The Regulatory Mechanism Of LSD1 On T-cell And Tumor-Associated Macrophages In The Immune Microenvironment Of Gastric Cancer

Gastric cancer is a malignant tumor originated from the gastric mucosal epithelium.The incidence and mortality rate are the third among all cancers.It is highly heterogeneous with limited diagnosis and treatment methods.It is prone to metastasis,which has a low five-year survival rate.Although the development of molecular targeting therapy and immunotherapy has benefited many cancer patients in recent years,the lack of effective therapeutic targets in gastric cancer and the low response rate of gastric cancer patients to immunotherapy make the treatment of gastric cancer still a great challenge.Therefore,the discovery of gastric cancer targeted drug targets and the study of their functions are of great significance for the treatment of gastric cancer.At the same time,the discovery of therapeutic targets involved in the immune regulation of gastric cancer or improving the response of gastric cancer immunotherapy is also an important field for gastric cancer treatment.Lysine-specific histone demethylase 1(LSD1)is the first histone demethylase discovered in 2004,which can remove the methylation of histone H3K4 and lysine of various non-histone substrates to perform many types of biological functions.LSD1 is highly expressed in a variety of solid tumors and hematological tumors,and promotes the stemness,differentiation and metastasis of tumor cells.Not only that,LSD1 is also involved in the regulation of tumor-associated fibroblasts,T cells,tumor-associated macrophages,natural killer(NK)cells and B cells in the tumor microenvironment.Inhibition of LSD1 can promote the infiltration of T cells in melanoma and breast cancer,thereby improving antitumor immunotherapy response.Previous studies of our group have shown that LSD1 is highly expressed in gastric cancer patient tissues,and LSD1 can promote the invasion and metastasis of gastric cancer cells,thereby promoting the development of gastric cancer.However,the function of LSD1 in the tumor microenvironment of gastric cancer is still unclear.Therefore,this project hopes to explore the function and mechanism of LSD1 in the immune microenvironment of gastric cancer by means of database analysis,sequencing analysis,animal model construction,and mechanism exploration in cells.In previous database analysis and animal level exploration,it was found that LSD1 affects the function of T cells in gastric cancer and promotes the development of gastric cancer.Single-cell sequencing was also used to analyze the regulatory function of LSD1 in the immune microenvironment of gastric cancer,which showed that macrophages have a huge impact.This topic has carried out in-depth exploration and research on this finding.Our study and key findings include:(1)LSD1 deletion inhibits gastric cancer tumor growth by promoting T cell-mediated immune responseWe firstly analyzed the correlation between LSD1 and the infiltration of various immune cells as well as related genes in the immune microenvironment of gastric cancer.The results showed that there is a correlation between LSD1 and infiltrating CD8+ T cells in gastric cancer.Then,the gastric cancer cell xenograft models of immune normal mice and T cell deficient mice were applied and results confirmed that LSD1 deletion can inhibit the development of gastric cancer depending on T cells in the tumor microenvironment.At the same time,the co-culture model of gastric cancer cells and T cells constructed in vitro also verified that LSD1 deletion can enhance the killing function of T cells on gastric cancer cells.All these experiments preliminarily determined the importance of LSD1 on tumor development through T cells in the immune microenvironment of gastric cancer.(2)LSD1 is correlated with PD-L1 expression in tumors from gastric cancer patientsBy analyzing the expression of LSD1 and T cell-related proteins in gastric cancer tissues,it was found that LSD1 was negatively correlated with T-cell function in gastric cancer tissues,and LSD1 was positively correlated with PD-L1.More than that,LSD1 deletion can reduce the m RNA level and total protein level of PD-L1,while the expression of PD-L1 in the cell membrane did not change significantly.In-depth exploration found that LSD1 deletion caused significant changes in PD-L1 transportation in endosomes,which was manifested as an increase in RAB11,a marker protein for recycling endosomes,and a decrease in TSG101,a marker protein for multivesicular bodies.Combined with the previous findings,it suggests that the inhibitory role of LSD1 on PD-L1-mediated T-cell function does not dependent on direct intercellular interactions,and there may be other regulatory mechanisms.(3)LSD1 deletion can reduce exosomal PD-L1 of gastric cancer cellsBased on the previous discovery,we used differential centrifugation to purify exosomes secreted by gastric cancer cells,and transmission electron microscopy,NTA,sucrose density gradient centrifugation and GW4869 blocking experiments were utilized jointly to verify that PD-L1 is harbored in gastric cancer cells derived exosomes.In addition,LSD1 deletion can significantly reduce the content of PD-L1 in exosomes as well as reduce the secretion of exosomes of gastric cancer cells.By blocking the exosome secretion pathway with the exosome secretion inhibitor GW4869,we found that LSD1 deletion can maintain the cell membrane PD-L1 expression by inhibiting the passage of PD-L1 through exosome secretion pathway.(4)LSD1 deletion blocks the inhibitory effect of gastric cancer cell derived exosomal PD-L1 on T-cell function as well as promotes the T-cell responseIn order to explore the regulation of LSD1 on the function of exosomal PD-L1,we first used the binding experiments of exosomes with PD-1 recombinant protein and T cells,and fluorescence imaging and transmission electron microscopy were applied to prove that gastric cancer cells derived exosomes can bind to PD-1 and T cells,while LSD1 deletion can reduce this binding ability.More than that,it was found that exosomes secreted by gastric cancer cells can inhibit the activation,proliferation and killing ability of T cells against gastric cancer cells,while LSD1 deletion can eliminate this function.Based on the material transfer function of exosomes,we co-incubated exosomes derived from gastric cancer cells with target cells,and used immunofluorescence,flow cytometry,western blotting,PD-1 binding experiments,T-cell killing experiments were used to verify that gastric cancer cells derived exosomes can fuse with target cells and deliver PD-L1 to target cells,thereby increasing the expression of PD-L1 in target cells and enhancing the binding ability of target cells to PD-1.At the same time,PD-L1 delivered to target cells can enhance the immune escape function of target cells,and induce the tolerance of target cells to T-cell killing,showing strong immunosuppressive function.When LSD1 was deleted,due to its down-regulation of exosomal PD-L1,exosomal PD-L1 was also decreased,leading to the functional elimination of exosomes as immunosuppressor,thereby blocking its effect on the immune escape function of target cells and restoring the gastric cancer cell responses to T cells.(5)LSD1 deleted gastric cancer cell derived exosomes promote T cell-mediated tumor immunity through PD-L1 in 615 miceLSD1 deletion significantly inhibited tumor growth in vivo,increased the proportion of tumor-infiltrating CD8+ T cells and the expression of CD3 and CD8 in tumor tissues as well as promoted the secretion of T-cell killing factors,while exosomes derived from MFC cells can promote tumor growth through PD-L1,reduce the proportion of tumorinfiltrating CD8+ T cells and the expression of CD3 and CD8 in tumor tissues,inhibit the secretion of T-cell killing factors.In contrast,exosomes derived from LSD1 deleted MFC cells showed the opposite results.And the effect of exosomes on these phenotypes can be blocked by PD-1 protein,demonstrating that LSD1 deletion plays a role in immune remodeling through the reduction of exosomal PD-L1.(6)LSD1 regulates tumor-associated macrophages in the immune microenvironment of gastric cancerWe used single-cell sequencing technology to analyze the role of LSD1 in immune microenvironment of gastric cancer and found that LSD1 significantly affects the infiltration and subtype distribution of tumor-associated macrophages,and LSD1 deletion can increase the infiltration of pro-inflammatory macrophages.We then used clodronate liposomes to erase mouse macrophages and further demonstrated that LSD1 deletion can inhibit the development of gastric cancer by modulating tumor-associated macrophages in the immune microenvironment of gastric cancer.Next,we constructed an interaction model between gastric cancer cells and M1-type,M2-type macrophages in vitro,and found that LSD1 deletion in gastric cancer cells can inhibit the polarization of M2-type macrophages as well as increase the recruitment of M1-type macrophages or decrease the recruitment of M2-type macrophages.Otherwise,LSD1 deletion can promote the pro-proliferative resistance of tumor cells to M2 macrophages,thereby inhibiting the proliferation of gastric cancer cells.It is shown that the deletion of LSD1 in gastric cancer cells can regulate the function of macrophages through the tumor microenvironment,thereby inhibiting the growth of tumor cells.(7)LSD1 affects the polarization of tumor-associated macrophages by regulating IL33In the RNA-seq analysis of MFC and MFC LSD1 KO cells and the enrichment analysis of their differential genes,it was found that LSD1 deletion can down-regulate the expression of inflammatory factor IL33,which has been reported to promote macrophages to M2-type.Based on this finding,we verified the function of IL33 on macrophage polarization and the regulation of IL33 by LSD1 at the cellular level,and confirmed that IL33 recombinant protein can induce macrophage polarization to M2 type,and LSD1 can indeed downregulate the level of IL33.These results indicate that LSD1 deletion can inhibit the expression and secretion of IL33 in gastric cancer cells,thereby inhibiting the polarization of M2 macrophages.In a nutshell,this subject found that LSD1 deletion can inhibit tumor development by regulating T cells and tumor-associated macrophages in gastric cancer for the first time.In the regulation of T cells,LSD1 deletion can inhibit the accumulation of exosomal PD-L1,thereby block the binding of exosomes to T cells and the inhibitory role of exosomes on T-cell activation and killing functions.Meanwhile,LSD1 deletion can reduce the delivery of exosomal PD-L1 to target cells and enhance the immune response of target cells to T cells.In tumor-associated macrophages,LSD1 deletion can inhibit tumor development by down-regulating IL33 expression and inhibiting the polarization of M2-type macrophages as well as reducing tumor recruitment of M2-type macrophages.This study discovered and revealed a new mechanism by which LSD1 regulates T cell activation,macrophage polarization and infiltration ratio of different functional subtypes through exosomal PD-L1 and IL33 in the immune microenvironment of gastric cancer.This provides strong evidence for the function of LSD1 as a potential target for immune regulation in gastric cancer.