The Clinical Study On Tumor Inflammatory Microenvironment And Vascularization In Sinonasal Malignant Melanoma

BackgroundPrimary mucosal melanomas are mainly arising from the nasal cavity, it is rare and comprises less than 2% of all malignant melanomas. Irrespective of their initial location, sinonasal malignant melanoma (SNM) is a group of representative aggressive and highly lethal tumors with 5-year survival rates less than 25%. Although the clinical stage and histological morphology are practical for prognosis and for guiding the treatment protocol in SNM, it still has been difficult to establish clear prognostic factors owing to a number of factors, including late presentation of the disease, limited number of cases, and poor clinical outcomes. Therefore, new biologic prognostic markers are needed to estimate the risk of disease progression.Vascularization is considered to play an essential role in the neoplastic progression leading to metastasis, and acted as a potential biologic marker in predicting clinical outcome. Traditionally, it's considered tha the main form of blood supply for tumor tissue is angiogenesis. Recently, several other mechanisms of vascularization including vasculogenic mimicry and lymphangiogenesis have been identified in tumors. Moreover, these different components of tumor microcirculation are not sharing similar signaling pathways. The term "vasculogenic mimicry" (VM) was introduced to describe the masquerade of tumor cells as endothelial cells. The tumor cell-lined vessels could bring cancer cells into broad contact with the blood stream, which predisposes the tumor to metastasis and contributes to the poor outcomes of tumors. Lymphatic vessels also belong to the vascular circulatory system. The lymphatic system is a network of capillaries, collecting vessels and ducts that drain most of the organs, and lymphangiogenesis also plays an important role in neovascularization. Although evidence of these three forms of neovascularization has been recognized for many years, its clinical significance and mechanisms has not been well studied.TAMs are the most prominent component of leukocytic infiltration in malignant melanoma tissue, and are key regulators of the link between inflammation and cancer. There mainly two subclassses of macrophages:M1 and M2. Some researches indicated that TAM could express several M2-associated protumoral functions. One of the most important characteristics of TAMs is that they would affect tumor growth through promoting the tumor angiogenesis directly. Different microenvironments may "educate" macrophages to carry out specific functions to support tumor cell requirements and activities in those different areas. Thus, discrimination of TAMs between intratumoral and peritumoral may be necessary. Recent studies also suggest that TAMs play a crucial role in lymphoangiogenesis and lymphatic vessel invasion. However, there is little evidence in the literature concerning that focal macrophage infiltration is associated with the rest of forms of neovascularization in SNM. Furthermore, some other researchers thought high TAMs infiltration means poor prognosis in SNM. We are eager to investigate the definite role of TAMs and their biologic effect in SNM, and the association between TAMs and the aforementioned vascularization markers in this tumor.There are significant differences between the microenvironment of tumor tissues and normal tissues, among which regional low oxygen (Hypoxia) is characteristic of tumor microenvironment. Celluar fragments and hypoxic tumor cells release high level macrophage chemotactic factor, which recruit macrophages to hypoxic regions. The recruitment of TAM in hypoxic regions of tumor tissues indicates its importance in the development of tumor. Furthermore, microenvironmental change in tumor tissues is considered to be the main reason leading to the phenotype changes of TAMs and vascularization. Up to date, it remained unclear which factor in tumor microenviroment play a crucial role in vascularization and phenotype changes of TAMs.In present study, we used immunohistochemistry to assess the density of TAMs in both intratumoral and peritumoral areas to investigate their clinicopathologic significance and prognostic value in SNM patients. We also observe three forms of tumor vascularization in SNM, and to evaluate their relationship with TAMs density. Furthermore, we compared the RAW264.7 macrophages migrating activities of under hypoxic to their normoxia counterparts in vitro and assessed the effect of these two factors on the migration. Thereafter, we further investigate the mechanisms leading to the suppression of migration under tumor microenvironment, and find a signaling pathway to inhibit RAW264.7 macrophages migration. So as to provide a new insight into the understanding of molecular mechanisms on how TAMs promote tumor progression and vascularization in tumor local-tissue microenvironments. The results will address the proposal of TAMs as a useful target to prevent tumor progression and vascularization, and might provide a clinical advantage in patients with SNM.PartⅠTumor-Associated Macrophage Infiltration in sinonasal malignant melanoma and its Prognostic SignificanceObjectiveTo assess TAM infiltration in both intratumoral and peritumoral areas in sinonasal malignant melanoma. And approach their clinicopathologic significance and prognostic value in pristine SNM by univariate analysis and multivariate analysisMethods1. Forty-five patients diagnosed as SNM who received surgery from April 1985 to November 2008 in Qilu Hospital, Shandong University were admitted to this study.2. We used immunohistochemistry to assess the density of TAMs (CD68+) in intratumoral and peritumoral area.3. Macrophage can be divided into M1 and M2 two sub-classes. We use immunofluorescence double staining marks the specificity of M1 marker iNOS (Inducible nitric oxide synthase) and M2 specific marker CD206 to clarify the percentage of M1 and M2 in both intratumoral and peritumoral areas, and their relationship with the development of tumor. To investigate TAM clinicopathologic significance and prognostic value in SNM patients and the activated phenotypes of TAM in in intratumoral and peritumoral area in SNM.Results1. TAMs counts in peritumoral area and intratumoral area were 13.3±7.3 and 20.1±10.0macrophages/0.155 mm2, respectively.2. Intratumoral macrophages (IT-TAM) infiltration was associated with tumor stage (P-0.041). The melanoma tissues were divided into thick category (>0.75 cm) and thin category (≤0.75cm) by the level of tumor thickness. A higher amount of intratumoral TAMs density(P= 0.044) was shown in patients with thick category. However, the IT-TAMs density was not affected by tumor location or vascular invasion. In addition, we also find there is no correlation between peritumoral macrophages (PT-TAMs) infiltration and tumor stage.3. Immunofluorescence staining showed high expression of tumor tissue within the M2-type macrophage-specific marker CD206, and peritumor tissues and normal mucosa of the M1-type macrophage markers significantly increased iNOS, indicating that human malignant melanoma tumors in macrophages mainly in M2-type mainly. For the distribution of TAM perspective above the tumor adjacent to tumor and normal mucosa tissues mainly M1-type macrophages. These results indicate that the incidence of tumor development, tumor type macrophages changed.4. Kaplan-Meier survival analysis of these subgroups revealed that IT-TAMs density correlated significantly with stage I/II patient OS (log-rank test, P= 0.036). Patients with low IT-TAMs density had longer survival (median,57 months) than did those with high IT-TAMs density (median,13 months). In contrast, PT-TAMs showed no prognostic significance for OS (log-rank test, P= 0.408)Conclusions1. Intratumoral TAM counts are obviously higher than those in peritumoral macrophages.2. TAM counts are associated with clinical stage, tumor Breslow tumor thickness and tumor aggressiveness.3. Tumor macrophages to M2-type dominated, while in tumor and normal adjacent mucosa, macrophages to M2-type dominated.4. The high IT-TAM count is one independent adverse prognosis factor of pristine SNM. PartⅡThe Influence and Mechanism of Tumor Microenvironment on nasal malignant melanomaObjectiveTo observe three of the most important patterns of blood vessels quantitatively on nasal malignant melanoma (angiogenesis, lymphangiogenesis and vascular mimicry). To analyze the expression of TAM infiltration, MMP-9 and HIF-2a in intraumoral and peritumoral SNM, and to explore the interaction between TAM infiltration, MMP-9, HIF-2a and SNM vascularization. To clarify the role of tumor microenvironment on SNM vascularization and its signaling mechanisms.Methods1. Retrospective analyze of clinical and pathological data on 45 patients diagnosed as SNM who received surgery from 1995 to 2008 in Qilu Hospital, Shandong University.2. The expression of macrophage markered by CD68 in intraumoral and peritumoral SNM were analyzed by immunohistochemistry (SP method).3. Three main vascular patterns in intraumoral and peritumoral SNM were analyzed quantitatively, including the angiogenesis density marked by CD34, lymphatic vessels labeled by D2-40 and vascular mimicry labeled by double PAS-CD34.4. The expression of MMP-9 and HIF-2αwere analyzed by cell and gene level, and to explore the interaction between MMP-9, HIF-2α, TAM infiltration and SNM vascularization.Results1. We found three blood supply models in intraumoral SNM, including endothelium dependent vessel, endothelium-dependent lymphatic vessel and vascular mimicry, 1. While only two blood supply patterns, endothelium-dependent vessel and lymphatic vessel, were discovered in peritumoral SNM.2. The MVD labeled by CD34 in intraumoral and peritumoral SNM was 17.7±2.3 and 23.2±7.3 respectively, the differences were significant, P<0.01. The vascular mimicry was found in 11 cases (24.4%) among of 45 cases SNM. LVD labeled by D2-40 in intraumoral and peritumoral SNM was 2.1±3.7 and 1.7±6.3 respectively, there is no significant difference, P>0.05.3. There is similarity among the expression sites of CD68, MMP-9 and HIF-2αin SNM. The total positive rate of MMP-9 was 75.6%(34/45), and the HIF-2αwas 40% (18/45). According to semi-quantitative comparison by Immunohistochemical image analysis software, compared to peritumoral area, the mean density of positive particles, positive areas and mean densityx positive areas of MMP-9 and HIF-2a in intraumoral area was increased significantly, P＜0.05.4. There is no differences between TAMs counts and MVD, VM, LVD density in tumor microenvironment. However, the IT-TAM was significantly correlated with MVD (r=0.871, P=0.01), and slightly correlated with VM (r=0.57, P=0.05) in those with positive HIF-2a.5. The expression of HIF-2a density in tumor microenvironment was correlated positively with MVD and VM (r=0.558, P=0.039), and no correlation with VM and LVD was found.6. In tumor microenvironment, the MMP-9 density was correlated positively with MVD (r=0.513, P=0.049), and no correlation with VM and LVD, while MMP-9 protein was correlated significantly with LVD (r= 0.671, P= 0.014)Conclusions1. There is synergistic reaction for HIF-2αand TAM in the process of vascularization in SNM. The expression of HIF-2a was up-regulated by TAM, which promotes angiogenesis and vascular mimicry.2. MMP-9 played an important role on the intraumoral angiogenesis, peritumoral capillary and lymphatic vessel neogensis in SNM tissue, which is an important factor for angiogenesis.3. SNM vascularization was influenced significantly by TAM-MMP9/HIF-2αpathway in the tumor microenvironment, which revealed that there is a new target for control tumor angiogenesis and growth by inhibition of tumor inflammatory microenvirment.