NSAIDs inhibit integrin-dependent squamous cell carcinoma invasion

Objectives: Oral squamous cell carcinoma (OSCC) is the sixth commonest cancer world-wide. 5 year survival rates for OSCC patients have remained at approximately 50% for 25 years hence novel therapeutic targets are desperately needed. We reported that the integrin αvβ6, (which is strongly expressed on OSCC but absent from normal mucosa) promotes invasion through Matrigel® by upregulating MMP-9. To extend these studies in vivo we have developed a quantifiable, physiologically-relevant invasion assay and used it to examine potential signalling inhibitors of avb6-dependent invasion. Methods: OSCC cells were overlaid onto synthetic stromal gels containing fibroblasts and grown for 7-14 days at an air-tissue interface. These organotypic gels were routinely processed for histology or transplanted onto the dorsal muscle fascia of nude mice for 6 weeks. Histologically, these tumours closely resembled clinical samples. Using Optilab Pro software we designed a digital image analysis method for objectively measuring OSCC invasion on cytokeratin-stained sections. Results: Invasion of OSCC in organotypic gels, both in vitro and in vivo, correlated with αvβ6 expression. To inhibit this invasion we used the selective cyclo-oxgenase 2 (COX-2) inhibitor NS398 that inhibited avβ6-dependent (but not αvβ6-independent) invasion of OSCC in organotypic cultures, both in vitro (p<0.001) and in vivo (p=0.004). The mechanism required prostaglandin E2 (a metabolic product of COX) since exogenous addition restored αvβ6-dependent invasion. COX-2 inhibition did not alter αvβ6 cell surface levels or ligand-binding affinity but did reduce MMP-9 secretion. The non-selective COX inhibitor indomethacin gave similar results to NS398, but the COX-1 inhibitor Resveratrol was ineffective. Conclusion: We have developed a quantifiable invasion assay for OSCC that can be easily transplanted onto mice and used this to show that increased αvβ6 expression promotes OSCC invasion in vivo. Moreover, since NS398 specifically inhibits αvβ6-dependent invasion, long-term treatment with NSAIDs may be effective inhibitors of OSCC invasion.