436 Macrophages promote growth of squamous cancer independent of T cells

Abstract

We assessed the role of macrophages in squamous cell carcinoma (SCC) behavior. We used mouse SCC cells derived from tumors harboring a Kras12D activation mutation and Smad4 deletion in keratin 15 (K15)-positive stem cells, and a human SCC cell line, FaDu. SCC cells were transplanted into immune-compromised or -competent (syngeneic) recipients. After tumors were established, we profiled tumor infiltrating leukocytes using CyTOF. CD45+ leukocytes comprised 10-25% of the total tumor cells, and tumor-associated macrophages (TAMs) were 5-10% of the CD45+ cells. There were typically 2-3 fold more M2 TAMs than M1 TAMs. We used clodronate liposomes to deplete TAMs. We found that the number of TAMs was not affected by the presence of T cells but differed among tumors derived from different SCC lines. Clodronate significantly reduced TAMs and macrophages in the spleen, resulting in reduced SCC volumes. Tumors with clodronate treatment did not show decreased proliferation, but exhibited 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 NFkB p65 and NFkB inhibitor attenuated FLIP protein levels in SCC cells. Further, TGFb1 serum levels and pSmad3 were reduced in clodronate-treated mice, but their reductions were insufficient to reverse epithelial-mesenchymal transition (EMT) or TGFb-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 (VEGFA), in clodronate-treated tumor cells. 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.