Analyzing the role of cancer-associated fibroblast activation on macrophage polarization.

Snail1 is a transcriptional factor required for cancer-associated fibroblast (CAF) activation, and mainly detected in CAFs in human tumors. In the mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT) model of murine mammary gland tumors, Snai1 gene deletion, besides increasing tumor-free lifespan, altered macrophage differentiation, with fewer expressing low levels of MHC class II. Snail1 was not expressed in macrophages, and in vitro polarization with interleukin-4 (IL4) or interferon-γ (IFNγ) was not altered by Snai1 gene depletion. We verified that CAF activation modified polarization of naïve bone-marrow-derived macrophages (BMDMΦs). When BMDMΦs were incubated with Snail1-expressing (active) CAFs or with conditioned medium derived from these cells, they exhibited a lower cytotoxic capability than when incubated with Snail1-deleted (inactive) CAFs. Gene expression analysis of BMDMΦs polarized by conditioned medium from wild-type or Snai1-deleted CAFs revealed that active CAFs differentially stimulated a complex combination of genes comprising genes that are normally induced by IL4, downregulated by IFNγ, or not altered during the two canonical differentiations. Levels of RNAs relating to this CAF-induced alternative polarization were sensitive to inhibitors of factors specifically released by active CAFs, such as prostaglandin E and TGFβ. Finally, CAF-polarized macrophages promoted the activation of the immunosuppressive regulatory T cells (T-regs). Our results imply that an active CAF-rich tumor microenvironment induces the polarization of macrophages to an immunosuppressive phenotype, preventing the macrophage cytotoxic activity on tumor cells and enhancing the activation of T-reg cells.