UMSCC1 and UMSCC47 invasion. Transwell assays were performed as described previously (19). Briefly, 50,000 UMSCC1 or UMSCC47 cells were resuspended in 250 μl 10% FBS/DMEM and plated in the upper chamber. The bottom chamber was filled with 1 ml 10% FBS/DMEM. Where indicated, 50,000 neutrophils (1:1 ratio) were added to the bottom chamber. A- Comparison between UMSCC1 and UMSCC47 baseline invasion without neutrophils. B- UMSCC1 invasion in the presence of neutrophils (indirect co-culture). Results shown are normalized to control.
ARTICLE ABSTRACT
Neutrophils have recently been shown to promote invasion and correlate with a poor prognosis in different cancers, including head and neck squamous cell carcinomas. In this study, we analyze the effects of neutrophils in the invasion of oral squamous cell carcinoma (OSCC) using a combination of conditioned media, direct and indirect coculture of human peripheral blood neutrophils, and UMSCC47 cells (OSCC cell line). Invasion and matrix degradation were determined using a modified in vitro invasion assay and an invadopodia assay, respectively. UMSCC47 and neutrophil cocultures or conditioned media from cocultures increased UMSCC47 invasion, invadopodia formation, and matrix degradation. Further analysis revealed an increase in TNFα and IL8 in supernatants of cocultures compared with neutrophil or UMSCC47 cultures alone and that inhibition of TNFα and IL8 significantly decreased OSCC invasion. Our results show that neutrophils increase the invasiveness of OSCC through the activation of invadopodia and matrix degradation, suggesting a paracrine activation loop between the two cells. Importantly, the presence of neutrophils in the oral environment may modulate the clinical behavior of OSCC. Cancer Immunol Res; 3(11); 1218–26. ©2015 AACR.
