Increased Expression of Pro-Angiogenic and Decreased Expression of Osteogenic Genes by Multipotential Stromal Cells after Stimulation with Myeloma-Derived Microvesicles.

Despite recent advances in the treatment protocols, Multiple Myeloma (MM) is still an incurable disease with median patients’ survival of five years. This dismal prognosis is mostly due to resistance of MM to conventional therapies. Recently, attention has been directed toward studying the role of bone marrow microenvironment in MM progression in order to find new ways to inhibit tumor growth by blocking interaction between tumor cells and their microenvironment. Microvesicles (MV) are small membrane fragments released by eukaryotic cells during cell membrane turnover or cell activation. Our group presented the evidence that PMV can facilitate haematopoietic stem cells engraftment in myeloablated recipients (Janowska-Wieczorek et al Blood 2001) and also that PMV could stimulate normal and malignant haematopoietic cells growth and survival (Baj-Krzywozeka et al Exp. Hematology 2001). Recently, it has been shown that PMV may transfer several platelet-derived adhesion molecules to the lung (Janowska-Wieczorek et al IJC 2005) or breast cancer cells (Janowska-Wieczorek et al Transfusion 2006) and thus increase their metastatic potential. In this study we assessed the influence of myeloma-derived microvesicles (MMV) on multipotential stromal cells (MSC). We also compared the gene expression profile of MSC from ten healthy donors (hMSC) and ten MM patients (mMSC). MMV were isolated from MM cell lines supernatants. Expression level of mRNA for genes involved in angiogenesis, invasion and MSC proliferation and osteoblastic differentiation were evaluated using qRT-PCR. hMSC were exposed to 20 ug/ml of MMV for 8 and 24 hours and changes in gene expression were quantified. We noticed increased level of IL8 expression: 4.5 fold after 8 hours and 2 fold after 24 hours stimulation. Upregulation of MMP9 expression was seen at 8 and 24 hours. HGF expression was decreased by approximately 2 folds at both 8 and 24 hours. Interestingly, 8-hour exposure of MSC to MMV resulted in downregulation of RUNX2, collagen1 and osteocalcin mRNA by 1.5, 3 and 2 folds, respectively. After 24 hours, level of downregulation of RUNX2 and collagen1 remained constant and level of osteocalcin decreased further to 3.5 folds. Western blot analysis revealed expression of Dickkopf 1 (Dkk1) protein by MMV. Dkk1 is a well know osteogenic inhibitor and it has been shown to be present in serum of MM patients (Tian E et at N Engl J Med 2003). We analyzed gene expression in mMSC in comparison to hMSC and observed increased level of IL8 (14 folds), VEGF and MMP9 (3 folds), and decreased level of HGF (2 folds). Difference in osteogenic genes expression in mMSC were also observed. RUNX2, collagen1 and osteocalcin were downegulated by 6, 11 and 5 folds respectively. Our study showed for the first time that MMV can induce expression of angiogenic and invasion promoting genes and inhibit expression of osteoblastic genes in MSC. This suggests presence of pro-tumorigenic activators and osteogenic inhibitors in MMV (including Dkk1). Moreover, MSC isolated from MM patients have skewed expression of several pro-tumorigenic and osteogenic genes in comparison to healthy controls. This data might suggest that MMV produced by MM cell in vivo could negatively influence bone marrow environment leading to bone lesions and pathological angiogenesis.