Colon Cancer Cell Secretes EGF to Promote M2 Polarization of TAM Through EGFR/PI3K/AKT/mTOR Pathway

Tumor environment has been recognized to affect cancer cell progression, such as tumor-associated macrophages. However, increasing evidences suggest that tumor cells are capable of regulating polarization of tumor-associated macrophages. In this study, we investigate the mechanism of how colon cancer cell impacts tumor-associated macrophages polarization.

We provide evidence that EGF which are secreted by colon cancer cells play contributory role in M2 polarization of macrophages, which support the notion that tumor environment, including tumor-associated macrophages, can be targeted to develop effective strategies for treating cancer.

We employed flow cytometry to detect marker molecules on macrophage membrane, such as CD68, CD16, and CD204. In addition, we used enzyme-linked immunosorbent assay to examine the level of these cytokines (interleukin-6, interleukin-1β, interleukin-10, and Arginase-1) secreted by colon cancer cells into the culture medium. Western blot was utilized to probe downstream proteins of epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway.

We cocultured colon cancer cell lines (HCT8 or HCT116) with human myeloid leukemia mononuclear cells (THP-1) and found that interleukin-6 and interleukin-1β levels were reduced, and instead, interleukin-10 and Arginase-1 levels were elevated, suggesting that colon cancer cells contributed to M2 polarization of THP-1. Meanwhile, high level of various growth factors (transforming growth factor-β [TGF-β], epidermal growth factor [EGF], and hepatocyte growth factor [HGF]) was observed in the medium of THP-1 cocultured with colon cancer cells. Furthermore, the protein level of phosphorylated PI3K, AKT, and mTOR significantly increased in THP-1 cell cocultured with colon cancer cells compared to THP-1 group. Besides, we established that colon cancer cells exerted their stimulatory effect on M2 polarization of macrophage from monocyte THP-1 using EGFR antibody mAb225 and PI3K inhibitor LY294002.