The Analysis of the Relationship between Multiple Myeloma Cells and Their Microenvironment

Abstract

The bone marrow microenvironment plays a key role in the stimulation of growth and survival of multiple myeloma (MM) cells. We investigated whether membrane microfragments (MFBs) exert a stimulatory effect on mesenchymal stem cell (MSC) gene expression or differentiation. MSCs from patients with multiple myeloma (MMBM-MSCs) proliferated at a slower rate than MSCs from healthy volunteers (BM-MSCs), and fewer MMBM-MSCs adhered to the substrate as compared to BM-MSCs. Phenotypic analysis revealed that MMBM-MSCs and BM-MSCs differed significantly in terms of their CD166 and CXCR4 expressions. In conclusion, our comparative analysis of mesenchymal cells from MM patients and healthy volunteers revealed differences in the genetic and phenotypic profiles of these two populations, their potential for osteodifferentiation, and expression of surface antigens. Moreover, we showed that membrane MFBs may alter the genetic profile of MSCs, leading to disorders of their osteodifferentiation, and interact with the WNT pathway via presentation of the DKK-1 protein.

Keywords: bone marrow; genotype; membrane microfragments; microenvironment; multiple myeloma; osteodifferentiation.

Figure 1

A. The rate of proliferation of BM-MSCs and MMBM-MSCs. 1-4 denotes number of cell passages. B. The growth rate of BM-MSCs and MMBM-MSCs after 24, 48, 72 and 96 h of cells culturing.

Figure 2

Comparison of surface antigens expressed by MMBM-MSCs and BM-MSCs. Cells were stained using the appropriate antigens and analysed using the FACS method. Statistically significant differences were documented for CD166 and CXCR4 antigens (n=5, p<0.05).

Figure 3

Genotyping of MMBM-MSCs and BM-MSCs from the third passage. Results were normalized, relative to the GAPDH control. The graphs show the level of mRNA for A) IL8, HGF, VEGF and β-FGF, B) TGF-β, IL-1 and IL-11, C) RUNX2, α1col, osteocalcin, PPAR-γ and PTHR, and D) MMP-2 and MMP-9 genes.

Figure 4

Expression of genes in MMBM-MSCs and BM-MSCs after 8-, 24- and 48-h stimulation with MM-MFBs. The cells were grown on 6-well plates in DMEM with 10% FBS for MSCs, 2 mM L-glutamine and antibiotics. Results were normalized, relative to the GAPDH control. The graphs show the level of mRNA for A) IL8, HGF, VEGF and β-FGF, B) TGF-β, IL-1 and IL-11, C) RUNX2, α1col, osteocalcin, PPAR-γ and PTHR, and D) MMP-2 and MMP-9 genes.

Figure 5

Osteoinduction of BM-MSCs in the presence of MM-MFBs. The cells from the third passage were cultured for 7, 14 and 21 days in the differentiation medium (DMEM with 10% FBS, dexamethasone, β-glycerophosphate and ascorbic acid). Subsequently, the cells were fixed in 4% paraformaldehyde, and calcium phosphate deposits were stained with alizarin red S. Legend: + MFB - differentiation in presence of 20 µl/ ml myeloma membrane microfragments, -MFB - differentiation in absence of any additional factors, control - cells that were not subjected to the differentiation. Pictures were taken at a 100 x (A) and 200 x (B) magnification. Representative images are shown.