Bone marrow-derived mesenchymal stromal cells in the tumor microenvironment of neuroblastoma

2015-04-21Bone marrow mesenchymal stromal cells (BMMSC) migrate from the bone marrow into the circulating blood stream where overtime they differentiate to adiposities, chondrocytes, myocyte, fibroblasts, osteoblasts or other cell derivatives. We hypothesized that BMMSC migrate from the bone marrow into the primary tumor and participate in the tumor microenvironment. Cells expressing characteristics of BMMSC were obtained from human and mouse bone marrow. Using a migration assay, human BMMSC were assessed for their ability to migrate toward five neuroblastoma cell lines. Higher migration rates were observed in co-culture conditions of CHLA-255 (P < 0.001), SK-N-BE(2) (P < 0.001), SK-N-SH (P < 0.001), SH-5YSY (P < 0.005) and NB-19 (P < 0.001), concluding a chemoattraction (Figure 2). To analyze the presence of small molecules, cytokines and chemokines with a potential role in chemoattraction, Luminex was used as a screening panel (Figure 3) and ELISA assays (Figure 4) validated increased expression levels of IL-6 (462 pg/mL), IL-8 (208 pg/mL) and MCP-1 (1,584 pg/mL), when human BMMSC were co-cultured with CHLA-255. Further analysis of MCP-1 as a recombinant protein did not show increased migration (P > 0.5) concluding an insignificant role in chemoattraction. In vivo, mouse BMMSC were placed in the femur (Figure 11) or intravenously (Figure 10) and tracked via bioluminescence for their ability to migrate towards neuroblastoma CHLA-255 tumors orthotopically injected into the kidney of immuno-deficient (nude) mice over a period of six weeks. On day 24, mouse BMMSC injected into the femur appeared to have migrated to the tumor kidney; however image may have presented a misleading signal based on additional analysis of the lysed tumor kidney levels of luminescence (P < 0.001). Preliminary data may suggest that some BMMSC may have migrated toward the tumor, but experiments to validate this observation are needed. NIH-3T3 murine skin fibroblasts injected via femur or intravenously were used as a control and did not migrate to the tumor kidney, as confirmed by ex vivo luminescence of lysed tumor kidney (P < 0.001). We demonstrated that BMMSC are attracted to neuroblastoma cells in vitro, and could migrate from the bone marrow to primary tumors in vivo while human fibroblasts and NIH-3T3 did not migrate to the tumor kidney. Overall, this data illustrates that BMMSC may be able to migrate from the bone marrow into the primary tumor. Future studies should assess BMMSC as being important mediatord of the tumor microenvironment and understanding the chemoattraction pathways