CONICET | Buscador de Institutos y Recursos Humanos

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 bellow 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.