A Study On EGFR Signaling Of Colorectal Cancer Modulating Macrophage Polarization

Tumor microenvironment is composed of tumor cells, endothelial cells, fibroblasts, immune cells, macrophages, in addition to the cytokines, matrix metalloproteinases (MMPs), and extracellular matrix (ECM) they produce. Tumor microenvironment not only plays an important role in tumor progression and metastasis, but also has profound effects on therapeutic efficacy. Moreover, radiotherapy and chemotherapy can also affect the tumor microenvironment. Tumor associated macrophages (TAMs) are major cellular components of the tumor microenvironment. TAMs can promote tumor growth and progression by the secretion of growth factors and matrix metalloproteinases, angiogenesis related factors, and inhibition of adaptive immunity. So, Targeting TAMs could improve therapeutic efficacy.Our researches focus on how EGFR-targeting modulates the tumor microenvironment, especially TAMs polarization, besides directly inhibiting tumor cell growth. Firstly, treated AOM/DSS mouse model with cetuximab, an epidermal growth factor receptor (EGFR) monoclonal antibody. Tumor growth was inhibited. Protein levels of p-EGFR (Y1068), EGFR, arginase-1 (Arg1) and inducible nitric oxide synthase (iNOS) in colons of AOM/DSS mice were increased compared to normal mice. While treated with cetuximab, these proteins were decreased except iNOS protein. Moreover, F4/80+/CD206+macrophage populations were downregulated in colons of AOM/DSS mice treated with cetuximab. Since EGFR protein was undetectable in macrophages, cetuximab might have an influence on macrophage through the secretion of tumor cells. Conditioned medium (CM) of HCT116 and SW480 colon cancer cell could induce Ana-1 cells polarization to M2-like phenotype, the mRNA levels of Arg1, IL-10 and IL-4 (M2 markers) were upregulated. When we knocked down EGFR of HCT116 and SW480, the ability of CMs to induce Ana-1 cells polarization to M2-like phenotype was partially abolished.In tumor xenograft model, when we injected macrophages with HCT116 cells, Ana-1 was polarized to M2 phenotype and promoted tumor growth, and knockout of EGFR in HCT116 cells could partially inhibit Ana-1 polarization to M2-like phenotype.To investigate how CM modulated macrophage polarization, we detected the CM of HCT116 cells by cytokines antibody array. The IGF-1 content significantly decreased when EGFR was knocked down. ELISA results also confirmed the expression of EGFR was positively correlated with the concentration of IGF-1. When treated Ana-1 cells with IGF-1, IGF-1R pathway was activated and Argl protein level was upregulated in a dose-dependent way. AG1024, an IGF1R inhibitor, could inhibit the polarization of Ana-1 cells to M2-like phenotype. These results indicated that IGF-1 secreted by cancer cells, might play a vital role in macrophage polarizationIn summary, EGFR-targeting could inhibit macrophage polarization to M2-like phenotype in vitro and in vivo, and IGF-1 secreted by colon cancer cells plays an important role in the process of polarization. Our present study reveals a possible mechanism of macrophage polarization influenced by EGFR-targeting. Furthermore, it also implies a potential strategy to inhibit macrophage polarization, which is a promising novel method for cancer treatment.