| Hepatocellular carcinoma (HCC) is one of the most common human cancers worldwide, ranking as the fifth in the morbidity of malignant tumor and the third leading cause of cancer related death in the world, resulting in 600,000-700,000 deaths each year, of which 55% occurred in China. In spite of the advances in diagnosis and treatment, the overall survival remains dismal due to the high recurrence rate after curative resection. Recently, the role of tumor microenvironment in tumor progression has gained extensive attention. Cumulative evidence has indicated HCC is a typical example of inflammation-related cancer mainly due to hepatitis viral infection and cirrhosis. The chronic inflammatory status in HCC leads to an intricate tumor microenvironment which is a complex and dynamic network composed of a large variety of inflammation/immune cells and mediators.Thus, to a large extent, HCC metastatic biologic behavior and poor prognosis may been determined and/or influenced by the local inflammatory or immune status. In this regards, these cells and some critical mediators in them therefore could provide us available target to establish a promising therapeutic strategy for HCC.Tumor microenvironment played an important role in the tumor progression. As a critical component of tumor immune microenvironment, the lymphocytes played dual roles in them, not only suppressing tumor growth and invasion but also promoting caner progression either by immunoselection or establishing an immune inhibition network within the tumor microenvironment that facilitated cancer development. Until now, much work has indicated CD8+ T, NKT and NK cells exert an tumor-suppressing effect, whereas T helper cells differs in dual effect via differentiating to various directions including Th1,Th2, Th17 and regulatory T cells within tumor microenvironment. Nonetheless, differential expression of infiltrating lymphocytes in tumor and paired peritumoral tissues was not well defined yet.In this study, firstly, we established and modified the technique of isolation of lymphocytes from tumor and peritumoral tissues, detecting types of major antitumoral effertor cells and the ratio of lymphocytes by flow cytometry. Secondly, we compared the differential genes expression profiles of infiltrating lymphocytes between tumor and paired peritumoral tissues from 15 HCC patients, which were further analyzed by the pathway and gene ontology analysis with divided groups by the tumor size and vascular invasion. Thirdly, based on the results of gene microarray of the infiltrating lymphocytes, forty immune genes were selected for expression verification by quantitative PCR on a total paried samples of 103 HCC patients. Finally, we evaluated the expression and prognositic value of CD39+Foxp3+regulatory T cells and CD39, which was an activated marker of regulatory T cells.PartⅠThe isolation and identification of infiltrating lymphocytes between tumor and peritumoral tissues of hepatocellular carcinomaPurpose:This part aimed to establish and modify the technique of isolation of lymphocytes from tumor and peritumoral tissues and to detect the types of major antitumoral effertor cells and the ratio of lymphocytes.Methods:Infiltrating lymphocytes were isolated and purified with discontinuous density-gradient centrifugation. The types of major antitumoral effertor cells and the ratio of lymphocytes were detected by flow cytometry.Results:The isolated and purified lymphocytes from the tumor and peritumoral tissues had high purity and viability (purity>95%, viability>90%). The ratio of CD3 postive T lymphocytes in peritumoral tissues was higher than that of peripheral blood (56.14±13.64 vs 43.17±7.38%,mean±SD, P= 0.0019), which meant the liver was the place where induced T lymphocytes differentiation and maturation. We further compared the ratio of CD4 postive T lymphocytes and found that the ratio of CD3+CD4+CD8- T cells in peripheral blood was the highest among them (52.47±15.94%). The ratios of CD3+CD4+CD8’T lymphocytes in peripheral blood and tumor were higher than those in peritumoral tissues(P<0.001,P=0.001), which were gradually increased in peripheral blood, tumor and peritumoral tissues (37.45±14.79 vs 41.39±13.63 vs 56.17±12.21%). Among them existed the difference between peritumoral tissues and peripheral blood, peritumoral tissues and tumor (P=0.003, P=0.003).The ratio of CD8 postive T lymphocytes was lowest among them, accompanying with reversed ratio of CD4/CD8 (1.585/0.499). The ratio of CD3"CD56+ NK was much lower in tumor than that of peritumoral tissues (12.66±7.62 vs 22.77±13.51%,P=0.026). The ratios of CD3+CD56+ NKT cells were gradually increased in peripheral blood, tumor and peritumoral tissues (3.35±2.22 vs 6.81±5.07 vs 16.05±8.52%).There was the difference between the ratio of CD3+CD56+ NKT cells in tumor and peritumoral tissues, even peripheral blood and the peritumoral tissues (P<0.001, P=0.006). The ratios of NK and NKT cells were lower in tumor than that of peritumoral tissues. Furthmore, the ratio of CD3+Va24+NKT cells was no more than 1% in peripheral blood, which was much lower than that of tumor and peritumoral tissues. However, There was no difference between tumor and peritumoral tissues.Conclusion:The isolated and purified lymphocytes from the tumor and peritumoral tissues had high purity and viability, which indicated that the technique of isolation was feasible. Compared with the peritumoral tissues, It was the situation that there were decreased ratio of CD8 T lymphocytes, NK cells and NKT cells and reversed ratio of CD4/CD8 in HCC, which indicated decreased major antitumoral efifertor cells in HCC.Part IIAnalysis of differential genes expression of infiltrating lymphocytes between tumor and peritumoral tissues in hepatocellular carcinomaPurpose:To analyze the differential genes expression of tumor infiltrating lymphocytes between tumor and paired peritumoral tissues in hepatocellular carcinoma.Methods:we compared the differential genes expression profiles of infiltrating lymphocytes between tumor and paired peritumor tissues from 15 HCC patients by hybridizing them on an Agilent whole genome. Functional analysis of the microarray data was performed using KEGG pathway and gene ontology analyses.Results:HCC patients were devided into two groups by tumor size (tumor diameter large than 5cm or not) and vascular invasion, which the corresponding case numbers were 9 vs 6 and 8 vs 7. We totally acquired about 19,786 gene expression. Compared with the peritumoral tissues, there were 521 increased and 456 decreased genes expression of infiltrating lymphocytes in tumor, which focused on the up-regulated complement and coagulation pathway and down-regulated natural killer cell cytotoxicity and antigen processing and presentation pathway, indicating impaired funtion of inate immunity and cell killing. There were 857 increased and 827 decreased genes expression of infiltrating lymphocytes in large tumor compared with the paired peritumoral tissues. The numbers of differential gene expression of tumor infiltrating lymphocytes between large and small tumor were 1501 increased and 1282 decreased, which meant that the tumor size was an important factor affected the differential gene expression. When the tumor was small, the fuction of natural killer cell cytotoxicity was impaired. With the tumor progression, the differential gene expression in tumor focused on the down-regultaed the Toll-like receptor pathway and Jak-STAT signaling pathway, which were the key pathway promoting the proinflammatory factor release. However, the differential gene expression in peritumoral tissues was increased with T helper 2 cytokines(interleukin 4 and interleukin 8).Conclusion:There were differential genes expression of infiltrating lymphocytes between tumor and peritumoral tissues in hepatocellular carcinoma, which focused on the up-regulated complement and coagulation pathway and down-regulated natural killer cell cytotoxicity and antigen processing and presentation pathway, indicating impaired funtion of inate immunity and cell killing. The tumor size was an important factor affected the number of differential genes expression.Part IIIValidation of the differential genes expression of infiltrating lymphocytes between tumor and peritumoral tissues in hepatocellular carcinomaPurposerTo validate the differential genes expression of tumor infiltrating lymphocytes between tumor and paired peritumor tissues in hepatocellular carcinoma.Methods:We selected forty immune genes for expression verification by quantitative PCR on a total paried samples of 103 HCC patients.Results:Above 90%(36 genes) of 40 differentially expressed genes were validated with the consistent up or down trend by us. These significant genes were focused on the imparied funtion of antitumor efforts cells (FASL, PRF1), natural killer cell cytotoxicity pathway (CD244, NKG2D), transcription factors of lymphocytes (EOMES, TBX21, TXK), costimulatory molecule and receptor (TNFRSF11A, CTLA4), cytokines, chemokines, integrin and receptors (IL8, XCL1, ITGAV), cell surface antigen and other immune molecules (CD160, BCOR). The majority of differentially expressed genes were associated with one or more of the tumor invasive or clinical chatacteristics such as tumor size, vascular invasion, incomplete encapsulation, poor differentiation, etc).Conclusion:There were differential genes expression of infiltrating lymphocytes between tumor and paired peritumor tissues in hepatocellular carcinoma. The data of whole genome microarray was reliable and repeatable.Part IVDifferential expression of CD39+Foxp3+Regulatory T cells between tumor and peritumoral tissues and their associations with prognosis of hepatocellular carcinomaPurpose:To evaluate the expression of CD39+Foxp3+Regulatory T cells between tumor and peritumoral tissues and their associations with prognosis of hepatocellular carcinoma. Furthermore we detemined the differential expression of CD39 andi the prognostic value in HCC.Methods:In this study, double immunohistochemistry (IHC) was used to analyze the expression of CD39 and Foxp3 in a cohort of 324 HCC patients. The expression of CD39 in various cells of tumor was also evaluated by double immunohistochemistry. The expression of CD39 in HCC cell lines with stepwise metastasis potential and human umbilical vein endothelial cell was determined by immunobloting and immunofluorescence, respectively.Results:Double IHC showed that tumoral tissues had significantly higher counts of positive Foxp3+and CD39+Foxp3+Treg (14.17 vs 4.99, P= 0.001; 11.53 vs 3.393, P< 0.001) and higher ratio CD39+Foxp3+/Foxp3+(83.34 vs 79.19%, P= 0.013). The levels of intratumoral Foxp3+and CD39+Foxp3+Treg showed prognostic role with OS (P= 0.035, P= 0.019,). IHC showed CD39 principally expressed on the vascular endothelial cells, macrophagocytes tumor cells and Treg in HCC. Compared with paired peritumoral tissues, tumoral tissues had significantly high positive CD39 expression (Mean Optical Density,0.0552 vs.0.0387, P< 0.0001). The levels of tumoral CD39 expression were shown to be related to TTR (P= 0.033) and OS (P= 0.021). Furthermore, The level of peritumoral CD39 expression showed prognostic role with TTR (P= 0.003).Conclusion:There were higher counts of Foxp3+ and CD39+Foxp3+ Treg and more expression of CD39 in tumor than those in paired peritumoral tissues of hepatocellular carcinoma. The levels of intratumoral Foxp3+ and CD39+Foxp3+Treg postive T cells showed prognostic role with OS, highlighting the suppressive role of CD39 positive Treg in HCC. High intrahepatic expression of CD39 reduced patient outcome of hepatocellular carcinoma after resection and can predict the postoperative recurrence and survival time of patients with HCC.Conclusions1. The isolated and purified lymphocytes from the tumor and peritumoral tissues had high purity and viability, which indicated that the technique of isolation was feasible. Compared with the peritumoral tissues, It was the situation that there were decreased ratio of CD8 T lymphocytes, NK cells and NKT cells and reversed ratio of CD4/CD8 in HCC, which indicated decreased major antitumoral effertor cells in tumor.2. There were differential genes expression of tumor infiltrating lymphocytes between tumor and peritumor tissues in hepatocellular carcinoma, which focused on the up-regulated complement and coagulation pathway and down-regulated natural killer cell cytotoxicity and antigen processing and presentation pathway, indicating impaired funtion of inate immunity and cell killing. The tumor size was an important factor affected the number of differential genes expression.3. There were differential genes expression of infiltrating lymphocytes between tumor and paired peritumoral tissues in hepatocellular carcinoma. The data of whole genome microarray was reliable and repeatable.4. The levels of intratumoral Foxp3+ and CD39+Foxp3+Treg postive T cells showed prognostic role with OS, highlighting the suppressive role of CD39 positive Treg in HCC. High intrahepatic expression of CD39 reduced patient outcome of hepatocellular carcinoma after resection and can predict the postoperative recurrence and survival time of patients with HCC.Novelty1. We firstly analyzed the differential genes expression with isolated infiltrating lymphocytes between tumor and paired peritumorral tissues in hepatocellular carcinoma and verify it reliable and repeatable.2. We firstly evaluated the expression and prognosis value of CD39+Foxp3+ regulatory T cells and CD39, which was the activated marker of regulatory T cells. |
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