Bone Marrow Microenvironment Of Advanced Breast Cancer Patients Without Bone Metastasis Favors The Cancer Cell Colonization

Bone metastasis is the major cause of death for advanced breast cancer patients (BCP). It is a multistep process that includes tumor cell mobilization, intravasation, survival in the circulation, extravasation and proliferation in bone marrow (BM) or bone. Accumulating evidence suggests that BM-mesenchymal stromal cells (MStC) play a critical role in BC-cell (BCC) colonization of the BM/bone. Despite increasing knowledge, the beginning of bone metastatic process in advanced BCP without BCC in the BM/bone has been poorly studied. So, this work was performed to evaluate the levels of OPG, RANKL, TRAIL, SDF-1, PDGF-AB, stanniocalcin-1 and MIF in peripheral blood (PB) and BM-plasma and BM-conditioned media (CM) of colony forming unit-fibroblastic cultures (CFU-F, day 14) from advanced BCP (IDC, without BM/bone metastasis) and healthy volunteers (HV). Also, we investigated the expression of membrane-RANKL (mRANKL) and SDF-1 in BM-MStC and their specific receptors in primary BC-tissue from these patients and MCF-7 and MDA-MB231 cells. At the end, we evaluated the effect of BM-plasma and CFU-F-CM over the migration and proliferation of both BCC lines. Methodology: Soluble factors were studied by ELISA. SDF-1, m-RANKL, CXCR-4 and RANK expression was analyzed by immunochemistry. Migration was performed over 14hs in transwells seeded with BCC exposed to BM-plasma and CFU-F-CM. Proliferation by MTS: after arrest, BCC were incubated for 48hs with 10% of BM-plasma or 100% or 50% of these CM with or without 1.25% FBS. Results: Significant difference in the OPG, RANKL, SDF-1 and MIF values in PB-plasma was found between both groups (BCP vs HV, X±SE, pg/ml): 2,005±195.90 vs 1,100±124.10 (p=0.001), 130.20±23.63 vs ≤31.25, 117±25 vs 254±28 (p<0.05) and 4,564±591 vs 2,265±402 (p<0.05), respectively. PDGF-AB level in BCP-BM-plasma was significant higher than HV-value (X±SE, pg/ml): 4,468±746 vs 2,528±421. 100% of BM-MStC expressed SDF-1 and mRANKL in both groups, but we observed higher mRANKL expression/MStC from BCP-BM compared to HV-values (++++vs++). The primary tissue-BCC expressed CXCR-4 and RANK (++) but we did not observe expression of them in the epithelial cells of non-malignan tissue. Moreover, 50% of BCC of both lines expressed CXCR-4 and 100% RANK. In addition, the BM-plasma and the CFU-F-CM from BCP induced a higher migration increase of MCF-7 and MDA-MB231cells compared with HV-values (p<0.05 in both lines and p<0.0001 and p=0.0356, respectively). We have not observed proliferation effect by CFU-F-CM over any of the lines, but we did observe a significant higher proliferation of MDA-MB231 cells when we used the BM-plasma from BCP compared with HV (p=0.0434). Conclusions: Data suggests that the high PB-RANKL and MIF levels in these BCP could play a role in the intravasation of BCC into the blood vasculature, binding to their R (RANK and CXCR-4, respectively) expressed in them. MIF and OPG not only are pro-angiogenic factors, but also, they have an anti-apoptotic effect over BCC favoring the circulating BCC survival. In addition, PDGF-AB could be responsible of higher proliferation of MDA-MB231 cells when we used the BM-plasma from BCP compared with HV, inducing a favorable BM microenvironment to seed and proliferation of circulating BCC. Moreover, the BM-MStC from BCP could enhance the migration of circulating BCC to BM/bone and the likely association between RANK present in BCC and the mRANKL in BM-MStC could promote the tumor cell proliferation favoring the bone metastatic process. Our findings suggest that the BM microenvironment of advanced BCP without BM/bone metastasis may induce a premetastatic niche to BCC colonization. Citation Format: Leandro M. Martinez, Valeria B. Fernandez Vallone, Vivian Labovsky, Hosoon Choi, Leonardo Feldman, Raul H. Bordenave, Emilio Batagelj, Federico Dimase, Ana Rodriguez Villafane, Norma A. Chasseing. Bone marrow microenvironment of advanced breast cancer patients without bone metastasis favors the cancer cell colonization. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B66.