Distribution And Clinical Significance Of T Regulatory Cells In The Colorectal Tumor Microenvironment

Background and ObjectiveColorectal cancer(CRC) is one of the most common malignant diseases worldwide. And according to the hypothesis of adenoma- adenocarcinoma, the majority of colorectal cancer is evolved by the adenoma, also known as the theory of colorectal adenomas- cancer sequence. In this evolution, a series of genetic and molecular changes associated with progress on histology. Adenoma is recognized as the most important of colorectal cancer precancerous lesions, but many of the specific mechanism in the evolution of colorectal adenomas- cancer sequence not yet fully understood. In recent years, studies of tumor immunology, in particular to study the regulation mechanism of tumor immune microenvironment for tumor biotherapy offers new ideas and approaches.T regulatory cells(Tregs) are a subpopulation of T cells that have been shown to be critical for the maintenance immunologic tolerance and homeostasis, The transcription factor forkhead box protein P3(Fox P3) is a member of the forkhead and winged helix family, and plays a key role in Tregs development and function, Fox P3+Tregs have been shown to modulate antitumour immune response and suppress the activity of cytotoxic T-cells. Therefore, Fox P3 has been considered as a specific characteristic marker of Tregs and frequently used in determining Tregs in humantumors. Including inhibition of expression of cell surface proteins such as cytotoxic T lymphocyte-associated antigen 4(CTLA-4), inhibiting the secretion of cytokines such as interleukin-10(IL-10) and tumor necrosis factor-?(TGF-?). However, previous studies regarding the role of Tregs were predominantly performed in patients with developed CRC. Of recent studies, have examined the Tregs in human colorectal adenomas and discussed the potential significance of Tregs in the precancerous lesions. Therefore, the aim of this study was to examine dynamic changes of Fox P3+Tregs and immunosuppressive cytokine IL-10 from the colorectal adenoma to CRC.Materials and Methods Thirty cases of CRC, 36 cases of colorectal adenoma(CRA) and 12 cases of normal tissues from health people were collected. Foxp3+Tregs and interleukin(IL)-10 positive cells were measured by immunohistochemistry(IHC),and messenger RNA(m RNA) levels of Foxp3 and IL-10 were quantified with real-time PCR in different tissues, beta-actin was applied as an internal control.Results1? The IHC results showed low density of Fox P3+ Tregs in the normal controls was predominately observed in the stroma, but rarely in the epithelium. In the adenomas, the Fox P3+ Tregs was frequently observed in the stroma, and some of Tregs were infiltrated in the adenomatous epithelium. In the CRCs, most of Tregs were located in the CRC tumor stroma and some of cells infiltrated in the CRC epithelium.The analysis of semi-quantitative results showed that the densities of Fox P3+ Tregs in both the adenomatous stroma and epithelium were significantly increased as compared with the normal controls(both P<0.05). The densities of Fox P3+ Tregs in the CRC stroma and epithelium were slightly increased as compared with adenomas, but the differences were not statistically significant(P>0.05).2? The IHC results showed that low density of Fox P3+ Tregs in the normal controls was observed in the stroma and epithelium. The IL-10 positive cells were observed in both the adenomatous stromal and epithelial. The expression pattern of IL-10 in the CRC is very similar to that in the adenoma, the IL-10 positive cellswas expressed in both the CRC stroma and epithelium. The semi-quantitative results also revealed that the density of IL-10 positive cells in the adenoma/CRC stroma was greatly increased as compared with the controls(both P<0.05). However, the density of IL-10 positive cells in the adenoma/CRC epithelium was not changed as compared with the controls(P>0.05).3? The expression of Fox P3 m RNA was quantified with real-time PCR. On the transcriptional level, the expression level of Fox P3 in the adenomas was slightly lower than that in the CRCs(P>0.05), but still significantly higher than that in the normal controls(P<0.05). The expression of Tregs in the adenomas was neither associated with the severity of dysplasia(LDG/MGD/HGD: 4.74±1.34 vs.4.72±0.96 vs. 6.05±3.23, P>0.05) nor the histological types(tubular?tubulovillous:4.15±1.12 vs 3.06±1.59, P>0.05). In addition, the expression of Tregs in the CRCs was not associated with the TNM stages(data not shown). The expression of IL-10 m RNA was quantified. The results showed that the level of IL-10 m RNA was greatly increased in both the adenomas and CRCs as compared with the normal controls(both P<0.05).However, the tissue m RNA levels of both Foxp3 and IL-10 demonstrated a non-statistical difference between the adenomas and the CRCs(both P>0.05).Conclusion The progression of adenoma-carcinoma sequence, Fox P3+ Tregs and IL-10 as a critical important factor in regulating immune tolerance and shaping immunosuppressive profile, is significantly increased in the adenomatous stage of the adenoma-carcinoma sequence. Such early activation of Tregs might inhibit the host immune response to the initiation of precancerous lesions and then contribute to the adenoma-CRC transition. Fox P3+ Tregs and IL-10 may be the molecular biological indicators for predicting malignant transformation of colorectal adenoma.