Interactions Between Stromal Fibroblasts And Human Colorectal Cancer Cells In Tumor Environment

BackgroundColorectal cancer was one of the most common digestive tract cancer globally with over 1200000 new cases diagnosed and 600000 deaths each year. According tostatistic results, cure rate of colorectal cancer improved slightly compared with its increasing morbidity and mortality.Many researches had shown that inflammatory bowel disease(IBD) especially ulcerative colitis(UC), the risk of deteriorating into colorectal cancer was positively correlated with their inflammatory degree.The mechanism has not been fully elucidated, the chronic inflammation state of local tissue microenvironment will facilitate the metastasis of cancer cell and the formation of tumor microvascular.All kinds of environmental factors, such as inflammation, stress, etc, affecting the stability of the tissue microenvironment contributing to the occurrence of disease.Tumorigenesis was closely related with its microenvironment, which was identified as "cancer cell nests" constituted by malignant cancer cells, immune cells,stromal cells and their secreted cytokines. Since the unlimited proliferation of tumor cells need to consume too much energy and materials metabolism of tumor and its microenvironment was confirmed to proceed through a special way differ from normal cells. According to recent studies, cancer associated fibroblasts(CAFs) of tumor microenvironment could promote tumor initiation, progression and metastasis by interfering cellular metabolism.Based on the mentioned research background, we used DSS-induced inflammatory model, DMH-induced colorectal cancer model and DSS+DMH-induced inflammation-cancer model, in vivo, to explore the role of inflammatory microenviro-nment in the development of colorectal cancer, and the changes in glucose metabolism of inflammatory microenvironment. And then,our research explored the interactions between cancer associated fibroblasts and colorectal cancer cells during tumorigenesis, and changes in the activity of metabolic assosiated key enzymes in the cell, by using a non-contact coculture system between fibroblasts and colonic cancer cells, in vitro. By exploring the underlying mechanism of CAFs metabolism reprogramming, we could make a better understanding of tumor growth environment, and provide new evidence for future treatment of colorectal cancer.Objective1. To explore the role of inflammatory microenvironment in the development of colorectal cancer, and the changes of glucose metabolism in inflammatory microenvironment.2. To explore the interactions between cancer associated fibroblasts and colorectal cancer cells during tumorigenesis, and its underlying molecular mechanism.Methods1. Establish inflammatory model, colorectal cancer model and inflammation cancer model.2. Co-culture CAF (CCD-18-co) to CRCs(colorectal cancer cells) by transwell.3.MTT assay measured the proliferation of cell.4. Western blot was performed to detect the expression of protains in co-cultured system.5. Immunofluorescence staining was performed to detect the expression of protains in co-cultured system.6. Immunohistochemistry was performed to detect the expression of PCNA,PKM2 and IDH2 in colorectal normal, inflammation and tumor tissures.7. Related kits tested the activity of PKM2 and IDH2.Results1. HE staining analysis indicate that animal model was induced successfully.2. Through the detection of TNF-a, PCNA proliferation index and HE staining,we found that there is a higher incidence of tumor and malignant degree in DSS+DMH-induced inflammation-cancer model than DMH-induced colorectal cancer model.3. The expression of proliferating cell nuclear antigen(PCNA) and PKM2 in DMH and DMH +DSS groups increased significantly in both their tumor cells and stroma cells, however the increased expression of IDH2 in DMH and DMH+DSS groups was observed only in their tumor cells but not stroma cells.4. Colorectal cancer cells were subsequently suspended with gradient concentration of CCD-medium diluted by fresh DMEM medium. And CCD-18-co cells were suspended with gradient concentration of colorectal cancer cell culture medium (LoVo, SW480, SW620) diluted by fresh DMEM medium. MTT assay showed that there was a mutually reinforcing relationship between tumor cells and fibroblasts.5. Both enzymes involved in KREBS CYCLE(IDH2) and glycolytic pathway (PKM2, GAPDH) were up-regulated in co-cultivated cancer cells, however the expression of IDH2 was significantly decreased in co-cultivated fibroblasts with the same increased PKM2 and GAPDH. The measurement of metabolic enzymes’ activities by using PKM2 and IDH2 activity assay kits obtained the same results.6. The oxidative stress key enzyme and autophagy related proteins were up-regulated in cocultured CCD-18-co.7. The abnormal expression of metabolism related proteins had been reversed by oxidative stress inhibitor and autophagy inhibitor.Conclusion1. Persistent inflammation could affect the glucose metabolism of cells in tissue microenvironment, promote cell proliferation,and then accelerate the occurrence and development of tumor.2. Fibroblasts existing in inflammatory microenvironment positively influenced the metabolism of colorectal cancer cells, through its autophage and oxidative stress pathway which were initially induced by neighboring tumor cells. Therefore, it’s possibility to develop fibroblasts as a potential target to treat colorectal cancer.