The Role of Cancer Associated Fibroblasts in Bone Invasive Oral Squamous Cell Carcinoma

Bone invasion is a common feature of oral squamous cell carcinoma (OSCC) and is associated with poor prognosis. In this study, we report a novel role for cancer associated fibroblasts (CAF) in OSCC bone invasion, and provide additional evidence that two subsets of CAF, myofibroblastic and senescent fibroblasts, can both promote bone destruction via a mechanism involving receptor activator of nuclear factor kappa-Β ligand (RANKL) and extracellular vesicles (EV). Our results show two populations of CAF, alpha smooth muscle actin (alphaSMA) myofibroblastic, and alphaSMA and p16INK4a positive senescent, are present and show increased expression of RANKL in ex vivo OSCC bone resections. To examine the mechanism underlying this observation, senescence was experimentally induced in normal oral fibroblasts (NOF) by culturing to replicative mitotic exhaustion (S-NOFRep), or exposure of proliferating cells to hydrogen peroxide (S-NOFH2O2) or cisplatin (S-NOFCis). Elevated expression of the molecular markers of senescence, p16INK4a and interleukin-6 (IL6), were seen; the latter is also a key component of the senescence-associated secretory phenotype (SASP) and mediator of bone absorption. Increased expression and secretion of RANKL was also detected in these cell cultures. Osteoclastogenesis and pit formation on a synthetic bone substrate were significantly increased in response to primary CAF, myofibroblastic and senescent fibroblast-derived conditioned media. This mechanism was significantly attenuated by the senolytic drugs Alvespimycin (17-DMAG) and Navitoclax (ABT263). Moreover, to understand the mechanism by which OSCC and surrounding CAF communicate in the tumour microenvironment, EV were isolated from H357 oral cancer cell line, primary CAF from fresh OSCC tissue and experimentally induced senescent fibroblasts (S-NOF). EV from all cells contained RANKL, and significantly promoted osteoclastogenesis. Osteoprotegrin (OPG), a RANKL decoy receptor, antagonist and bone resorption regulator reduced osteoclastogenesis, providing further evidence of a role of RANKL in CAF-mediated bone destruction. In conclusion, this work provides novel evidence that CAF play a functional role in bone invasion. The ability of senolytic drugs to reduce senescent CAF burden and inhibit osteoclastogenesis holds promise as a novel therapeutic approach in bone invasive OSCC.