are characterized phenotypically by the co-expression of a certain number of markers such as e.g. CD73, CD90, CD105, CD146, and the absence of expression of other markers, more particularly CD45, CD31 and CD34. Same can be derived from bone marrow, adipose tissue, or umbilical cord blood. Mesenchymal stem or stromal cells are of human origin and come from a patient with MM or from a healthy subject. In a preferential mode, the mesenchymal stem/stromal cells cultured in step a. are primary cells. “Endothelial progenitors” refer to cells engaged in endothelial differentiation but which are not yet recognizable as endothelial cells under the microscope. Same are characterized phenotypically by the expression of a certain number of markers such as e.g. CD133, CD34, CD31, VEGFR2. “Endothelial c