Endothelial Progenitor Cells within the Bone Marrow Microenvironment of Multiple Myeloma Patients Display Stem Cell Traits.

Multiple myeloma (MM) is an incurable disease of clonal plasma cells that accumulate in the bone marrow (BM). Tumor growth and progression are promoted in MM through paracrine interactions between tumor cells, stromal cells, and matricellular components of the BM microenvironment. Endothelial progenitor cells (EPCs) are a key component of this microenvironment that mediate neovascularization. EPCs in MM patients contribute to neoangiogenesis and enhance the growth of tumor cells through the secretion of VEGF and IL-6. We found that EPC levels are increased in MM patients and covary with disease severity. Further, EPCs from MM patients were found by X chromosome inactivation studies to be clonally restricted, and to display clonotypic IG heavy-chain gene rearrangements identical to the same patients' tumor cells. Based on new data regarding clonal restriction and on maintenance of MM EPCs in long-term culture compared both to EPCs obtained from normal controls and to human umbilical vein endothelial cells, the present study explored the hypothesis that EPCs in the vascular MM niche may be a source of MM-initiating cells with stem cell traits. The existence of MM stem cells is consistent with the finding of inevitable relapse of MM patients following response to treatment. We now show that clonal MM EPCs can be maintained in long term culture, unlike EPCs from healthy controls. In addition, our gene expression data show that MM EPCs have a higher level of expression of genes associated with stem cell function (e.g., GREM1, RUNX2, HOXC6, FOXF2) compared to the same patients' MM tumor cells, as well as to control EPCs. Genes such as WNT5A, SOX9, and ADAM12, which are involved in differentiation, an integral stem cell process, were also highly expressed in MM EPCs (manuscript in preparation). Stem cells have the abilities of self-renewal and differentiation. Cancer stem cells (CSCs), such as those found in acute myeloid leukemia, share these characteristics. Similar to hematopoietic stem cells (HSCs), CSCs are known to engraft to spleen and BM, where they proliferate and are capable of forming spleen colonies. To determine whether MM EPCs were able to home and proliferate in a similar fashion, we intravenously injected them into non-lethally irradiated mice. Using flow cytometry we examined the prevalence of human EPCs that expressed differentiation markers. The formation of spleen colonies was also assessed.

EPCs (>98% vWF/CD133/ KDR+ and CD38-) from BM aspirates of MM patients were outgrown on laminin-coated flasks as previously described. Nine irradiated NOD/SCID mice were injected (i.v.) with 10⁶ EPCs derived from 6 patients newly diagnosed with advanced MM. Spleens and BM were harvested 2-4 weeks later. The cells were analyzed by immunofluorescence and flow cytometry to determine the presence of human cell differentiation markers, including CD45 (a hematopoietic differentiation marker) and CXCR4 (a differentiation/migration marker).

EPCs cultured from 4 newly diagnosed patients were maintained in culture up to 7 months. Flow cytometry showed the engraftment of human cells to the spleen and BM of all mice 2-4 weeks following injection. The mean number of CD45+ cells in the experimental group increased within the spleen by 54.1 fold and in the BM by 35.9 fold compared to vehicle-injected controls. Similarly, CXCR4+ cells in the experimental group increased within the spleen by 15.9 fold and in the BM by 12.5 fold. In addition, of the 9 spleens harvested, 1 spleen showed colony formation, while none of the vehicle-treated animals displayed colony formation.

Results show a high degree of proliferation of CD45+ cells within the spleen and BM following injection of MM EPCs. Since CD45 is a marker of hematopoietic differentiation, the proliferation of cells expressing this marker suggests that human hematopoietic cells expanded after EPCs were injected. Furthermore, the proliferation of cells expressing CXCR4+, which is required for HSC quiescence and which also plays a role in EPC differentiation, indicates that EPCs are part of the stem cell niche in MM. In addition to the sizeable increase seen in the number of cells expressing these differentiation markers, 1 of 9 mice injected, formed spleen colonies. Taken together, these data suggest that a portion of the MM EPC population possesses traits similar to those of HSCs and CSCs, and this thesis warrants further investigation.

No relevant conflicts of interest to declare.