Tumor Infiltrating Cd4⁺Cd25⁺Cd127^Low/-Cd49D^-Regulatory T Cells Related To Prognosis Of HCC Patients

Part I Increased prevalence of CD4+CD25+CD127low/-CD49d FoxP3+regulatory T cells in HCC tumor microenvironmentObjective Expansion of regulatory T cells (Tregs) in tumor microenvironment was one of the mechanisms by which cancer cells escaped host defense. The purpose of this study was to investigate the increasing prevalence of Tregs in HCC cancer microenvironment. Methods Levels of CD4+CD25+CD127low/-CD49d-FoxP3+Tregs in peripheral blood mononuclear cells (PBMCs) from HCC patients (n=58) and healthy donors (n=12), tumor infiltrating lymphocytes (TILs) extracted from HCC (n=20), matched peri-tumor and PBMCs were measured by flow cytometry. Their suppression were determined by using activation marker expression (CD154). Superana levels of interleukin (IL)-10and transforming growth factor (TGF)-β1were measured by enzyme linked immunosorbent assay. Results The prevalence of Tregs in PBMCs from HCC patients was significantly higher than that from healthy donors and expressed high levels of IL-10and TGF-β1Similarly, the frequency of Tregs in tumor tissues was increased compared with that in matched peri-tumor and PBMCs. TIL Tregs were more suppressible by approximately30%than their peri-tumor and PBMCs counterparts. CTLA-4, GITR and MFI of FoxP3at higher levels on tumour-infiltrating Tregs compared with matched peri-tumor and periphery. Conclusions This study identified an increased frequency of CD4+CD25+CD127low/-CD49d-FoxP3+Tregs in patients with HCC. Further effort is needed to establish new immunotherapeutic strategies to modulate Tregs to promote a competent antitumor response. Part Ⅱ Comparative gene expression profile of CD4+CD25+CD127low/-CD49d-regulatory T cells in human hepatocellular carcinomaObjective The aim of this study was to compare gene expression profile of regulatory T cell (Treg) in HCC tumor sits with that of peri-tumor by Agilent whole genome oligo microarray. Methods Levels of CD4+CD25+CD127low/-CD49d-Tregs in HCC tumor (n=9), matched peri-tumor and PBMCs were measured by flow cytometry. Tregs from tumor and peri-tumor were hybridized on an Agilent whole genome microarray. Functional analysis of the microarray data was performed using KEGG PATHWAY and Gene Ontology (GO) analyses. Results The prevalence of Tregs in HCC tumor was significantly higher than that in peri-tumor. There were1032genes up-regualtion and1357genes down-regulation in tumor Tregs compared to peri-tumor. KEGG PATHWAY showed that7pathway up-regulation and9down-regulation of intratumoral Tregs compared to peri-tumor. GO showed that431terms up-regulation and237terms down-regulation. Microarray gene expression analysis showed that CD46, BCL6, TNFRSF11A, MDM4, P2RX7, CD276, TNFRSF18and IL6R were among the most upregulated genes in tumor Tregs compared with peri-tumor Tregs. The selected genes were validated by RT-PCR. Functional analysis showed that the complement and p53pathways were significantly upregulated in tumor Tregs, and that the GO terms related to cell differentiation, proliferation, apoptosis and migration were significantly affected. Conclusions This study identified IL6/IL6R, RANK/RANKL signalling might play an impotant role in intratumoral Tregs between HCC tumor cells. Part III Lectin Microarray based glycan profiling change of periphery regulatory T cell in patients with HCCObjective The aims of this section was to assess the applicability of application of lectin microarray to screen the glycosylation changes in complex biological systems. Based on that, glycosylation profilings were screened to search for the characteristic glycan structures associated with periphery regulatory T cells in HCC patients. Methods Firstly, we selected one pair of model protein:FET and its sialic acid defective strains ASF, of which characteristic glycan structures on the cell surface were well defined. Integrated protein was labeled with Cy3, then frozen in liquid nitrogen to terminatate the labeling. Next, membrane protein of Tregs was extracted by natural membrane protein extraction kit.10μg of protein was added to each chip and incubated for2hours, after washing, scanning and statistical analysis, lectin affinity profiling was obtained from the chip. Different batches of samples were loaded to detect the reproducibility of lectin microarray. HCC cell lines HCCLM3, MHCC97H, MHCC97L and Hep3B were used to hydratied with lectinarray and glycotranferase PCR microarray. Results The results showed that the characteristic glycosylation difference between ASF and FET were dtermined by defined lectin microarray. Comparing with ASF, FET showed increased affinity and decreased signals for SNA, MAL-Ⅰ and MAL-Ⅱ. HCC patients periphery Tregs presented elevated signals for LCA, UEA-Ⅰ, VVA, SBA, ACA, LEL and decreased signals for RCA-I with contrast to healthy controls. MAL-Ⅰ, MAL-Ⅱ, WGA, PHA-L were correlated with ST6GALNAC1、ST3Ga11、FUT8and MGAT5. Conclusions Our results proved that lectin microarray is an applicable glcan analysis tool, and could be used in clinical to screen HCC related Tregs glycan profiling, in search of better glycol-biomarkers for tumor immuno-escape. Besides, our study also presented reference and technology platform for the evaluation of the relationship between complex biological systems and glycan alteration. Part IV Overexpression of Galectin-1Associates with Poor Prognosis in Human Hepatocellular Carcinoma Following ResectionObjective High expression of the galectin-1predicted poor patient outcome in several tumors. The aim of this study was to investigate its prognostic value in patients with hepatocellular carcinoma (HCC) after resection. Methods Galectin-1and tumor-infiltrating FoxP3+regulatory T cells (Tregs) were validated by tissue microarrays from HCC patients (n=386) and statistically assessed for correlations with the clinical profiles and the prognosis of the patients. Serum galectin-1levels and frequency of FoxP3+Tregs in circulation was measured by ELISA and flowcyte, respectively. And serum levels of galectin-1was validated by ELISA in147HCC patients. Results We found that galectin-1, prevalently up-regulated in HCC, was significantly associated with tumor invasive characteristics (such as vascular invasion, incomplete encapsulation, poor differentiation, multiple number and large tumor size). Patients with high galectin-1expression had a significantly poorer tumor recurrence (p=0.025) and overall survival (p=0.021) than those with low galectin-1expression. Even in early-stage disease, high galectin-1expression also independently associated with shortened survival (p<0.001) and increased tumor recurrence (p=0.005). Multivariate Cox proportional hazards analysis showed that galectin-1was an independent marker for predicting poor prognosis of HCC. Galectin-llevel was positively related to number of tumor-infiltrating FoxP3+Tregs (r=0.416,p<0.001) and their combination served as a better prognosticator. The postoperative tumor recurrence and survival of HCC patients with galectin-1high and FoxP3high were significantly poorer than the other groups (both p<0.001). Conclusions Galectin-1may be a new prognostic factor for HCC after resection and potentially be a high-priority therapeutic target.