Are Stromal Cells of Adipose Tissue From Multiple Myeloma Patients Are Normal ? A Comparison of Adipose Derived Stromals Cells From Healthy Donors and Multiple Myeloma Patients

One of the main feature of MM is bone disease resulting from an increased osteoclastic activity and inhibition of osteoblast function; this imbalance also promotes MM growth leading to a vicious circle. Interestingly mature osteoblasts reduce osteoclastogenesis via high levels of OPG and reduced levels of RANKL and have anti-MM effect too, especially via decorin causing MM cells apoptosis. Hence, therapeutic approaches restoring osteoblast function could reduce osteoclastogenesis and MM growth. Mature osteoblasts originate from bone marrow mesenchymal stromal cells (MSC) that are key component of MM microenvironment. We and others showed that MSC from patients with MM are abnormal. In particular, they have an impaired capacity to diffentiate into osteoblasts. Hence we studied mesenchymal stromal cells from adipose tissue (AT) called the Adipose derived Stromal Cells (ASC). They have very similar properties than MSC, in particular osteoblastic differentiation capacity. The aim of our work is to demonstrate that ASC from patients with MM are normal, contrary to their MSC; we propose the first study comparing ASC from MM patients and ASC from Healthy Donor (HD) with functional and genomics tests.

We studied ASC from 15 patients with newly diagnosed MM (MM ASC) and CRAB symptoms and from 15 HD (HD ASC) between 18 and 65 years. The stromal vascular fraction was isolated from subcutaneous AT by centrifugation and enzymatic digestion. The ASC were sorted by adhesion to the plastic flask and expanded for 3 passages of 21 days in culture medium MEMa containing 10% FBS and ciprofloxacine. We perform:

• CFU-F test

• Flow cytometry 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 media with qualitative and quantitative analysis for osteogenic, chondrogenic and adipogenic lineages;

• ELISA measurement of IL-6, DKK-1 and GDF15 in culture supernantant

• Co-culture of MOLP-6 (stroma-dependant MM cell line) with ASC in serum free medium

• Hematopoietic support assay using CD34 positive cells co-cultured ASC

• Gene Expression Profiling on GeneChip Human Gene 1.0 ST Array (RNA was extracted using the RNeasy Kit Quiagen)

• miRNA profile on SmartChip miRNAv3 (miRNA were extracted with miRNeasy Mini Kit Quiagen)

Our data confirm the stromal nature of MM and HD ASC. The cells were adherent and proliferate into plastic flasks; able to differentiate into osteogenic, chondrogenic and adipogenic lineage; positive for CD90, CD73, CD105 and negative for CD14, CD45. We next found that MM and HD ASC have the same 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=0.166). Phenotype didn't show any significant difference except for CD200. Osteogenic, chondrogenic and adipogenic differentiation assays didn't show any difference according to the origin of ASC; alizarin (mmol/l): HD 2.66±0.6, MM 2.15±2.1 p=0.15, chondroitin sulfate (ng/ml): HD 0.5±0.17, MM 0.58±0.58 p=0.2, glycerol (mg/well): HD 2.7±1.6, MM 3.28±1.2 p=0.26. Measurement of IL-6 (HD 7143±10029, MM 10192±6379 pg/ml p=0.15), DKK1 (HD 16698±2432, MM 15948±6558 pg/ml p=0.98), and GDF15 (HD 0.29±0.3, MM 0.43±0.37 ng/ml p=0.13) shows similar levels in both population. We then analysed proliferation rate of MOLP-6 in co-culture with ASC and observed no difference (proliferation rate HD 1.49±0.34, MM 1.66±0.80 p=0,33). MM ASC and HD ASC had the same capacity to support haematopoiesis. Finally, genomic analysis performed by GEP did not identify any difference between the two groups. miRNA analysis tested 1036 targets and found only one, HSA-MIR-196A, differentially expressed between HD and MM ASC(p=0,014).

To our knowledge, this is the first exhaustive study that compare ASC from MM patients and HD. Our results strongly suggest that MM ASC are normal and could potentially be used in autologous stem cell transplantation in order to restore the patients' osteoblastic function.

Roussel:celgene: Honoraria; janssen: Honoraria. Attal:celgene: Membership on an entity's Board of Directors or advisory committees; janssen: Membership on an entity's Board of Directors or advisory committees.