Abstract
Oral cancers, primarily squamous cell carcinomas (SCCs), progress either slowly or aggressively. Here we assessed the role of macrophages in SCC behavior. We used mouse SCC cells derived from tumors harboring a KrasG12D activation mutation and Smad4 deletion in keratin 15-positive stem cells and a human oral SCC cell line, FaDu, which has NRAS amplification and SMAD4 deletion. SCC cells were transplanted into immune-compromised or immune-competent (syngeneic) recipients. After tumors were established, we used clodronate liposomes to ablate macrophages. We found that the number of tumor-associated macrophages (TAMs) was not affected by the presence of T cells but differed considerably among tumors derived from different SCC lines. Clodronate significantly reduced TAMs and splenic macrophages, resulting in reduced SCC volumes. Tumors with clodronate treatment did not show decreased proliferation but did exhibit increased apoptosis and reduced vascular density. FLIP (Fas-associated via death domain-like interleukin 1β-converting enzyme inhibitory protein), an apoptosis inhibitor abundantly produced in tumor cells and TAMs, was reduced in tumor cells of clodronate-treated mice. Reduced FLIP levels correlated with reductions in phosphorylated nuclear NFκB p65 and NFκB inhibitor attenuated FLIP protein levels in SCC cells. Furthermore, TGFβ1 serum levels and pSmad3 were reduced in clodronate-treated mice, but their reductions were insufficient to reverse epithelial-mesenchymal transition or TGFβ-mediated angiogenesis in endothelial cells. Consequently, metastasis was not significantly reduced by macrophage reduction. However, reduced pSmad3 correlated with reduction of its transcriptional target, vascular endothelial growth factor A, in clodronate-treated tumor cells, which correlated with reduced vascular density in clodronate-treated tumors. Taken together, our study revealed that macrophages contribute to SCC expansion through interactions with tumor cells but are dispensable for SCC metastasis. Our study provides novel insights into understanding the contributions and limitations of TAMs in SCC progression.