The Role Of Soluble TIM-4 In Inhibiting NSCLC

[Background]Lung cancer includes small cell lung cancer and non-small cell lung cancer(NSCLC),and NSCLC accounts for 85%.Immune checkpoints promote tumor progression by inhibiting immune system.Emerging evidences have shown that immune checkpoint inhibitors could significantly improve the survival rate and prolong the survival time of lung cancer patients.However,treatment tolerance,resistance and toxicity of immune checkpoint inhibitors continue to emerge.Thus,it is necessary to explore new treatment strategies.In recent years,soluble immune checkpoints have been gradually paid attention to.Many studies have reported that soluble immune checkpoints could be detected in tumor patients and inflammatory diseases.Evidences show that the levels of soluble immune checkpoints might be correlated to disease severity,treatment effect and prognosis of tumor patients.In lung cancer patients,soluble PD-L1 levels negatively correlate with the effect of targeting therapy.In oral squamous cell carcinoma,increased soluble TIM-3 is negatively correlated with patient survival.These studies indicate that soluble immune checkpoints have the potential to predict the effect of tumor treatment and patient prognosis.As an immune checkpoint,TIM-4 is selectively highly expressed on the surface of antigen-presenting cells.TIM-4 could bind to phosphatidylserine and promote antigen-presenting cells to phagocytose apoptotic cells.Recently,it has been found that TIM-4 is also expressed in non-small cell lung cancer,nasopharyngeal cancer,cervical cancer and other tumor cells,as well as tumor-related immunocytes.TIM-4 promotes the progression of multiple tumors,indicating that TIM-4 has the properties of an oncogene.TIM-4 belongs to type I transmembrane glycoprotein.After hydrolyzing by ADAM 10 or ADAM 17,membrane TIM-4 produces soluble TIM-4(sTIM-4).It has been reported that sTIM-4 level is significantly increased in ischemic stroke and ankylosing spondylitis patients.And serum sTIM-4 levels are positively correlated with disease severity and negatively correlated with patient prognosis.In addition,increased sTIM-4 has been reported in cervical cancer patients.However,the levels of sTIM-4 in lung cancer patients and its role remain completely unknown so far.[Aims]1.To determine the levels of sTIM-4 in NSCLC patients.2.To verify the role of sTIM-4 in lung cancer progression.3.To explore the roles of sTIM-4 in other tumor progression and anti-tumor immunity.[Methods and Results]ELISA was performed to detect the sTIM-4 in serum from NSCLC patients and healthy controls.The results showed that sTIM-4 levels were significantly increased in NSCLC patients,indicating the potential correlation between sTIM-4 and lung cancer development.However,the levels of sTIM-4 in NSCLC patients showed no correlation to the treatment response to PD-1 antibody.To verify the effect of sTIM-4 on lung cancer progression,we constructed the eukaryotic expression vector of sTIM-4 and lung cell lines overexpressing sTIM-4.The CCK-8 and EdU assay showed that sTIM-4 could significantly inhibit the proliferation of lung cancer cells,and conditional medium containing sTIM-4 was used to verify its role.In addition,the results of RT-qPCR and flow cytometry showed that sTIM-4 significantly reduced the expression of proliferation-related molecules PCNA and Ki67 in lung cancer cells.Subsequently,wound healing and transwell assay were performed to evaluate the effect of sTIM-4 on the migration of lung cancer cells.The results showed that sTIM-4 could inhibit the migration of lung cancer cells.In addition,A549-LV-sTIM-4 cells were injected subcutaneously into nude mice to address the role of sTIM-4 in vivo.The results showed that tumor-forming ability of lung cancer cells overexpressing sTIM-4 was significantly inhibited.In order to explore the role of sTIM-4 in other types of tumors,we set up melanoma-bearing mouse model and sTIM-4 was expressed in vivo.We found that sTIM-4 could significantly prolong the survival time of tumor-bearing mice and inhibit tumor growth.To determine the effect of sTIM-4 on anti-tumor immunity,we isolated intratumoral mononuclear cells and performed flow cytometry analysis.The results showed that sTIM-4 significantly increased the proportion of CD8+T cells and NK cells,indicating that sTIM-4 could improve the capacity of anti-tumor immunity.Meanwhile,we monitored the body weight of tumor-bearing mice,observed the change of main organs,and no obvious toxic and side effects were found in sTIM-4 administered mice.[Conclusion]sTIM-4 could inhibit the proliferation and migration of lung cancer cells.In addition,sTIM-4 also could inhibit melanoma progression,and improve the anti-tumor immunity of host.