Characterization of Hemangioblastic Traits within Endothelial Progenitor Cells of Multiple Myeloma Patients

Multiple myeloma (MM), a neoplasm of committed B-lymphocytes within the bone marrow (BM), is the second most common hematologic malignancy in the U.S. Despite prolonged median survival with anti-myeloma strategies aimed at the tumor and its BM microenvironment, MM remains invariably fatal. Endothelial progenitor cells (EPCs) are CD133+/KDR+ cells that originate in the BM and play a key role in supporting tumor growth and MM progression. Using X-chromosome inactivation and RT-PCR analyses, we previously found EPCs from MM patients to be clonally restricted and to display clonotypic IG heavy-chain gene rearrangements identical to the same patients' tumor cells (Braunstein et al., 2006). Based on the shared genetic identity that we and others have demonstrated between tumor cells and EPCs in MM patients, the present study explored the hypothesis that, similar to hemangioblasts, which are CD133-expressing precursors to adult hematopoietic and endothelial cells, EPCs may be a source of vascular and MM progenitor cells. Since hemangioblasts are known to exist predominately in the quiescent phases of the cell cycle, in this study we also examined the cell cycle status of CD133-expressing BM cells from MM patients in order to gain insight into their hemangioblastic traits.

BM aspirates were acquired from MM patients under IRB approval. EPCs (>98% vWF/CD133/KDR+ and CD38-) from BM aspirates of MM patients were outgrown on laminin-coated flasks as previously described. The spleen colony assay was used to determine the stem cell capacity within BM-derived EPCs by i.v. injection into NOD/SCID mice. The spleens and BM of mice were harvested 2–4 weeks later. Cells were analyzed by immunofluorescence (IF) and flow cytometry. Hoechst 33342 (Hst) and Pyronin Y (PY) were used to measure the cell cycle status of CD133+ cells using FACS analysis.

Two to four weeks following i.v. injection of MM EPCs, human cell surface marker expression detected by flow cytometry within mouse BM and spleen cells shifted from CD133+/CD45-lo, a progenitor cell phenotype, to CD133−/CD45-hi, a more differentiated phenotype, suggesting the ability of MM EPCs to differentiate in vivo. Cell cycle analysis of the CD133+ population in BM cells of MM patients showed that these cells were predominantly non-cycling. On average, 60.5% of CD133+ cells were found to be in the G0/G1 phase of the cell cycle, as indicated by low levels of IF staining with Hst and PY.

CD133+ cells are strong candidates as precursors to MM tumor and vascular progenitor cells. As quiescent cells are non-dividing, they often escape cytotoxic effects of chemotherapy, resulting in relapse, and therefore, identification of these cells is critical. Ongoing studies include the engraftment of CD133+ cells in the subcutaneous NOD/SCID gamma xenotransplant model and their growth in response to anti-myeloma strategies; results of these studies will be discussed.

No relevant conflicts of interest to declare.