Myeloid-derived Suppressor Cell And Macrophage Exert Distinct Angiogenic And Immunosuppressive Effects In Breast Cancer

Background:Although cancer immunotherapy is,currently,a novel cancer treatment modality with a potential to cure a cancer,it is still facing multiple challenges.Among them,the immunosuppressive tumor microenvironment is a key obstacle to hinder a cancer immunotherapy.Myeloid-derived suppressor cell(MDSC)is considered as a major player in peripheral tumor-associated immunosuppression,however,what is the role of MDSC in the tumor microenvironment remains not fully understood.Objective:To analyze the components and properties of tumor-infiltrating myeloid cells and get insight into their roles in the formation of the tumor microenvironment.Methods:MMTV-PyVT breast tumor tissues,MCaP0008 and E0771 tumor cells were orthotopically inoculated into the mammary fat pads of FVB/N and C57BL/6 mice respectively.When tumors reached 6-8 mm in diameter,tumor tissues were harvested and cut into two pieces.One portion was fixed with 4%paraformaldehyde for histological analysis.The remaining piece was dissociated to obtain single cell suspensions.Single cell suspensions were immunostained and used for flow sorting or flow cytometric analysis.Mixed leukocyte reaction(MLR)was used to evaluate the immunosuppression of myeloid cell populations.Tube formation assay was used to assess the pro-angiogenic effect of myeloid cell populations was assessed by tube formation assay.Result:In the spontaneous MMTV-PyVT as well as orthotopically inoculated MCaP0008 and E0771 breast tumor models,CD45+CD11b+Gr1hiF4/80-,CD45+CD11b+Gr1-F4/80+ and CD45+CD11b+Gr1int/lowF4/80int/low were three major tumor-infiltrating myeloid cell populations.CD45+CD11b+Gr1hiF4/80-cells were also Ly6G+Ly6Clow,an equivalent phenotype of tumor-associated neutrophils(TANs).Then,we analyzed the functional characteristics of tiMDSC and TAM isolated from breast tumor tissuess.TiMDSCs and TAMs were co-cultured with splenocytes under anti-CD3/CD28 antibody treatment.We found that TAMs were more immunosuppressive than tiMDSCs in breast tumors.To reveal molecular mediators involved in immune suppression,TiMDSCs had significantly higher levels of pro-inflammatory factors,such as IL12α,IL1β,CXCL9 and CXCL10,relative to TAMs.Conversely,TiMDSCs have significantly lower levels of anti-inflammatory factors,including IL10,Argl,CCL17,and CCL22,compared to TAM cells.In addition,we found that tiMDSCs were more frequently located in hypoxic/necrotic areas,compare to TAMs.In flow sorted tiMDSC and TAM cells derived from MCaP0008 breast cancer tissue,tiMDSCs had significantly higher levels of SDF1α,MMP9,VEGF-a,and PIGF,compared to TAMs.Currently,TiMDSCs were more potent than TAMs in their ability to induce tube formation in spontaneous MMTV-PyVT breast tumors.The differentially immunosuppressive and angiogenic capabilities of tiMDSCs and TAMs indicated that they may play different roles in tumor initiation and progression.To answer this question,we analyzed tiMDSCs and TAMs on day 7 and 14 after MCaP0008 breast cancer cell inoculation.These data show that the initiation and progression of MCaP0008 breast tumors were accompanied by a shift of tiMDSCs to TAMs.In addition,we found that neutrophils were increased with lung colonization by breast cancer after 4T1 primary tumors were removed.Furthermore,the proportion of tiMDSCs in lung metastases was much higher than in that observed in primary breast tumors.The proportion of tiMDSCs in small lung metastases was also higher then larger lung metastases.These data suggested that lung colonization by breast cancer was associated with the accumulation of neutrophils.Conclusion and significance:In this study,we found that MDSC within the tumor microenvironment was different from MDSC in the peripheral immune organs.TAM was much potent than tiMDSC in suppressing T cell proliferation in multiple murine orthotopic breast tumor models.In line with their differential angiogenic and immunosuppressive capability,Breast tumor initiation and metastatic colonization was accompanied by a shift of tiMDSC to TAM.Our findings suggest that tiMDSC is more pro-angiogenic and promotes tumor initiation,while TAM is more immunosuppressive and facilitates tumor immune evasion.Thus,selectively targeting on TAM,rather than tiMDSC,could alleviate the immunosuppressive tumor microenvironment and potentiate cancer immunotherapy.