Streptococcus Pneumoniae Endopeptidase O(PepO) Suppresses Triple Negative Breast Cancer Growth By Inducing Tumor Associated Macrophage To Reprogram Into Tumoricidal M1-like Macrophage

Background:Breast cancer is the most common malignant tumor that causes women’s death.It is a heterogeneous disease with different molecular biological characteristics,clinical characteristics and pathological characteristics.Triple negative breast cancer（TNBC）is a subtype of breast cancer,which refers to breast cancer with negative ER,PR and HER2.The clinical characteristics of TNBC are easy invasion,high recurrence rate,poor prognosis and short survival.Clinically,surgery and chemotherapy are the most widely used treatment methods,but once chemotherapy is resistant,it is easy to relapse and metastasis.In addition,because TNBC lacks corresponding receptors,traditional endocrine therapy and targeted therapy are ineffective,and immunotherapy is considered as a promising treatment strategy.However,immunotherapy,including immune checkpoint therapy and car T cell therapy,is costly and time-consuming,and cannot meet a large number of clinical needs.Therefore,it is still an urgent problem to seek a more economical,safe and effective treatment for TNBC.Streptococcus pneumoniae endopeptidase O（PepO）is a 72 KD virulence protein of Streptococcus pneumoniae,which induces a strong innate immune response in a TLR2/TLR4 dependent manner.Our previous study found that PepO can induce M2-like macrophages to reprogram into M1-like macrophages.In breast cancer’s microenvironment,macrophages,vital innate immune cells,can be polarized into M1-like macrophages that inhibit tumors or M2-like macrophages that promote tumors.Obviously,reprogramming M2-like macrophages into M1-like macrophages in the tumor microenvironment can enhance the anti-tumor properties of M1 like macrophages.However,what molecular mechanism does PepO induce in M2-like macrophages to reprogram into M1-like macrophages?Does PepO induce M2 like macrophages to reprogram into M1 like macrophages and activate their tumoricidal properties?In vivo,it is not clear whether PepO can induce tumor associated macrophages to reprogram into M1 like macrophages and inhibit tumor growth.Objective:The purpose of this study is to clarify the anti-tumor effect of PepO in vivo and in vitro,to clarify the molecular mechanism of PepO inducing tumor associated macrophages to reprogram into tumoricidal M1-like macrophage,and provide a theoretical for the further usage of PepO in clinical treatment,and to provide a new idea for the immunotherapy of triple negative breast cancer.Methods:in vitro,triple negative breast cancer cells py8119 or 4T1were co-cultured with M2 like bone marrow macrophages or M2 like macrophages reprogramed by PepO using Transwell assay.Flow cytometry（FCM）was used to count the apoptotic tumor cell to clarify whether M2 like macrophages induced by PepO were reprogrammed into tumor killing macrophages;Transcriptome sequencing（RNA-seq）was used to preliminarily evaluate the effect of PepO on the antitumor function of macrophages;In vivo,the mouse triple negative breast cancer cancer cell line py8119 was selected to establish a triple negative breast cancer in situ model in wild（WT C57BL/6）mice to clarify the anti-tumor effect of PepO in vivo;Immunohistochemistry（IHC）,immunofluorescence（IF）and FCM were used to detect the density and proportion of M1 and M2 like macrophages in the tumor microenvironment,as well as the number of CD8⁺T cells and the proportion of activated cytotoxic T cells;TUNEL stain and IF were used to evaluate the level of tumor cell apoptosis,the ability of tumor cell proliferation（KI-67）and the process of epithelial mesenchymal transition（EMT）;Transcriptome sequencing（RNA-seq）was used to explore the impact of PepO treatment on the tumor microenvironment at the early stage and the gene changes after PepO reprogrammed M2 like-macrophages;In TNBC-bearing mice,clodronate liposomes were injected through tail vein to deplete macrophages and to explore whether the anti-tumor effect of PepO in vivo depends on macrophages.Py8119 cells and macrophages were mixed and injected into the breast pad of Nude mice to evaluate the tumoricidal ability of macrophages induced by PepO in vivo;TLR2（Toll like receptor 2）-deficient mice(TLR2^-/-)and TLR4（Toll like receptor 4）-deficient mice(TLR4^-/-)were used to clarify whether the antitumor effect of macrophages induced by PepO depends on TLR2 and TLR4.RT q PCR and ELISA were used to detect the expression of phenotypic marker genes of macrophage induced by PepO;Western blot was used to explore the molecular mechanism of PepO inducing M2-like macrophages to reprogram into M1-like tumor tumoricidal macrophages;The combination therapy of doxorubicin and PepO was used to clarify the potential value of clinical application of PepO.results:1.PepO induces M2-like macrophages to reprogram into M1-like tumoricidal macrophagesIn vitro,M2-like macrophages were treated by PepO for 24 hours,and their M1 macrophage markers were up-regulated and M2 like macrophage markers were down-regulated.In addition,M2-like macrophages treated by PepO co-cultured with triple negative breast cancer cells for 48h,which significantly promoted tumor cell apoptosis.Transcriptome data showed that PepO switched M2 phenotype macrophages into M1 phenotype,meanwhile enhancing the cell killing,phagocytosis,and nitric oxide biosynthesis of macrophage.2.In vivo,PepO significantly inhibited tumor growthCompared with the control group,after PepO treatment,the tumor growth was slow and the tumor weight was reduced.TUNEL staining of histopathological sections showed that there was a large area of apoptotic field in the tumor tissue.Ki-67 staining showed that the proliferation of tumor cells was inhibited by PepO.and the cell cycle was blocked in S phase.Immunofluorescence staining results showed that PepO inhibited the epithelial mesenchymal transformation process of tumor tissue.After PepO treatment,the tumorigenicity rate of tumor cells was significantly reduced.3.In the tumor microenvironment,PepO significantly activates the innate and adaptive immunitytranscriptome sequencing of tumor tissue showed that treating by PepO enhanced the expression of T cell activation genes IKZF1,Rac2,IGF1,Gas6,S100A9 and CD4 in the early stage.Reduce the expression of car9,VEGFA and M2-like macrophage markers retnla and Arg-1.Go analysis results showed that PepO enhanced adaptive immune response and caused immune cell differentiation and activation（such as regulation of leukocyte differentiation,leukocyte activation and T cell activation）.According to KEGG analysis,PepO activates PI3K-Akt pathway.4.In vivo,the anti-tumor effect of PepO depends on macrophage reprogramming.IHC staining and IF staining of tumor histopathological sections showed that after PepO treatment,the distribution density of tumoricidal M1-like macrophages in tumor tissue was significantly increased,while the distribution density of M2-like macrophages was decreased.ELISA detection showed that the secretion of proinflammatory factor IL-6 of M1-like macrophages in tumor tissue was increased,and the secretion of anti-inflammatory factor IL-10 of M2-like macrophages was decreased.Flow cytometry results showed that the proportion of M1-like macrophages（F4/80⁺CD86⁺or F4/80⁺i NOS⁺）increased significantly,while the proportion of M2 like macrophages（F4/80⁺CD206⁺）decreased.In vivo,PepO failed to suppress tumor growth after using Clodronate lipoomes to exhaust macrophages in mice.In Nude mouse model of TNBC,M2 like macrophages induced by PepO can effectively inhibit tumor growth.5.PepO promotes the recruitment of CD8⁺T cells to tumor tissues and activates cytotoxic T cells by stimulating macrophages to secrete cytokines.IHC staining and FCM of tumor histopathological sections showed that after PepO treatment,the density and cell number of CD8⁺T cells in tumor tissues was increased,and the proportion of activated cytotoxic T cells（CD45⁺CD3⁺CD8⁺CD69⁺CD107a⁺PD-1⁺）increasedsignificantly.Macrophage transcriptome sequencing showed that the cytokines that recruit T cells gene expression of Cxcl9 and Cxcl10 was up-regulated after PepO induction,and the gene expression of Ccl12,which promotes CD8⁺T cells to activate into cytotoxic T cells was increased.Go analysis of macrophages upregulated genes after PepO induction showed:regulate leukocyte activation,cytokine mediated signaling pathway,leukocyte migration,positively regulate cytokine production,and regulate cell activation.High throughput detection of cytokines trip found that the secretion of proinflammatory factors,cytokines and chemokines that chemotactic and activated T cells increased significantly.6.PepO reprogrammes macrophages into M1 like macrophages by activating PI3K-AKT signaling pathway,and inhibits JAK2-STAT3signaling pathway to inhibit the polarization of tumor associated macrophages into M2 like macrophages.7.PepO reprogram M2-like macrophages to polarization into M1 like macrophages depends on TLR2 and TLR4.8.PepO combined with doxorubicin further inhibited triple negative breast tumor growth.Conclusion:This study confirms that PepO can reprogram tumor associated macrophages（M2）into M1 macrophages with tumor killing effect in vivo and in vitro,and PepO can effectively inhibit the progression of triple negative breast cancer in vivo.The molecule mechanism was preliminarily clarified:PepO was recognized by TLR4 and TLR2,and then activated PI3K-AKT signaling pathway,inhibited JAK2-STAT3 signaling pathway.Thereby reprogramming tumor associated M2 macrophages to polarize into M1 like tumoricidal macrophages.And activate the adaptive immunity（T cell activation）and play an anti-tumor role.This study provides a theoretical basis for the further usage of PepO as a clinical anti-tumor drug.