Effect And Mechanism Of Claudin-3 Protein Deletion On Lymphatic Metastasis Of Melanoma

BackgroundMelanoma has a high incidence in Europe and the United States,with-a mortality rate of 39%.Malignant melanoma metastasis is the main reason for the increased death rate of patients.Studies have shown that melanoma metastasis is mainly via the lymphatic vessels into regional lymph nodes.It has been demonstrated that dissection of sentinel lymph node is the optimal approach to prevent melanoma metastasis in clinical treatment.However,the underlying mechanism related to melanoma lymphatic metastasis is still elusive.Claudins are a family of membrane tight junctions(TJs)that play critical roles in regulating cell growth and adhesion,the permeability of cell bypass and maintain the polarity of epithelial cells.The loss of TJs integrity affects tumor cells' migration and dissemination during the metastatic process.Studies show that loss of Claudin-1 protein promotes brain metastasis of melanoma.Previous studies have shown that the expression of Claudin-3 varies in different human tumors that correlates with tumor stage and-metastasis in different tumors.For example,Claudin-3 was reported to be upregulated in lung adenocarcinoma while downregulated in lung squamous cell carcinoma.In addition,the loss of Claudin-3 can elevate the metastasis of lung squamous cell carcinoma by promoting tumor cell survival and invasion.It has also been found that the expression level of Claudin-3 protein in colon cancer,pancreatic cancer,prostate cancer,gastric cancer and breast cancer have been confirmed positively correlated with tumor stage.However,few studies have shown the relationship between Claudin-3 and melanoma metastasis.In the present study,we built a B16F10 footpad transplantation tumor model in Claudin-3 knockout(KO)mice and found that the loss of Claudin-3 promoted melanoma cell B16F10 metastasis into the draining lymph nodes.The results may provide a new target of restricting melanoma metastasis that benefits the treatment strategies.ObjectiveThis project aims to study the effect of Claudin-3 protein deletion on lymph node metastasis of melanoma in the tumor microenvironment.Methods1.To determine whether Claudin-3 knockout(KO)mice are homozygous that can be used to construct the animal model,nucleic acid gel electrophoresis is used for gene identification.2.The foot pad tumor animal model was constructed on Claudin-3 KO mice and Claudin-3+/+control mice.B16F10 melanoma cells with m-Cherry fluorescent label were injected into the footpad of mice.Then the survival time,body weight and primary tumor size of foot-bearing animal model was closed observed.3.At day 25,mice were sacrificed,and ipsilateral popliteal lymph node of tumor(dpLN),ipsilateral inguinal lymph node of tumor(diLN),contralateral popliteal lymph nodes(pLN)and primary tumors were removed to observe the tumor metastasis and other changes.4.All lymph nodes were analyzed by flow cytometry to observe the metastasis of B16F10 melanoma cells with m-Cherry fluorescent label.5.Tumor tissues were stained to detect the infiltration of immune cells and secreted factors by flow cytometry and PCR analysis.6.Immunofluorescence was conducted to observe the density of lymphatic vessels and blood vessels in primary tumor tissues.7.The expression of Claudin-3 protein on melanoma were identified by staining the melanoma tissue chip.8.The expression of Claudin-3 protein on lymphatic endothelial cells was detected by RT-PCR using lymphatic endothelial cell line SVEC4-10.9.To study the functional changes of newly formed lymphatic vessels,mice were injected with Evans blue dye on the feet,and permeability of lymphatic vessels was observed and analyzed.Results1.We confirmed the homozygous Claudin-3 knockout mice and constructed the melanoma metastasis model successfully.2.In the tumor bearing model,the survival time of Claudin-3 KO mice was significantly shorter than that of control mice.The body weight,tumor volume and tumor weight of Claudin-3 KO mice were not significantly different from control.3.Claudin-3 KO mice had more metastatic melanoma cells in the ipsilateral popliteal lymph nodes and ipsilateral inguinal lymph nodes of tumor injection sites than control.There was no difference in the amount of metastatic melanoma cells in contralateral popliteal lymph nodes and other tissues such as lung,brain and bones.4.After immunofluorescence staining of primary tumor tissues with LYVE-1 and CD31,it showed that more lymphatic vessels in Claudin-3 KO mice than control,while no statistical difference of blood vessels between Claudin-3 KO mice and control.5.Claudin-3 protein is expressed on melanoma tissues and lymphatic endothelial cells.6.More macrophages and increased VEGF-C level in tumor tissues of Claudin-3 KO mice was detected compared with control.While the presences of B cells,CD4+T cells,and CD8+T cells were the same.7.The lymphatic permeability of Claudin-3 KO mice was higher than control mice.ConclusionThis study reveals that loss of Claudin-3 protein promotes lymphatic metastasis of B16F10 melanoma cells in the foot pad tumor transplantation model.The number of lymphatic vessels in the tumors of Claudin-3-/-mice was significantly more than that of control wild-type mice.Further research found increased number of pro-lymphangiogenic factors VEGF-C in the primary tumor site of Claudin-3-/-mice than control.Therefore,the results of our study could provide insights into the mechanism of melanoma metastasis in the absence of Claudin-3 protein.Further studies may reveal Claudin-3 as a potential target to improve the management of tumor progression and metastasis.