Upregulation of RANKL in Bone Metastatic Breast Cancer Cells after Direct Co-Culture with Osteogenically Differentiated Human MSCs

Bone metastases arise in nearly 70 percent of patients with advanced breast cancer, leading to massive bone lysis. RANKL/RANK/OPG pathway is the key molecular axis for osteoclasts formation, regulating both normal bone resorption and metastatic bone lysis. This work aims at investigating the reciprocal interactions between fluorescent human bone metastatic breast cancer cells (BOKL) and bone-derived cells (MSCs). MSCs were harvested from 3 different donors, cultured for 14 days in osteogenic medium and tested for differentiation. BOKL were cultured in growth medium (CTR), in direct co-culture and in medium conditioned by the same MSCs (CM). After enzymatic detachment and FACS sorting of the two cell types, real time PCR for proliferation and migration related genes was performed. Alizarin red staining and assay for calcium content confirmed osteogenic differentiation of MSCs. PCR demonstrated RANKL up-regulation up to 17 fold in BOKL in direct co-culture with MSCs as compared to CTR. On the contrary, we did non observe significant up or down regulation for genes of BOKL cultured in CM. Furthermore, RANKL decoy receptor OPG was 2-fold up-regulated in BOKL directly co-cultured with MSCs from each of the 3 patients. In conclusion, through gene expression analysis in accurately separated cell populations following direct co-culture, we reliably showed that direct but not indirect co-culture between BOKL and bone differentiated MSCs increased expression of key genes in metastatic cells. Thus demonstrating the fundamental role of direct contact between bone metastatic breast cancer cells and bone cells in the initiation of the vicious cycle causing bone resorption and consequent metastatic cells growth.