Crucial Involvement Of Inflammatory Microenvironment In The Regulation Of Colorectal Carcinogenesis

[Background] Chronic inflammation is a major driving force in the development of cancer in many tissues. Ulcerative colitis (UC) is one of the major forms of chronic inflammatory bowel disease (IBD) in humans. The risk of developing colorectal cancer (CRC) in human with Ulcerative colitis is higher than in the general population, which is related to the duration, extent, and severity of inflammatory disease. The infiltrated immune cells, inflammatory cytokines, chemokines, matrix metalloproteinase and so on consisited the inflammatory microenvironment which plays a key role in the development of colon cancer. Our preliminary results indicate that tumor-associated neutrophils mediated by CXCR2-CXCL2 chemokine axis can promote tumorigenesis of colorectal cancer. We also found a large infiltration of fibroblasts in the mouse model, some report suggested cancer-associated fibroblasts as a main component of tumor stroma play a very important role in the formation of tumor and the effect and mechanism of CAFs in colon cancer is unclear.[Purpose] Further to validate the role of tumor-associated neutrophils (TAN) in the initiation and progression of CAC through treating chronic colitis-associated carcinogenesis in mice with Ly6G. Explore the effect of cancer-associated fibroblasts (CAFs) on the development of colon cancer, revealed the role of FGF signaling pathway during the formation of colon cancer, carified the mechanisms that cancer-associated fibroblasts promote tumorigenesis colon cancer.[Methods] (1) Establish AOM / DSS mouse model,The mouse treated with AOM/DSS were randomly divided into two groups at 56 day, continuous intravenous injection of anti-neutrophil antibodies Ly6G for a week was regarded as the treatment group, another group of continuous intravenous injection of control IgG antibody for 7 days was regarded as control group. At 67 day, the mouse were anesthesia and euthanized, then the colorectal tissue was preserved to use. qRT-PCR to detect the mRNA level of CXCL2, CXCR2, MMP-9 and neutrophil elastase NE in colon tissue with or without treatment of Ly6G. HE staining to detect the dynamic changes of colorectal organizational structure with or without treatment of Ly6G, Immunofluorescence and immunohistochemical staining to determine the infiltration of neutrophils, the protein levels of NE, and the CXCL2, CXCR2, CD31, PCNA, MMP-9 protein levels with or without Ly6G treatment of CAC mouse model. (2) Infiltration of fibroblasts, macrophages and T lymphocytes as well as the proangiogenesis gene MMP-7 protein level were detected at 0,14,28,35,56 day through immunohistochemical staining in CAC model,qRT- PCR was used to detect various stages changes of chemokine cytokine mRNA levels, Western-blot to detect various stages of p-Erk, Erk protein levels which are related to cell proliferation. (3) Human colon tissue specimens were obtained from 5 individual patients upon surgery for colon cancer with his informed consent and with approval from the Human Subjects Research Ethical Committee of Fudan University Shanghai Cancer Center. We separated the tumor, adjacent the tumor, and normal tissue, which was at least 2 cm apart from the edge of tumor foci and was judged histologically to be free from adenocarcinoma cells and immediately isolated the fibroblasts. qRT-PCR was used to detect the mRNA expression level of cell growth factors such as FGF mRNA levels, the supernatant of three kinds of fibroblasts were co-cultured with colon cancer cells, then Western-Blot to detect the phosphorylation level of signal molecule Mek / Erk,and the protein level of MMP-7. Meanwhile, Western-Blot to detect the related signaling molecules expression after treatment with FGFR4 inhibitor and the recombinant protein FGF-1 or FGF-3. CCK8 and tube formation assay to determin the effects of cancer-associated fibroblasts CAFs on endothelial cells.[Results] 1. Compared with the control group, the tumor burden was decreased in the Ly6G treated group, HE staining suggested morphology and structure was revered of in colonic tumors after Ly6G injection.2. Compared with the control group, the neutrophil infiltration was significantly reduced, the expression of CXCL2, CXCL5 and CXCR2 significantly decreased, MMP-9 expression was remarkedly reduced accompany with the reduced neovascularization in the Ly6G treatment group. NE expression was also significantly reduced together with decreased cell proliferation. These results suggest that treatment of anti-neutrophil antibodies can reverse the formation and development of colon cancer.3. in the CAC model mouse, the infiltration of fibroblasts,macrophages and T lymphocytes were enhanced and the expression of inflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α were increased in the procession of colon cancer, in addition, CXCL-2 and PDGF-a expression were also significantly increased. Moreover, proangiogenesis gene MMP-7, proliferation-related signaling molecules p-Erk, Erk expression was also significantly increased.4. the purity of fibroblasts extracted in vitro was more than 90%, there were more FGF1 and FGF3 in CAFs compared with NFs and PFs. The supernatant of three kinds of fibroblasts were co-cultured with colon cancer cells, compared with NFs and PFs, CAFs can upregulate the expression of p-FGFR4, p-Erk and MMP-7 while FGFR4 expression has no change significantly. The supernatant of three kinds of fibroblasts were co-cultured with endothelial cells, CCK8 and tubule formation assay showed that CAFs can promote the proliferation and migration of vascular endothelial cells.5. The phosphorylation level of FGFR4 and Mek/Erk and MMP-7 expression were decreased after adding FGFR4 inhibitor in colon cancer cells which were cultured with the supernatant of CAFs. 6. Adding cytokines FGF-1 or FGF-3 in colon cancer cells, the results showed the upregulation of p-Mek, p-Erk and MMP-7.[Conclusion] In summary, administration of anti-neutrophil antibodies can reverse the development of colon cancer which is further to validated tumor-associated neutrophils promote the occurrence and development of colorectal cancer. Cytokines or/and growth factors secreted by inflammatory cells and tumor cells maybe make normal fibroblasts (NFs) into cancer-associated fibroblasts CAFs, the FGF-1 and FGF-3 secreted by CAFs can activate their receptor FGFR4 expressed in colon cancer cells, then activated the down signaling molecule Mek / Erk and increased the expression of MMP-7, promoted the occurrence and development of colon cancer through the cell proliferation and tumor angiogenesis.