Bone Abstracts

The microenvironment of osteolytic metastases includes carcinoma cells derived from the primary lesion as well as bone-forming and bone-resorbing cells, namely osteoblasts (OB) and osteoclasts (OC). At this site, both the high glycolysis of cancer cells, and the bone resorption process result in a very acidic milieu. This, in turn, induces the surrounding stroma and OB to secrete pro-inflammatory cytokines and growth factors that promote tumorigenesis and cancer-associated osteolysis via OC activation. Here, we investigated the role of acidic microenvironment in osteolytic carcinomas and its effect on the interplay between cancer and stromal cells.

The glycolytic rate and capacity of carcinoma cells were determined by Seahorse Extracellular Flux (XF-96) analyzer. The level of expression of the main pumps and transporters involved in the regulation of cell metabolism and acidification, under both normoxic and hypoxic conditions, were determined by Real-time PCR. To evaluate the effect of tumor acidification on the stroma of bone metastases, mRNA expression of IL6, IL8, RANKL, and M-CSF was assayed by Real-time PCR in OB exposed to acidic medium. Additionally, to evaluate the effect of the secretome of tumor-associated stroma on OC differentiation and activity, human OC derived from peripheral blood mononuclear cell (PBMC) were cultured with the supernatant of OB pre-conditioned in acidic medium.

Our results confirmed a high glycolitic and acidification activity in carcinoma cell lines, an increased gene expression of the proton pump Vacuolar ATPase (V-ATPase), of the carbonic anhydrase CA-9, and of the GLUT-1 transporter, both in mammary and in renal carcinoma cells. Furthermore, acidity induced an increase of IL6 and IL8 expression in OB that in turn promoted OC formation. Our results confirm that, in bone metastases, extracellular acidification by tumor cells promote the release of pro-mitogenic and pro-osteoclastogenic factors from the surrounding stroma.