The Relationship Between TAMs Induced By IL-6 And Lymphatic Metastasis In Gastric Cancer

Gastric cancer is one of the most common malignant gastrointestinal tumor, its incidence ranked fifth in the neoplastic disease, and the mortality rate ranked the second, the cause of death is invasion and metastasis of cancer cells in patients. The priority metastasis of gastric cancer is lymphatic metastasis, and the formation of lymphatic vessels is the basis of the lymph node metastases. Therefore, researching the mechanism of lymphatic vessels formation is an important approach to explore new therapeutic target and inhibit the lymph node metastases.Tumor microenvironment is constituted of the tumor cells, mesenchymal cells, as well as the infiltration of immune cells, etc. Infiltration of inflammatory cells play important role in the process of invasion and metastasis of tumor cell and macrophages are one of the main inflammatory cells in the tumor microenvironment. Macrophages are differentiated by circulation of monocyte in peripheral blood, including proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. Macrophages infiltrated in tumor microenvironment are named tumor associated macrophage(TAMs), which exhibit M2 macrophages-like phenotype and closely related to the progression of many tumors.Tumor microenvironment is the execution venues of tumor immune effector, where many factors involved in the interaction with tumor tissue and immune system. As a potent and pleiotropic inflammatory cytokines in tumor microenvironment, IL-6 composes the inhibited tumor immune microenvironment that provides a basis for tumorigenesis, progression and metastasis. Now we focus on gastric cancer and explore the relationship among IL-6, TAMs and lymphatic metastasis in gastric cancer environment.Objectives:1. To explore the distribution of IL-6, TAMs and VEGF-C in gastric cancer microenvironment and their potential role in tumor progression and lymphatic metastasis.2. To induce TAMs with IL-6 and investigate the effect of TAMs on tumor progression and lymphatic metastasis.Methods:1. Detection and analysis the expression of IL-6, TAMs, VEGF- C and lymphatic vessel density in gastric cancer tissues and normal gastric tissuesGastric cancer tissues and normal gastric tissues were collected from 40 patients with gastrectomy, subsequently embedded in paraffin, and diagnosed at the Southwest Hospital of Third Military Medical University from July 2008 to July 2009. The sections were made immunohistochemical double staining with rabbit anti human IL–6, rat anti human CD68, rat against human D2-40 monoclonal antibody and rabbit anti human VEGF- C polyclonal antibody, respectively. The sections were first evaluated at x100 magnification, and five representative areas where marker accumulated at high density were identified. They were then counted at x200 magnification in each case, and the average value was used. The relationship between clinical data and the expression of IL- 6, TAMs, VEGF-C and lymphatic vessels was analysised with T-test and chi-square test. The correlation among IL- 6, TAMs, VEGF-C and lymphatic vessels in gastric cancer tissue was test with linear regression analysis. The median number of IL-6, CD68, VEGF-C and D2-40 was made as dividing line, then the divided groups were made postoperative follow-up for survival analysis.2. Establishing the model of monocyte differentiated to TAMs with M2 phenotypesCD14+ monocytes were extracted from normal human peripheral blood monocyte(PBMC) with magnetic bead separation, then the monocytes were cultured with 100 ng/ml M – CSF. After 6 days, the cells were test the macrophage phenotypes CD68 and CD11 b with flow cytometry instrument, then the immature macrophages were cultured with different concentration of IL-6 for 24 h. Total RNA were extracted from cultured cells, then the gene change of IL-10, IL-12 and VEGF-C was test with RT-PCR.3. The effect of TAMs induced by IL-6 on the invasion ability of MGC803 gastric cancer cell linesMGC803 gastric cancer cell was separately co-cultured with the blank control group only with culture medium, the negative control group with macrophages induced by M-csf and the experimental group with macrophages induced by IL-6+M-csf for 48 h. After crystal violet staining, the number of gastric cancer cells penetrated through Matrigel was counted with optical microscope.Results:1. IL-6、CD68、VEGF-C and D2-40 expression in gastric cancer and normal gastric tissueStatistical analysis showed that the mean number of IL-6, TAMs, VEGF-C and lymphatic vessels in tumor tissues is higher than that in adjacent normal tissues(48.0±10.3 vs 11.1±2.3, 26.0±5.5 vs 5.9±1.6, 26.7±6.5 vs 9.7±2.8, 7.6±3.8 vs 2.5±1.2,P<0.05)。2. The relationships among IL-6, TAMs, VEGF-C and LVD with clinical pathological features of gastric cancerThe expression of IL-6, TAMs, VEGF-C and LVD is positively associated with depth of invasion, the expression of them in the T3+T4 group are higher than that in the T1+T2 group(50.1±8.8 vs 41.5±12.2, 27.3±5.1 vs 22.2±5.1, 28.0±6.3 vs 22.8±5.8, 8.3±3.9 vs 5.5±2.6, P < 0.05); The expression of IL-6, TAMs, VEGF-C and LVD is positively associated with TNM stages, the expression of them in the advanced stage TNM group(III+IV stage)are higher than that in the early stage TNM group(I+II stage)( 5 7.3±2.6 vs 38.6±5.3, 31.1±1.9 vs 21.0±2.2, 32.6±2.9 vs 20.8±2.3, 10.6±2.9 vs 4.7±1.6, P<0.05); The expression of IL-6, TAMs, VEGF-C and LVD is positively associated with lymph node metastasis, the expression of them in the group with lymph node metastasis are higher than that in the group with non-lymph node metastasis(55.2±6.4 vs 39.2±6.7, 29.9±3.8 vs 21.3±3.0, 31.3±4.5 vs 21.2±3.4, 9.6±2.9 vs 5.2±3.3, P<0.05). However the expression of IL-6, TAMs, VEGF-C and LVD is non-associated with gender, age, tumor location, tumor size and distant metastasis.3. The correlation of IL- 6, TAMs, VEGF- C and LVD in gastric cancerThe expression of IL-6 was positively correlated with TAMs number in GC(r=0.941, P<0.05); TAMs number was positively correlated with the expression of VEGF-C(r=0.911, P<0.05); the expression of VEG-C was also positively correlated with LVD(r=0.741, P < 0.05).4. The relationship among IL-6, TAMs, VEGF-C and LVD with the prognosis of gastric cancerOne year cumulative survival rate in high expression of IL-6 group, high expression of CD68 group, high expression of VEGF-C group and high expression of D2-40 group respectively was 70.0%, 70.0%, 70%, 65.0%, and it in low expression of IL-6 group, low expression of CD68 group, low expression of VEGF-C group and low expression of D2-40 group respectively was 90.0%, 90.0%, 90.0%, 95.0%. The difference was statistically significant(χ2=4.410,χ2=4.075,χ2=4.353,χ2=4.705,P<0.05).5. The effect of IL-6 induce in TAMsCD14+ human blood monocytes were grown with 1640 culture medium and MCS-F. After 6 days of induction, these cells were stimulated with a different concentration of IL-6. Total RNA were extracted from cultured cells, and the expression of IL-10, IL-12, VEGF-C and TGF-β were test by RT-PCR. The result suggested that the expression of IL-10, VEGF-C and TGF-β in macrophages induced by M-csf+IL-6 group is higher than that in macrophages induced by M-csf group(1.1±0.21 vs 0.1±0.01, 1.1±0.04 vs 0.1±0.01, 0.9±0.32 vs 0.1±0.02, p<0.05), while the expression of IL-12 in macrophages induced by M-csf+IL-6 group is lower than that in macrophages induced by M-csf group(0.1±0.02 vs 1.0±0.39, p<0.05). Results showed that TAMs induced by IL-6 have a phenotype of IL-10 high and IL-12 low, which express high level of VEGF-C and TGF-β6. TAMs induced by IL-6 affect the invasive ability of gastric cancer cellsThe blank control group only with culture medium, the negative control group with macrophages induced by M-csf and the experimental group with macrophages induced by IL-6+M-csf were separately co-cultured with MGC803 gastric cancer cell for 48 h. After crystal violet staining, the cells were counted with optical microscope. Results suggested that the number of gastric cancer cell penetrated through Matrigel in the experimental group was more than that in the negative control group and in the blank control group(124.0±8.9 vs 68.7±12.1 vs 67.0±7.5, p<0.05).Conclusion:1. The expression of IL-6, TAMs, VEGF-C and D2-40 is both in tumor tissues and normal gastric tissues, which in tumor tissues is higher than in normal gastric tissues. The expression of IL-6, TAMs, VEGF-C and LVD is positively associated with depth of invasion, lymph node metastasis and TNM stages2. IL-6, TAMs, VEGF-C and LVD are positively correlated with each other in gastric cancer microenvironment and the expression of them is positively correlation with the prognosis of patients. Patients with high expression of IL-6, TAMs, VEGF-C and LVD have poor prognosis. Results show that IL-6 plays an important role in the polarization of TAMs, lymphangiogenesis and the progress of gastric cancer.3. TAMs induced by IL-6 have a phenotype of M2 macrophages, which express high level of VEGF-C, with VEGF-C acting on the lymphatic endothelial cells, the processes of lymphangiogenesis are speed up. With the number of TAMs increasing, the invasive ability of gastric cancer cells is enhanced. Results indicate that the M2-like polarization of macrophages induced by IL-6 promote lymphatic metastasis of gastric cancer.