Arsenic Trioxide Inhibits M2 Tumor-associated Macrophages Polarization In Lung Adenocarcinoma By Targeting The Notch Signaling Pathway

【Background】Lung cancer is one of the most common causes of cancer-related death worldwide,and lung adenocarcinoma(LUAD)accounts for approximately 40%of all lung cancer cases.Despite the long-term commitment to the development of cytotoxic drugs and targeted drugs targeting lung cancer cells at home and abroad,the overall effective rate and patient survival indicators of advanced lung adenocarcinoma treatment have not been significantly improved,and the 5-year survival rate is only 4.5%.Studies have shown that immune escape is an important reason for the rapid progression and distant metastasis of advanced lung adenocarcinoma,and immune checkpoint inhibitors can effectively inhibit tumor immune escape and restart immune surveillance,bringing hope to overcome advanced lung adenocarcinoma.The degree of benefit obviously depends on the expression level of CTLA-4 or PD-1/PD-L1 molecules,which limits its wide application in patients with advanced lung adenocarcinoma.Tumor-associated macrophages(TAMs)are the most abundant immune cells in the tumor microenvironment(tumor microenvironment,TME).Studies have confirmed that M2 TAMs(immunosuppressive subtype)are widely present in all types of solid tumors and participate in the process of tumor immune escape.Therefore,inhibiting the polarization of TAMs in the TME to the M2 subtype to reverse the tumor immunosuppressive microenvironment and restart immune surveillance is expected to be a breakthrough in overcoming immune escape in advanced lung adenocarcinoma.The Notch signaling pathway is a classical embryonic development and cell differentiation regulation pathway.Many recent studies have confirmed that the Notch signaling pathway plays a key role in the polarization process of TAMs.Wang et al.found that the activation of the Notch signaling pathway in breast cancer directly promotes the secretion of chemokines such as CCL2 and IL-1βby tumor cells,inducing monocytes derived from bone marrow to migrate to tumor tissues and differentiate into M2 TAMs.Overexpression of Notch1 levels in human head and neck squamous cell carcinoma by gene intervention can upregulate the proportion of CD68+/CD163+M2 TAMs in the TME.In addition,the Notch pathway also mediates the formation of a positive feedback loop between tumor cells and TAMs,exacerbating cancer progression.These studies suggest a close relationship between the Notch signaling pathway and the immune suppression phenotype of TAMs.Therefore,establishing a safe and effective Notch signaling pathway inhibitor to block the positive feedback loop between tumor cells and TAMs and reverse the tumor immune suppression microenvironment has significant clinical implications.Arsenic trioxide(ATO),the main component of the traditional Chinese medicine arsenic,was first used by Chinese scholars to treat acute promyelocytic leukemia(APL)and has demonstrated effective and safe anti-tumor activity.It has been approved by the U.S.Food and Drug Administration(FDA)for the treatment of APL.My research group has been exploring the anti-tumor effects and mechanisms of ATO in lung cancer for many years.In my master’s research project,I found that intrapleural injection of ATO can effectively reduce the production of malignant pleural effusion in lung adenocarcinoma patients with pleural metastasis.This effect is related to the decrease in pleural microvessel density and vascular permeability.We further confirmed in a mouse lung adenocarcinoma model that ATO reduces microvessel density in tumor tissue and target organs,inhibits tumor growth and metastasis by downregulating the vascular endothelial growth factor(VEGF)pathway.In our investigation of the anti-vascular effect of ATO,we found that in addition to affecting VEGF,ATO blocks the Notch pathway,disrupts endothelial cell ultrastructure,and leads to"de-functionalization"of tumor neovessels,shifting the tumor microenvironment(TME)towards an anti-vascular direction.The anti-vascular effect of ATO that we discovered is likely only part of the role of Notch pathway regulation in TME.Whether ATO can directly regulate the immune phenotype of tumor-associated macrophages(TAMs)through the Notch pathway is still unknown.To explore the pharmacological efficacy and potential mechanisms of ATO in regulating tumor immunity,we first successfully constructed an in vitro model of TAMs by co-culturing tumor cells with macrophages.We demonstrated that ATO targets and inhibits the polarization of M2-type TAMs by blocking the Notch pathway in this model.We then further validated the inhibitory effect of ATO on M2-type TAMs polarization and lung adenocarcinoma growth in a C57BL/6 mouse tumor model.Finally,we explored the relationship between Notch signaling pathway activation and TAMs infiltration and M2-type TAMs polarization in 19 human lung adenocarcinoma tissues and further observed the impact of TAMs phenotype on patient prognosis.Our research has fully demonstrated,from in vitro,in vivo,and clinical perspectives,the role and mechanism of ATO in regulating TAMs polarization to inhibit the progression of lung adenocarcinoma.【Contents and Methods】Part 1:In vitro study of ATO inhibiting M2-polarized TAMs by blocking the Notch pathwayFirstly,an in vitro TAMs model was established by co-culturing lung adenocarcinoma cells and macrophages.The inhibitory effect of ATO on the expression of M2-polarized TAMs related markers was confirmed using flow cytometry,Western blot,and RT-PCR.Then,the key molecules of the Notch pathway,Jagged1 and Notch,were overexpressed using lentiviral transfection,and the targeting effect of ATO on blocking the Notch signaling pathway was verified in reverse.ELISA and si RNA technology were then used to determine that the induction of CCL2 and IL-1βmediated the reduction of M2-polarized TAMs by ATO.Finally,cell proliferation experiments,Transwell migration,invasion experiments,scratch experiments,and clone formation experiments confirmed that ATO reduced M2-polarized TAMs and inhibited the progression of lung adenocarcinoma in vitro.Part 2:In vivo study of ATO inhibiting M2-polarized TAMs by blocking the Notch pathwayFirstly,the subcutaneous transplantation tumor model of C57BL/6 mice was established,and the inhibitory effect of ATO on lung adenocarcinoma growth in vivo was evaluated with Notch pathway inhibitor DAPT as a positive control.Then,the effects of ATO on macrophage recruitment and M2-polarized TAMs were detected by immunofluorescence staining and Western blotting in the transplanted tumor.Finally,the safety of ATO was preliminarily evaluated by monitoring the body weight of the mice.Part 3:The effect of Notch pathway activation in human lung adenocarcinoma tissue on TAMs polarization and patients prognosisFirstly,clinical information and biopsy specimens of late-stage lung adenocarcinoma patients were collected to describe their clinical and pathological characteristics.The effect of Notch pathway activation on tumor malignancy and chemotherapy sensitivity was analyzed by immunohistochemical staining of lung adenocarcinoma tissue.The positive correlation between the expression of key molecules of the Notch signaling pathway and M2-polarized TAMs was confirmed using Pearson correlation test.Finally,the effects of Notch activation,TAMs infiltration,and M2 polarization on survival time were further clarified through bioinformatics analysis of 719 lung adenocarcinoma patients.【Results】Part 1:In vitro study of ATO inhibiting M2-type TAM polarization through blocking the Notch pathway1、ATO inhibits M2-type TAM polarization in the co-culture system.2、ATO inhibits M2-type TAM polarization in the co-culture system by blocking the Notch signaling pathway in lung adenocarcinoma cells.3、ATO inhibits M2-type TAM polarization by downregulating the secretion of Notch pathway-dependent CCL2 and IL-1β.4、ATO inhibits Notch-TGF-β1 signaling pathway activation in TAMs by blocking lung adenocarcinoma CCL2 secretion.5、ATO inhibits lung adenocarcinoma cell proliferation,migration,invasion,clonogenicity,and epithelial-mesenchymal transition(EMT)by reducing M2-type TAM polarization.Part 2:In vivo study of ATO inhibiting M2-type TAM polarization through blocking the Notch pathway1、ATO inhibits the growth of subcutaneous lung adenocarcinoma tumors in mice.2、ATO reduces TAM recruitment and M2-type TAM polarization in subcutaneous lung adenocarcinoma tumors in mice.3、ATO blocks activation of the Notch and TGF-βsignaling pathways in subcutaneous lung adenocarcinoma tumors in mice.4、ATO exhibits good safety in the mouse subcutaneous lung adenocarcinoma tumor model.Part 3:The impact of Notch signaling pathway activation in human lung adenocarcinoma tissue on TAM polarization and patient prognosis1、Expression of the Notch pathway key ligand Jagged1 and receptor Notch1 in human lung adenocarcinoma tissue is positively correlated with infiltration of CD68~+total TAMs,and activation of the Notch signaling pathway promotes macrophage polarization towards CD206~+M2-type TAMs.2、Expression of Jagged1 and Notch1 in human lung adenocarcinoma tissue is closely related to serum tumor marker levels in patients,with higher expression of Jagged1 and Notch1 associated with higher levels of CEA,CA19-9,and CYFRA21-1 in patient serum.3、Activation of the Notch pathway and TAMs infiltration in human lung adenocarcinoma tissue indicate poorer chemotherapy efficacy and shorter survival time in patients.【Conclusion】This study investigated the relationship between the activation of the Notch signaling pathway and M2 polarization of tumor-associated macrophages(TAMs),as well as the clinical prognosis of lung adenocarcinoma patients,using lung adenocarcinoma cells,TAMs,a mouse model of lung adenocarcinoma,and clinical specimens of lung adenocarcinoma patients at three levels:in vitro,in vivo,and clinical.The study demonstrated that ATO can inhibit M2 polarization of TAMs by blocking the Notch signaling pathway,thereby exerting an anti-tumor effect.This provides a theoretical basis for the clinical application of ATO in immunotherapy for lung adenocarcinoma.