Galectin-9 Expression And Function On NK Cells Infiltration In Colorectal Cancer

Background:Colorectal cancer (CRC) is one of the leading causes of cancer related deaths in the world. During the last decades, in spite of the undoubted improvement in surgery and pharmaceutical strategies in the field of cancer treatment, current data point out that CRC is still far from being therapeutically controllable. The present dearth of knowledge regarding the immunological and molecular basis of CRC is a major obstacle to improving the diagnosis and treatment of this disease. Thus, identifying new biomarkers is necessary for the future development of targeted therapies for CRC. The development of cancer is a multi-step process, which is governed not only by numerous cell intrinsic factors, but also by the cell extrinsic factors in the tumor microenvironment. The former involves the gradual loss of regulation over the growth and functional malignant process of normal cells, and the later appear equally critical to the progression and treatment of cancer, such as immune cell, pro-inflammatory factor and chemokines. Based on the influence of immune microenvironment on tumor progression and prognosis, researchers have found more evidence that certain type leucocytes in the microenvironment of colorectal cancer are characterized by immune regulation mediated by the mediators from tumor site. Galectin-9, as a member of the galectins family, is widely distributed in the tissues involved in the immune system (such as the spleen, thymus, and peripheral blood lymphocytes) and in tissues of endodermal origin (such as liver, small intestine, stomach and lungs). Numerous studies have demonstrated the expression of galectin-9 differences in tumor tissues and corresponding normal tissues. Most studies suggest that tumor cells express galectin-9 was significantly lower than their normal controls. And in breast, lung, kidney, adrenal and prostate cancer cell lines, galectin-9 expression was reduced or absent. For cell lines derived from tumors of the the ovaries and nervous system, the difference galectin-9 expression is dependent on tumor cell subtypes. Galectin-9 involved in tumor development through a variety of mechanisms, such as regulation of apoptosis and cell cycle, adhesion and metastasis, angiogenesis and tumor immune escape. These studies suggest that galectin-9 may be as a potential tumor prognostic biomarker. However, the expression of the protein and its clinical significance in colon cancer has not been adequately studied.As an important component of the tumor microenvironment, certain types of leukocytes influence the tumor progress and prognosis. Natural killer (NK) cells are one of the major cell types in the innate immune system. In CRC, extensive intratumoral infiltration by NK cells is associated with a better prognosis, depending on their cytotoxic effects on cancer cells. However, a recent study found that NK cells are generally scarcer in the CRC microenvironment than in adjacent normal mucosa despite the presence of relatively high levels of NK cell-responding chemokines in tumor tissues. This contradiction suggested that chemokines alone might not be sufficient to recruit NK cells to the tumor. In tumor-bearing mice, galectin-9 increased the number of NK cells in the peritoneal exudate, indicating that it plays a potential regulatory role that involves NK cells during tumor progression. Given the close association between galectin-9 expression and NK cell numbers, it is reasonable to speculate that a reduced level of galectin-9 in a tumor contributes to the poor infiltration of NK cells into the tumor microenvironment. The coordinated reconstitution of the actin cytoskeleton is required for cell migration. The actin cytoskeleton senses and integrates chemical and physical signals into force-generating structures to control motility. Rho GTPase family proteins are key regulatory molecules that link surface receptors to the organization of the actin cytoskeleton in all the eukaryotic cells. Rho/ROCK signaling is necessary for many cytoskeleton-dependent processes, including the regulation of the cytoskeleton by the phosphorylation of downstream substrates, increases in actin filament stabilization and the generation of actin-myosin contractility. Galectin-8, another tandem-repeat galectin similar to the galectin-9 protein, induces cytoskeleton rearrangement in Jurkat T cells. Based on the above research results, we speculate that the galectin-9 may regulate the F-actin cytoskeleton in NK cells.T cell immunoglobulin mucin-3 (Tim-3), as a member of Tim family, is also one of the major receptors for galectin-9. As an important regulatory factor, Tim-3 plays an important role in a variety of lymphocytes and tumors. There is growing evidence that Tim-3 can function as a determinant of both the anti-tumor immune response and the development of cancer at multiple levels. Several reports suggested that PD-1(hi) Tim-3(+) T cells are strongly associated with leukemia relapse post transplantation in acute myelocytic leukemia (AML) patients. In renal cell carcinoma, levels of Tim-3 were significantly increased on both CD4+T cells and CD8+T cells and were associated with higher stages. Moreover, unregulated Tim-3 expression in NK cells is associated with the stage of the disease and predicts a poorer prognosis in melanoma and lung adenocarcinoma. Thus far, the expressions and clinical value of Tim-3 on various lymphocyte subsets in CRC patients has not been elucidated.In this study, we found that galectin-9 expression was reduced in colon tumor tissues, which is associated with poor prognosis in these patients. We also provide evidence using in vitro studies that galectin-9 enhances NK cell migration by exerting effects on F-actin polarization via the Rho/ROCK1 signaling pathway. Furthermore, we characterize Tim-3 expression on peripheral lymphocyte and clinical significance in CRC patients to further complement the regulatory mechanism of galectin-9 in NK cell-mediated immunity and tumor progression in colorectal cancer.SECTION I Galectin-9 expression in colon cancerOBJECTIVE:To analysis the galectin-9 expression in colon tumor tissues and explore the clinical significance of galectin-9.METHODS:A total of 128 colon tumor patients were enrolled in this study. Among these patients,38 colon cancer and matched para-tumor tissue samples were obtained to evaluate differences in galectin-9 expression and NK cell distribution between the two sites. Another group of 90 patients were used for survival analysis. Basic information, including sex, age, and clinical data, was retrieved from the medical records of each patient. An experienced pathologist reviewed the histopathological criteria, including TNM stage (7th edition, Union for International Cancer Control). The galectin-9 expression was assessed by immunohistochemistry. For galectin-9, the immunohistochemical score was determined based on the percentage of positive cells and staining intensity. The expression intensity was represented as a histological score (∑pi), where p is the percentage of galectin-9 positive cancer cells (scored as 1,1-10%; 2,11-50%; 3,51-80%; or 4,81-100%) and i represents the staining intensity (scored as 0, no staining; 1, weak staining; 2, moderate staining; or 3, strong staining). Strong (++++), moderate (+++), mild (++), weak (+) and negative (-) staining were 10-12,7-9,4-6,1-3 and 0, respectively, and the score was reduced to a 2-tier system (-/+:low vs.++/+++/++++:high) for statistical analysis according to the median.RESULTS:Among these 128 individuals,78.91% cases were positive for galectin-9 expression. During the evaluation of tumor and para-tumor tissue samples obtained from 38 patients, we found that galectin-9 was primarily expressed in the cytoplasm of both tumor and normal glandular cells but was not expressed in the nucleus or on the surface. The median immunohistochemistry score for galectin-9 in colon carcinoma was lower than that in the normal mucosa (p<0.001). Significant differences in galectin-9 expression were observed among TNM stage, lymph node metastasis, and histological grade but not gender, age, or distant metastasis. Kaplan-Meier analyses show the median survival time of patients with galectin-9high is longer than patients with galectin-9low expression (p=0.012).In addition, early TNM stage (Ⅰ/Ⅱ) and no lymph node metastasis (N-) were significantly associated with good outcomes. Univariate and multivariate analysis reveal that galectin-9 expression in colon tumor tissues is a positive prognostic factor for overall survival.CONCLUSIONS:Our data provided the first evidence that galectin-9 is down-regulated in colon tumor tissue and suggests a poor outcome.SECTION II The function and mechanism of galectin-9 in regulation NK cell infiltrationOBJECTIVE:To detect CD56+NK cell infiltration characteristics in colon tumor tissue and the corresponding normal tissues, and analyze whether there is a correlation between galectin-9 expression and NK cell infiltration. In addition, we explore the mechanism of galectin-9 regulate NK cell chemotaxis.METHODS:The galectin-9 expression was assessed by immunohistochemistry in 38 colon cancer and matched para-tumor tissue samples. The degree of CD56+NK distribution was determined for more than 10 high-power (200×) microscopic fields in each tissue sample. A total of 5 areas with the densest lymphocyte distribution were selected, and microphotographs were obtained. In each case, the mean index of CD56+cells per single high-power field was counted, and this number was used for statistical analysis. Based on the median cell number (cut-off point of quartile), the patients were divided into CD56 negative-, low-, mild-and high-intensity groups to examine the correlation between galectin-9 expression and CD56+NK cell distribution in the same case. SiRNA-galectin-9 and the corresponding negative control were transfected into HT29 cells through Lipo 2000. RT-PCR, western blot analysis and ELISA were used to detect efficiency of transfection. Tumor culture supernatants were also collected. NK-92 cells were plated in the top chambers. Colon tumor cell culture supernatant or varying concentrations of rh-galectin-9 were added to the bottom chamber. NK-92 cells were permitted to migrate into the bottom chamber for 4 h at 37℃. They were then harvested and counted using cell-count boards. Y-27632 dihydrochloride and C3 transferase were added to test the role of Rho/ROCK signaling in NK92 migration. NK92 cells treated with or without galectin-9 for 4h were incubated with rhodamine-labeled phalloidin. The role of rhGalectin-9 in F-actin polarization in NK92 cells was investigated using laser scanning confocal microscopy.RESULTS:The CD56+cell infiltration in para-tumor tissues was significant higher than that in tumor tissue (p<0.001). Moreover, the percentage of CD56+cell intratumoral infiltration was significantly higher in galectin-9hlgh tissue than in galectin-9low tissue (p<0.0001). Galectin-9 expression significantly correlated with CD56+NK cell infiltration in colon cancer and para-tumor samples (p<0.0001; R2=0.658). The HT29 culture supernatant significantly enhanced NK92 cell chemotaxis. The promoting effects of HT29 supernatant on NK92 chemotaxis were significantly reversed by galectin-9 siRNA and the effects were statistically significant in both siRNA #378 and siRNA #690 groups. The recombinant galectin-9 can significantly enhance the NK92 chemotaxis. Y27632 and C3 transferase significantly inhibited NK cell migration. Rhgalectin-9 significantly increased the expression of multiple members of the Rho family, including RhoA, RhoB, RhoC, RhoF, RhoG, RhoH, and RhoTl. ROCK1 protein levels in NK cells stimulated with rhGalectin-9 for 24 h were also up-regulated, which reach statistical significance. Rhgalectin-9 also influences the rearrangement of the F-actin cytoskeleton in NK92 cells. Galectin-9 also enhances primary NK cell chemotaxis by enhancing Rho/ROCK expression.CONCLUSIONS:Our data show that galectin-9 is down-regulated in colon tumor tissue and suggests a poor outcome. Furthermore, we found that galectin-9 has a novel function in immunological surveillance by regulating F-actin polarization in NK cells, and this regulation is associated with the activation of Rho/ROCK1 signaling. These results provide a foundation for the future exploration of a potential role for galectin-9 in the regulation of NK cell trafficking, potentially representing a new target for the immune treatment of colon cancer.SECTION III Expression and clinical significance of Tim-3 in peripheral lymphocytes in colorectal patientsOBJECTIVE:To explore the frequency of T cells and NK cells and NKT cells expressing Tim-3 in peripheral blood of preoperative CRC patients and paired blood. We also investigated the expression of Tim-3 in lymphocyte subsets from postoperative blood samples of CRC patients. Data were correlated with clinic-pathological parameters of patients.METHODS:127 Chinese individuals including 89 colorectal cancer patients and 38 age-and gender-matched healthy volunteers (designed as the healthy control group (HC)) were recruited from Qilu Hospital of Shandong University from May 2013 to October 2013(Jinan, China). Peripheral blood samples were obtained from the participants after they provided written informed consent and processed within 6 hours of collection. The frequency of T cells and NK cells and NKT cells expressing Tim-3 was assessed by multicolor flow cytometry. The associations between Tim-3+ lymphocytes and clinical pathological factors were analyzed.RESULTS:Compared to healthy individuals the percentage of circulating NK cells from CRC patients was significantly decreased (p=0.0027); whereas the levels of T cells and NKT cells from untreated CRC patients and healthy controls had no significant difference (p>0.05). The expression of Tim-3 on NK cells is higher than on NKT cells and T cells. Tim-3+NK cells was significantly decreased in CRC cases compared to HCs (p=0.0239). It was found that the frequency of circulating NK cells and Tim-3+NK cells were negatively correlated with clinical cancer stage compared to the healthy controls but not with other clinicopathological parameters. The patients with stage Ⅰ/Ⅱ/Ⅲ cancer contained the remarkably increased percentage of NK cells which reach the statistical significance when compared with IV stage (p= 0.0014). Significant difference was also seen for percentage of Tim-3+NK cells between non-metastatic patients and metastatic groups (p= 0.0218). There was no difference in percentage of lymphocytes between patients untreated and accept surgery. Strikingly, Tim-3+NK cells in patients with surgery was significantly higher than those without surgery (P= 0.0003). Surgery elevated Tim-3 expression to baseline levels on NK cells in nearly every patient with reduced pre-surgical levels (P= 0.0126). No relationships were observed between altered levels of NK cells and Tim-3 expression patterns on NK cells in relation to serum concentration of cancer biomarker tested pre-treatment.CONCLUSIONS:Our data suggests that lower Tim-3 expression on NK cells is associated with CRC progress and with poor prognostic clinical parameters. We show that Tim-3 is a bioactivity marker expressed in NK cells from CRC patients. Whether galectin-9 affect NK cells by Tim-3 still need to be confirmed by future experiments.Summary:1. Low expression of galectin-9 in colon tumors can predict poor prognosis and can regulate NK cell infiltration by Rho/ROCK1 pathway;2. Levels and Tim-3 expression of peripheral NK cells in colorectal cancer patients reduced significantly, and predict CRC progress and poor TNM staging.Innovation:1. We investigated the clinical significance of colon tumor cells derived galectin-9 and its influence on NK cell infiltration from the point of view of immunology. The results suggest that galectin-9 regulate NK cell chemotaxis through affecting Rho/ ROCK1 pathway and F-actin polarization. And the specific mechanism remains to be further studied.2. We discussed the expression level and clinical significance of Tim-3 of different lymphocyte subtypes in peripheral blood of colorectal cancer patients, and found that Tim-3 on the NK cells was lower and indicated later clinical stage. This suggests that it could be another potential mechanism for galectin-9 regulation of NK cells and immune escape in CRC patients.