Correlation Between Tumor-associated Macrophage And VEGF, MMP-9and MVD Expression In Esophageal Squamous Cell Carcinoma

Background and ObjectiveEsophageal cancer ranks sixth common malignant tumor in the world. Treatment of esophageal cancer is mainly by surgery, using radiotherapy, and combination with chemotherapy, cavity built-tube and cavity laser microwave resection. Because of high rate of surgical resection, efficacy is satisfied for early esophageal cancer. But the majority of patients with esophageal cancer treatment locally advanced, with low rate of surgical resection. Surgical treatment only is difficult to change the stagnant situation over the years of long-term survival of patients with esophageal cancer.5-year survival rate by surgery alone in the treatment of esophageal cancer is only22-25%. How to detect other treatment measures, such as targeted drug, to improve the quality of life and prognosis of patients with esophageal cancer is imperative.One hundred years ago, Rudolf Virchow first noticed that there was a lot of leukocyte infiltration in malignant tissue. Some scholars believed that there were a certain link between inflammation and cancer. Macrophage is an important component of inflammatory cells, playing essential mediator of the relationship between inflammation and cancer. However, the phenotype of the macrophage has great heterogeneity. In response to microenvironmental signal and the different physiological and pathological conditions, they exhibit distinct phenotypes with diverse functions. The adjacent cells and different cytokines in the microenvironment could have an impact on the function of the macrophages.Recent studies have found that tumor-asociated macrophage(TAM) in the brain glial, ovarian cancer, intrahepatic bile duct epithelial cancer, angioimmunoblastic T-cell lymphoma, lung adenocarcinoma, pancreatic cancer and melanoma showed M2polarized phenotypic characteristics, and the M2macrophage (or M2/M1ratio) was negatively correlated with survival rate of cancer patients. In experimental mice tumors, there was also evidence that the majority of TAM exhibit characteristics of M2macrophage that could promote tumor invasion, angiogenesis and hematopoietic cell recruitment. TAM may be converted to M2polarized macrophage in phenotypic characteristics, which has no anti-tumor effect, but to participate in the process of tumor development, invasion and metastasis. Therefore, it has become a potential targeting in cancer treatment. However, macrophage polarized phenotype and its clinical significance in esophageal squamous cell carcinoma have not been relevant research.Materials and MethodsTumor specimens from61esophageal squamous carcinoma patients were confirmed by HE stating, Including56men and5women with a mean age of60.4years. None of the patients had received treatment, without rheumatoid autoimmune diseases, heart disease and diabetes, etc. The lesions of each patient were classified into stages according to the UICC2010pTNM classification scheme (7th edition); stage Ⅰ, Ⅱ, Ⅲ, and IV lesions were present in12,32,17and0patients, respectively. Histologically,18tumors were graded as well-differentiated squamous cell carcinoma,15as moderately differentiated, and28as poorly differentiated. Lymph node metastasis occurred in22patients; no metastatic lymph nodes were observed in the remaining39patients. The polarized phenotype (M1or M2) of tumor-associated macrophages was determined utilizing immunofluorescence staining. The VEGF, MMP-9and MVD expression in cancer tissue were examined using immunohistochemical methods. The correlation between tumor-associated macrophage numbers and clinical pathological characteristics was analyzed using SPSS17.0(SPSS Inc., Chicago, IL, USA). Statistical differences between the means were analyzed using the independent samples t-test. Rates were compared using a χ2 test. The relationship between M2counts and VEGF, MMP-9and MVD expression was assessed using the Spearman correlation test. A p-value of0.05was considered statistically significant.Results1) To identify and quantify the infiltrated TAM associated with the M1or M2phenotype, we utilized anti-CD16/32, anti-CD206, and anti-CD68antibodies for immunofluorescence staining. CD68-positive macrophages were detected in varying concentrations in all61esophageal squamous carcinoma cases and in all adjacent normal lesion samples. In esophageal squamous cell carcinomas, CD68-positive TAM was predominately located in the peritumoral stroma and tumor tissues; in particular, TAM was observed along the invasive tumor front. Ml-polarized TAM was defined as CD68+CD16/32+, whereas M2-polarized TAM was distinguished by the expression of CD68+CD206+. M1and M2-polarized TAM were detected in all61esophageal squamous cell carcinoma tissues. Overall, the percentage of M2-polarized TAM was81.17±2.93%, and the percentage of M1-polarized TAM was18.13±2.93%.2) We divided all of the cases into two groups according to their CD68grade (cutoff value=103); the low-TAM group included32cases, and the high-TAM group was comprised of29cases. There was a significant difference in VEGF, MMP-9and MVD expression between the two groups (p<0.01). Next, we divided all of the cases into two groups according to their levels of CD68/CD206expression (cutoff values=83):the low M2-polarized TAM group (32cases) and the high M2-polarized TAM group (29cases). The VEGF, MMP-9and MVD was significantly higher in the high M2-polarized TAM group than in the low M2-polarized TAM group (p<0.01). Lastly, we divided all of the cases into two groups according to their levels of CD68/CD16/32expression:the lowM1-polarized TAM group (31cases) and the high M1-polarized TAM group (30cases). There was no significant difference in VEGF, MMP-9and MVD between the two groups (p>0.05).3) TAM number and M2-polarized TAM number were significantly associated with p-TNM staging and lymph node metastasis (p<0.01); however, these factors were not associated with gender, age or tumor differentiation (p>0.05). The number of M1-polarized TAM failed to correlate with any of the clinicopathologic features included in this study (p>0.05).Conclusions1) In present study, M1and M2macrophages coexisted in tumor microenvironment, which showed the heterogeneity in esophageal squamous cell carcinoma. However, a larger number of M2-polarized TAM is detected than that of Ml-polarized TAM.2) M2-polarized TAM is closely related to VEGF, MMP-9expression and microvessel density in esophageal squamous cell carcinoma, however, M1-polarized TAM is not related to these.3) M2-polarized TAM is closely related to lymph node metastasis and p TNM stage in esophageal squamous cell carcinoma, however, M1-polarized TAM is not related to these.