TIM-4 In Lung Cancer Cells Promotes Tumor Immune Escape By Educating Macrophages

BackgroundAt present,the incidence of lung cancer is the highest among all tumor diseases worldwide,and the prognosis of lung cancer is very poor due to low early diagnosis.Therefore,lung cancer is one of the malignant tumors with the fastest increase in morbidity and mortality,which has become the greatest threat to people’s health and life.The pathological type of lung cancer is diverse.The high incidence of non-small cell,especially adenocarcinoma,tends to grow outside the tube,but it can also spread through the bubble wall.It can invade blood vessels and lymphatic vessels in the early stage and often appear metastasis before obvious symptoms.Therefore,exploring the pathogenesis of non-small cell lung cancer is of great significance for the early diagnosis and effective treatment of lung cancer patients.The etiology of lung cancer is very complex and has not yet been fully elucidated.It is generally believed that it is associated with smoking,occupational carcinogenic factors,air pollution,ionizing radiation,dietary nutrition and other factors that cause malignant transformation of cells and irreversible genetic changes,including proto-oncogenes activation,inhibition of tumor suppressor genes,activation of self-feedback and secretion loops,and inhibition of apoptosis,eventually lead to uncontrolled cell growth.The occurrence,development and invasion and metastasis of non-small cell lung cancer are not only related to the characteristics of lung cancer cells,but also closely related to the immune microenvironment of lung cancer cells.The monitoring function of the immune system plays an important role in the process of clearing lung cancer cells,but lung cancer cells can escape from immune surveillance through some mechanisms and tumor develops in vivo.The break-up of the balance between the two aspects is critical for tumor cell growth.Therefore,revealing the immune escape mechanism of lung cancer has become a key scientific problem to be solved urgently.Lung cancer cell induced immune tolerance is one of the main mechanisms of immune escape of lung cancer,but many immune cells in the microenvironment also play an important role in the process of immune escape of lung cancer,among which tumor-associated macrophages have become the current research hotspot.The complex interaction between tumor-associated macrophages(TAMs)and tumor cells plays an important role in the development of non-small cell lung cancer,and the poor prognosis of patients is closely related with infiltrating macrophages in multiple malignancies.The source of these TAMs is mononuclear cells in the blood,which accumulate around the tumor tissue under the chemotaxis of various cytokines such as CCL2,CCL3,CCL4,CCL5,CCL8,and VEGF.Macrophages are devided into two major categories:classical activation macrophages,also known as M1 macrophages,usually exhibit antibacterial activity and pro-inflammatory phenotypes;alternative activation pathway macrophages,also known as M2 giants phagocytes mainly involved in promoting angiogenesis and tissue remodeling and repair.The tumor microenvironment generally converts pro-inflammatory and anti-tumor Ml macrophages towards M2 macrophages to increase the release of pro-tumor cytokines,so tumor-associated macrophages often have the phenotypic characteristics of M2 macrophages.Normally,the specific phenotype of macrophages changes with different stages of tumor progression.In the early stage of the tumor,macrophages exhibit anti-tumor characteristics of M1 macrophages due to activation of the immune system.With the proliferation and growth of tumor,the advanced tumor microenvironment shows immunosuppressive properties,and macrophages also exhibit M2 macrophage characteristics that promote tumorigenesis and enhance tumor angiogenesis.In general,M2 macrophages appear more commonly in the tumor microenvironment,and this phenotypic change is affected by hypoxia and tumors and various cytokines secreted by stromal cells.The classification and remodeling of macrophages are closely related to the immune microenvironment of lung cancer.It is important to clarify the relevant mechanisms for the prognosis of lung cancer.The T cell immunoglobulin domain and mucin domain protein(TIM)family is widely expressed on the surface of various immune cells and participates in various metabolic activities in vivo.The human TIM family has three major members:TIM-1,TIM-3,and TIM-4,which are expressed on the surface of Th2 cells,Thl cells,and APCs,respectively.The biological essence of TIM-4 is the phosphatidylserine receptor,which is mainly expressed on the surface of activated monocyte-macrophages and myeloid-derived dendritic cells.TIM-4 can mediate the phagocytosis of apoptotic cells by macrophages and can act as a ligand for the TIM-1 molecule to cooperatively regulate the proliferation of T cells.By marking and tracking TIM-4hi CD169+ cells in mice,they are mainly composed of tissue-resident macrophages distributed in various organs of the body,and have the characteristics of maintaining high activation and immune regulation after migrating to secondary lymphoid organs.TIM-4hi CD169+ cells promote antigen induced Tregs cells,and are highly sensitive to apoptosis.Some current studies have shown that TIM-4 can affect a variety of autoimmune-related diseases such as asthma,rheumatoid arthritis and systemic lupus erythematosus.Recently reports show chemotherapy can increase the expression of TIM-4 in TAM.Apoptotic tumor cells are phagocytosed by TAM expressing TIM-4,and TIM-4 binds to AMPK to induce autophagy to degrade tumor antigens and weaken antigen presentation,as well as specific CLT response to reduce the anti-tumor effect of chemotherapy.In addition,M2-like macrophages express high levels of TIM-4,while M2 macrophages can promote tumor progression,thus blocking TIM-4 can effectively enhance anti-tumor immune response.Thus,TIM-4 is used as a promising candidate for anti-tumor target.Recent studies show that TIM-4 is also expressed in gliomas,Langerhans cell sarcoma and multiple tissue sarcomas derived from dendritic cells,parapharyngeal liposarcoma,but its specific biological functions is unclear.Our laboratory reported the biological role of TIM-4 in the development of lung cancer for the first time,and found that the expression of TIM-4 in lung cancer was significantly higher than that in adjacent tissues,and the expression in lung cancer was negatively correlated with its prognosis.In addition,multiple inflammatory factors could induce the expression of TIM-4 in lung cancer cells,and TIM-4 overexpression could promote the progression of lung cancer through the RGD domain.Recently,it has been reported that the expression of TIM-4 in gliomas is significantly higher than that adjacent to tumors.Interfering with the expression of TIM-4 can inhibit the growth of glioma cells,induce apoptosis,and reduce the ability of colony formation.The high expression of TIM-4 in renal clear cell carcinoma is associated with a poor prognosis.In vitro studies have confirmed that interference with TIM-4 can increase the sensitivity of renal cancer cells to sorafenib.Combined blocking of TIM-3 and TIM-4 can enhance the efficacy of melanoma tumor vaccine.Combined blocking of TIM-1 and TIM-4 can enhance the role of DC vaccine in the treatment of lung cancer.The above studies showed that TIM-4 expressed in macrophages can promote immune escape of tumors,and TIM-4 expressed in tumor cells can also increase the malignant biological behavior of tumor cells.However,whether TIM-4 expressed in tumor cells also involved immune escape is not yet clear.This article intends to investigate the role of TIM-4 expressed in lung cancer cells in the induction of immune tolerance of lung cancer cells based on the previous research in our laboratory through in vitro and in vivo experiments by elucidating its effects on lung cancer cells and macrophages.TThis study will provide a fuller basis for TIM-4 as an anti-tumor target,with important research value and potential clinical application prospects.AimTo explore the role of TIM-4 expressed in lung cancer cells by regulating the lung cancer cells and educating macrophages in the immune escape of lung cancer,and to elucidate its relevant mechanisms,which would provide new candidate targets for early diagnosis and targeted treatment of lung cancer.Methods1.Immunohistochemistry staining and immunofluorescence assay Immunohistochemistry or immunofluorescence staining was performed to detect TIM-4 or CD86 expression in tumor tissues for tumor-bearing mouse and lung cancer tissue microarrays.Explore the relationship between TIM-4 expression and macrophage infiltration in lung cancer tissues.2.Cell experiments2.1 Construction of A549 cell line overexpressing TIM-4 Design and synthesis of TIM-4 Lentiviral LV-TIM-4 and control empty vector lentivirus.A549 cells were infected at the same titer(MOI=10).The green fluorescence of A549 cells after infection was observed by fluorescence microscopy.After confirming the effect of infection,RNA and protein were extracted,and the remaining cells were cultured to expand continuely.2.2 Detection of TIM-4 and cytokines in tissues and cells by RT-PCR’Lentivirus-infected A549 cells,THP-1 cells,and tumor-bearing mouse tumor tissues were colllected.RNA was extracted from cells and tissues using Trizol by one step method.Reverse transcription and PCR were performed to detect TIM-4,TNF-a,TGF-P,and IL-6,IL-10,IL-12,MCP-1,HAS2,etc.The expression and the changing trend of various cytokines in different tissues and cells were assayed.2.3 Construction of co-culture systemUsing a Transwell chamber with a diameter of 0.4 μm,A549 cells overexpressing TIM-4 and their controls were added to the upper chamber,and activated THP-1 cells were added to the lower chamber to induce activation of the THP-1 cells for 48 h.The Transwell chamber was carefully removed and collected.Co-cultured THP-1 cells were collected to test the expression of various cytokines.2.4 Detection of surface molecules on A549 and THP-1 cells by flow cytometryA549 cells overexpressing TIM-4 or control cells and THP-1 cells were stained with fluorescent conjugated antibodies.Then fixed with paraformaldehyde and the HLA-A/B/C,HLA-DR,CD80,CD86 expression on A549 cells THP-4 were detected separately.The relative fluorescence intensity and expression level of HLA-DR,CD80,CD86 and other molecules were analyzed by flow analysis software.3.Animal experimentNude mice tumor-bearing experiment:Twelve 4-6-w male BALB/c nude mice were randomly divided into two groups.A549 cells infected with the TIM-4 lentivirus and their control cells were injected subcutaneously in the right subdiaphragmatic zone.7-10 days later,the length and width of the tumor body were meassured every day,and the tumor growth curve was recorded.After the mice were sacrificed,the tumor weights were weighed and recorded.The photographs of the tumors were taken.A small amount of tumor tissue was obtained to extract RNA by Trizol lysis.Expression of TIM-4,HLA-A,B,C,and TNF-α,etc.in tumor tissues were detected.In addition,some tissues were fixed in paraformaldehyde and paraffin sections were made.The macrophage infiltration and phenotype were detected by、immunohistochemistry.Results1.To clarify the role of TIM-4 in immune escape of lung cancer cells,we set up A549 cell line over-expressing TIM-4.Phenotype A549 cells over expressing TIM-4 was analyzed.Fluorescence microscopy,western blot and RT-PCR detection showed that the expression of TIM-4 in cells infected with TIM-4 lentivirus was significantly higher than in control cells,indicating that the virus-infected A549 cell line was successfully constructed.2.We found that overexpression of TIM-4 in A549 cells significantly reduced the level of HLA-I molecule expression on the surface and promoted the high expression of monocyte chemoattractant protein-1(MCP-1)and TNF-a,IL-6,VEGF-A,implying that TIM-4 might induce lung cancer immune tolerance and thus accelerate tumor progression by regulating the above factors.3.In Transwell experiments,we demonstrated that TIM-4 overexpression in A549 cells did increase the number of macrophage migration.Co-culture system of A549 cell with THP-1 cells was performed to elucidate effects of TIM-4 overexpression on A549 cells on macrophage function.The results showed that in the co-culture system of macrophage and lung cancer cells,the number of M2-type macrophages was greater than that of M1-type macrophages.At the same time,TIM-4 overexpression in A549 cells promoted the secretion of HAS2 by macrophages.We also found that overexpression of TIM-4 in A549 cells could reduce the expression of CD80,CD86 and HLA-DR on the surface of macrophages.This result suggests that TIM-4 molecule might promote immune escape by inhibiting the antigen presentation function of macrophages and the progress of lung cancer.4.Results from lung cancer tissue array showed that TIM-4 expression in lung cancer tissues were significantly higher than those in paracancerous tissues.TIM-4 expression in lung cancer tissues increased along with clinical stage increased,and CD68 expression showed similar trend.In addition,single TIM-4 positive cells in lung cancer tissues was positively related with CD68 positive cells.5.In tumor bearing model of A549 overexpressing TIM-4,we verified that TIM-4 expression was higher in tumor tissues.We found that TIM-4 overexpression promoted lung cancer growth,and more F4/80 positive cells were observed in TIM-4 overexpression group.In addition,we further demonstrated that HLA-I expression decreased and TGF-β,Ym-1 expression,M2 macrophage marker,increased inTIM-4 overexpression group.Conclusion1.TIM-4 overexpression inhibits HLA-I molecule expression in A549 cells,but promotes cytokines production by A549 cells.2.TIM-4 overexpression in A549 cells promotes macrophage migration and promote the conversion of macrophages to TAMs.3.TIM-4 overexpression in A549 cells can reduce the expression of CD80,CD86 and HLA-DR on the surface of macrophages,indicating that TIM-4 overexpression in lung cancer cells might inhibit antigen presentation of macrophages.Innovation and SignificanceIn the study of lung cancer,we first propose that TIM-4 molecules expressed in tumor cells promote tumor immune escape by regulating tumor cells and macrophages.Ectopic expression of TIM-4 in tumor cells might weaken anti-tumor immune response by decreasing antigen presentation function of macrophages and promoting their polarization to TAMs.Our results confirme that high expression of TIM-4 in lung cancer cells plays a key role in remodeling macrophages.This finding broadens the biological function of TIM-4 in the development of lung cancer and can provides full evidences for TIM-4 used as new candidate in targeted immune therapy of lung cancer.