The Mechanism And Clinical Significance Of Costimulatory Molecule B7-H3 In Colorectal Carcinoma Microenvironment

The normal cells are balanced with the around tissue environment. The interactions between them regulate cell proliferation, differentiation, apoptosis and the secretion of cell cytokines. However, the balance is broken during the tumor genesis. The rapid proliferation of tumor cells needs to build their own tissue environment, named tumor microenvironment. In the tumor microenvironment, both positive and negative immune cells exist, including macrophages, leukocytes, effective T cell, regulative T cells, anergic T cell, etc. During the different stages of the tumor development, the changes of the number and function of the immune cell subsets, as well as the production of various cell cytokines and the different immune responses their involved, result in the coexist of immune surveillances and tolerance. This is how the immune escape of the tumor finally formed. Thus, the tumor infiltrated immune cells act dual characters during the genesis and development of tumor. The major factors involved in the tumor immune escape in the tumor microenvironment are the abnormal APC cells (DC and macrophages), inhibitive myeloid cells, regulative T cells and the negative molecules of B7 costimulatory family, which are highly expressed in the tumor cells and interspace cells.The generation of"costimulator signal"is based on the dual-signal model of T cell activation, first described by Bretscher and Cohn in 1970. Due to the increasingly research on the co-stimulatory signal, the costimulatory molecules are now the popular research targets in the immunology study. Recently, some costimulatory molecules are detected extraordinarily expressed in various tumors. The abnormal expression of costimulators on the tumor cells, including the decrease of positive costimulatory molecules and the abnormal increase of the negative costimulatory molecules, which are involved in the progress of escape, metastasis and migration, etc., are important elements to construct the tumor microenvironment. The newly discover of the extraordinary expression of B7 family members in tumor has attracted more and more attention to the research on B7 costimulatory family.B7-H3, one of the B7 family was cloned form the human DC cDNA library in 2001. The human B7-H3 gene, located in chromosome 15q24, belongs to I type transmembrane protein of the Ig super family. The mRNA of human B7-H3 is generally expressed in the human tissues, while the protein is rarely expressed in the normal tissues. Mouse has only one transcript of B7-H3, which contain one IgV-domain and one IgC domain (2IgB7-H3), while human and other primates have an addition transcript, which contains four domains: IgV-IgC-IgV-IgC (4IgB7-H3). The initial researches suggested B7-H3 to be a positive cosimulator, for it could stimulate the proliferation of T cell, and improve the CTL activity as well as the secretion and expression of IFN-. However, the subsequent researches indicated that B7-H3 fusion protein could inhibit the T cell proliferation in vitro, and the mouse B7-H3 was mainly involved in the negative regulation of immune response, thus B7-H3 might be a negative costimulator. The uncertain of its receptor hold-up the research of B7-H3 function. Up to now, B7-H3 protein has been detected to be highly expressed in many tumor tissues through the histochemistry analysis. The B7-H3 molecule expressed in the cell surface and cytoplasm as well as in the tumor vessel, has a relationship with the clinical patho-factor and prognosis, and thus suggested to be important in the tumor genesis and development. However, the regulatory mechanism of the abnormal expression of B7-H3 in tumor tissues and how the B7-H3 molecule regulate the tumor immune response, remain unclear.Colorectal carcinoma is one of the frequently occurred cancer in human, the fourth cause of death leading in worldwide. The tumor genesis of CRC started from normal tissues to polypi, adenoma, intraepithelial neoplasm and cancer. It is a long and slow period. The long-term of interaction between immune cells, cytokines and tumor cells play important roles in the tumor progression.This study is to explore the costimulatory molecule B7-H3 expression and function mechanism in CRC microenviroment and tumor immue response.PartⅠThe expression and clinical significance of B7-H3 in Colorectal carcinomaObjective: To investigate the expression of costimulatory molecule B7-H3 in CRC tissues and analysis the correlation to pathological parameters and survival rate; to explore the significance of B7-H3 expression in the tumor genesis of CRC.Methods: 219 cases of CRC tissues were collected with particular pathological data, and tissues from each precancerous lesion stage of CRC (polypi,adenoma, intraepithelial neoplasm) were collected as well. B7-H3 expression was detected by IHC, and the correlation was analyzed with statistic software.Results: The IHC staining showed that low B7-H3expression was found in 99 cases of tissues, and high B7-H3 expression was found in120 cases of tisses. B7-H3 expression was uncorrelated with the patients'gender, tumor location, lymphatic metastasis, distant metastasis and mucinous adenocarcinoma, while correlations were found between B7-H3 expresion and patients'age, size of tumor, differentiation and tumor stages. B7-H3 expression has no relevance to patients'survival time. Further, we found that B7-H3 expression occurred in early stage of precancerous stage, adenoma and intraepithelial neoplasm, while other members of negative B7 family molecules such as B7-H1 and B7-H4 expression were found only in tumor tissues.Conclusion:①.Costimulatory molecule B7-H3 was abnormally expressed in CRC tissues, correlating to CRC stages and tumor progression.②.B7-H3 was the only B7 family molecule involved in CRC tumor genesis with the early expression in precancerous lesions.PartⅡT he regulation of soluble B7-H3 and significance in CRC patients'serumObjective:To detect the expression of soluble B7-H3 in CRC patients'serum, and analysis the correlation to clinical parameters; to explore the regulation and production mechanism of soluble B7-H3 in CRC cells.Methods: 56 cases of CRC patients'and 60 cases of healthy person's serum were collected with pathological data. ELISA was performed to detect to soluble B7-H3 expression, FCM were used to analyze the membrane B7-H3 on CRC cell lines. Cytokines TNF-a,IFN-γwere used to stimulate CRC cell lines, and MMPI was used to inhibit the production of soluble B7-H3.The B7-H3 expression changed was determinate by FCM, ELISA and RT-PCR. The expression of two B7-H3 isoforms were analyzed by RT-PCR.Results: ELISA visualization show that soluble B7-H3 existed in CRC patients and healthy person's sera. The expression of soluble B7-H3 was remarkably increased in CRC patients'serum (30.34±11.93ng/ml) compared to healthy donors (16.8±7.48ng/ml). the soluble expression of B7-H3 was closely correlated to ymphatic metastasis and distant metastasis. Membrane B7-H3 could be expressed on all of the five CRC cell lines, Neither TNF-a nor IFN-γcould affect the membrane expression. Whereas, TNF-a could up-regulate the 2IgB7-H3 mRNA expression and soluble B7-H3 secretion in CRC cell lines. The secretion of soluble B7-H3 by CRC cells could be inhibited by MMPI.Conclusion:The soluble B7-H3 expression was increased in CRC patients'serum, and involved in tumor metastasis. This revealed that the membrane B7-H3 and soluble B7-H3 might play different roles in CRC progression, through functions in different location, tissue and periphery. The soluble B7-H3 expression was regulated by TNF-a, the important inflammatory cytokine involved in CRC. TNF-a could promote the soluble B7-H3 secretion of CRC cells, by up-regulating the 2IgB7-H3 transcription, which might be the main source of soluble B7-H3. MMPs coud promte the CRC cells to shed the membrane B7-H3 molecule into soluble B7-H3.PartⅢThe Effect and mechanism of tumor-associated B7-H3 on tumor infiltrated immune cellsObjective: To investigate the infiltrating of immune cells in CRC tumor microenvironment and the clinical significance, and study that how B7-H3 regulate the infiltrated immue cell and influent the tumor immune response.Methods: IHC staining was performed to investigate the T cell subset and macrophages by marker molecules. The correlations were analyzed between immune cells amounts and B7-h3 expression, clinical parameters. Monocyte was separated from whole blood, and stimulated by LPS, and the B7-H3Ig was used to analyze the possible receptor. CW-2, a CRC cell line expressing high level of B7-H3 molecule, was adopted to induce monocyte differentiation. The membrane marker of macrophage was detected by FCM, and the intracellular molecules was analyzed by PT-PCR, the secretion of cytokines were determinate by ELISA. A neutralizing antibody against B7-H3 was used to block the B7-H3 signal in the above system.Results: IHC staining show that a large amount of CD3+T, CD8+T and CD68+TAM were found in CRC tissues. The amount of CD3+T was correlated to CRC patients'survival time, while CD8+T made no sense at this point. B7-H3 expression was unrelated to the T cell infiltrating. The amount of CD68+TAM was negatively related to CRC patients'survival time and positively related to B7-H3 expression. With LPS stimulation, the possible receptor of B7-H3 was induced on monocytes. With the co-effect of B7-H3 and tumor cytokines, monocyte polarized into M2 macrophages, with low expression of MHC-Ⅱ(DR) and high expression of MMR (CD206), and barely expressed iNOS while produced high level of IL-10 and TNF-a. Using the neutralized B7-H3 antibody, the MHC-Ⅱ(DR) and iNOS were up-regulated and the MMR (CD206) and cytokines were decreased.Conclusion: This part of study manifested thatCD3+T , correlated t survival times, played a important role in tumor immune response, while CD8+T, unrelated to survival time, might be composed by some heterogeneity CD8+T cells such as anergic cells in tumor microenvironment.B7-H3 was not supposed to regulate tumor immune response by targeting T cell but macrophaged, and the possible receptor of B7-H3 could be induced on monocyte. B7-H3 expression was related to the amount of infiltrated macrophages, tumor-associated B7-H3 could promote the monocyte polarization into tumor associated macrophages, M2 macrophages. The neutralization of B7-H3 signal could inhibit the differentiation of M2 macrophages, which indicated that B7-H3 could participate the tumor escape by induction of M2 macrophages in tumor microenvironment. It's a new thought of how B7-H3 was involved in tumor immne response.Taken together, this study investigated the B7-H3 expression and clinical significance in CRCs, and further explored the mechanism of how B7-H3 regulate the tumor response in microenvironment, which offered the new backgrounds for search of the diagnosis bio-marker and means to intervene the CRC progression. Our study is original and of potential application value.