The Role Of Tumor-infiltrating T Cells In Colon Cancer Microenvironment

Background:Colon cancer is a common gastrointestinal malignant tumor with increasing incidence in China. Recently, it was postulated that the immune response at the primary tumor microenvironment may influence the outcome of patients with colon cancer. Tumor-infiltrating T lymphocytes play an important role by dominating such an immune process. Volumes have been written about the relationship between gene mutation and tumor genesis, however, nowadays further studies still need to be done to explore the complicated interaction between genetic background and tumor immunity.Objective:1. To determine the distribution and expression of tumour-infiltrating T-cells in tumor and peficancerous sites in surgical samples of colon cancer, and to investigate the correlation of tumour-infiltrating T-cells with clinical and pathological features of these patients.2. To explore the mechanisms of immune evasion by Treg cells.(1) To analyze the infiltration of tumor macrophage (CD68) and dendritic Cell (S-100) in tumor sites and to investigate whether they can influence the distribution of regulatory T cells.(2) To study whether Treg cells exert immune evasion by using granzyme B in the tumor microenvironment.3. To determine whether the expression of tumor-infiltrating T-cells in colon cancer may differ given K-ras gene mutated.Materials and Methods:27patients who had undergone curative resection of colon cancer at PUMCH were enrolled in this study. Samples were prepared with standard pathological procedures. The relevant clinical and pathological information was collected. Immunohistochemical staining was used to identify tumour-infiltrating T-cells, granzyme B, DC and APC in colon cancer. PCR-sequencing method was employed to detect K-ras mutations. The potential correlation of clinical and pathological features to tumor-infiltrating T cells was also analyzed.Results:(1) The expression levels of CD4+T cells,CD45RO+T cells and Thl(T-bet) in tumor sites were lower than those in peficancerous sites (P<0.001, respectively). The percentages of Treg (FOXP3) in tumor tissues were higher than in peficancerous sites (P<0.001). FOXP3/T-bet in tumor sites were higher than that in peficancerous sites (P<0.001). CD8/FOXP3in tumor sites were lower than that in peficancerous sites (P=0.021)(2) There was no significant correlation between tumor-infiltrating T cells subset in tumor sites and clinical and pathological features of these patients, however, the expression levels of CD8+T cells in peficancerous sites were lower in vascular invasion group than those in non-vascular invasion group (P=0.000) The expression of Thl(T-bet) in peficancerous tissues were higher in TMN stage I and II than it in stage III and IV (P=0.002) and also had a significant difference in different stage of Duke (A, B vs C+D, P=0.011), the higher the Duke stage, the less the expression.(3) The expression of S-100in tumor sites were lower than that in peficancerous sites (P=0.000). We found a significant correlation between S-100-positive DC and FOXP3-positive Tregs (r=0.839, P=0.000). Granzyme B in tumor sites, however, was not correlated with FOXP3.(4) PCR-sequencing was used to detect the first exon of K-ras in19specimens of colon cancer, with the mutation rate42.11%. K-ras mutation had no effect on the expression of tumor-infiltrating T-cells.Summary:(1) The expressions of CD4+T, CD8+T, CD45RO+T cells and T-bet in tumor sites were lower than those in peficancerous sites, on the contrary, the expression of FOXP3was higher in tumor sites.(2) FOXP3/T-bet in tumor sites were higher than that in peficancerous sites, while CD8/FOXP3appeared lower in primary tumor sites.(3) We found a significant positive correlation between S-100-positive DC and FOXP3-positive Tregs. Granzyme B in tumor sites had no significant correlation with FOXP3.Conclusions:(1) The abundance of tumor infiltrating T cells including CD4+T cells,CD8+T cells,CD45RO+T cells,Treg and Thl cells is the hallmark of the tumor immune microenvironment in colon cancer. Tumor immune evasion is dominated in primary tumor sites, while up-regulated immunity is present in peficancerous sites.(2) Treg (FOXP3) expression was related with dendritic cells (S-100protein), suggesting that DC in the tumor microenvironment might be involved in the regulation of Treg cell formation. Macrophages (CD68) expression and Granzyme B did not play a role in the formation and activity of Treg cells.(3) The mutation of K-ras didn’t have effects on the expression of tumor-infiltrating T-cells.