Comparison of Stromal Cells of Adipose Tissue From Multiple Myeloma Patients and Healthy Donors

Multiple myeloma (MM) is a B-cell neoplasia characterized by the accumulation of malignant plasma cells in the bone marrow (BM) patients causing severe bone disease (with osteolytic lesions, pain, pathological fractures) and cytopenias. These lesions are irreversible, even for patients in complete response to treatment. We have proposed to treat those lesions with mesenchymal stem cells (MSC) of the BM using their ability to differentiate into osteoblasts and to support haematopoiesis. But our previous results demonstrated that MM MSC are abnormal. We thus studied cells from adipose tissue (AT) with similar properties than MSC; the Adipose derived Stromal Cells (ASC). The aim of this study is to demonstrate the normality of ASC in MM context for a potential use in autologous stem cell transplantation. Here we propose the first study comparing ASC issue from MM patients and healthy donors (HD).

We studied ASC from 15 patients with newly diagnosed MM and 15 HD between 18 and 65 years. All gave their informed consent. The stromal vascular fraction was isolated from subcutaneous AT by and centrifugation. The ASC were sorted by adhesion to the plastic flask and expanded for 3 passages. We then performed the following assays to compare ASC from MM and HD:

• Cell culture assay in MEMa medium containing 10% FBS and ciprofloxacine for 3 passages (P) of 21 days;

• CFU-F test to evaluate the progenitor frequency for each P;

• Flow cytometry at the end of the first P for CD73, CD90, CD45, CD34, CD14, CD49a, CD13, CD105, CD31, CD146, CD29, CD164, HLA ABC, CD166, TLR4, CD80, CD83, CD10, CD38, CD138, CD26, CD157, CD106, CXCR4;

• Differentiation assay in specific induction media with qualitative and quantitative analysis for osteogenic (red alizarin coloration and quantification), chondrogenic (blue alcian coloration and chondroitin sulfate dosage) and adipogenic (Nile red coloration and glycerol measurement) lineages;

• ELISA measurement of IL-6, DKK-1 and GDF15 in culture supernatant at the end of the first P;

• Determination of the number of MOLP-6 (stroma-dependant MM cell line) after 7 days of co-culture with ASC in serum free medium.

The cell culture assay of ASC didn't show any difference between MM and HD for all the studied parameters: expansion capacity (HD 470± 45, MM 208±194; p=0.13), cell population doubling (HD 16.0±0.3, MM 15.0±1.3; p=0.17) and progenitor frequency (HD 3.4%±3, MM 3.7%±5.5; p=0.165). ASC phenotype didn't show any significant difference for all the checked markers and confirm their stromal feature: positive for CD90, CD73 and CD105, and negative for CD14 and CD45. The differentiation assay was evaluated for osteogenic, chondrogenic and adipogenic lineages. We did not underline any difference comparing ASC from MM or HD (Table 1). We previously showed that MM MSC secreted increased amount of IL-6, GDF15 and DKK-1 when compared with HD MSC. Interestingly, no difference was observed between MM ASC and HD ASC (Table 2). In the same way, contrary to MM MSC, MM ASC didn't promote the proliferation of MOLP-6 cell line better than HD ASC (proliferation rate: 1.49±0.34 for HD and 1.66±0.80 for MM; p=0.33).

To our knowledge, this is the first study to compare ASC from MM patient and HD. These preliminary data suggest that ASC from MM patients are normal and could potentially be used in autologous stem cell transplantation for MM patients. We are currently completing this study by performing haematopoiesis support and microarrays assays.

No relevant conflicts of interest to declare.