Mechanism Of Tumor-associated Macrophages Promote Tumor Stromal Fibrosis Through Tumor Cell–derived Osteopontin

Background and Objective:Breast cancer ranks first in incidence and second in mortality among female cancer patients.Triple-negative breast cancer(TNBC)is a refractory type of breast cancer.In the event of distant metastasis,chemotherapy resistance or tumor recurrence,the available treatment options are very limited.The median survival time is less than 18 months,which is life-threatening.Although the treatment of immune checkpoint inhibitors in recent years has benefited some tumor patients,according to multi-center randomized controlled trials,the treatment effect of PD-1 antibody in TNBC patients is unsatisfactory.At present,TNBC is still treated with chemotherapy and surgical treatment.In recent years,with the application of single-cell RNA sequencing(sc RNA-seq)and advancement on the tumor microenvironment(TME),it has been acknowledged that the refractory TNBC is not only related to the characteristics of tumor cells,but also related to the crosstalk of complex cellular components in the TME.The cellular components in the TME of TNBC include:tumor-associated macrophages(TAMs),cancer-associated fibroblasts(CAFs),endothelial cells and pericytes that constitute the tumor vasculature,inflammatory cells,bone marrow-derived cells,etc.,among which TAMs and CAFs are the main components of TME.TAMs are involved in chemoresistance and cancer development by stimulating angiogenesis,releasing pro-inflammatory cytokines,suppressing cytotoxic T cell responses,and remodeling the extracellular matrix(ECM).CAFs can produce collagen in the TME to build a physical barrier to protect tumor cells from drug killing.In breast cancer,the high expression ofα-smooth muscle actin(α-SMA)in the tumor stroma is often associated with poor prognosis.But surprisingly,in pancreatic ductal carcinoma(PDAC)model,targetingα-SMA positive CAFs showed immunosuppression and shorten survival.Thus,a full understanding of the bidirectional communication between tumor cells and associated stroma is expected to enhance therapeutic sensitivity and improve patient outcomes.Osteopontin(OPN)is a multifunctional phosphoglycoprotein encoded by the SPP1 gene.It is widely involved in physiological and pathological processes of the body.OPN is overexpressed in a variety of cancers,including lung cancer,prostate cancer,breast cancer,colorectal carcinoma,and hepatocellular carcinoma(HCC).Previous studies have shown that OPN can maintain tumor cells’stemness,recruit macrophages,reprogram normal fibroblasts to CAFs,and promote remote metastasis through binding to integrin and/or CD44 receptors.However,in the TNBC model,whether the regulation of CAFs activation by tumor cell-derived OPN is completely dependent on integrin and CD44 receptors?In addition to these two receptors,whether OPN can participate in CAFs activation through other signal-mediated pathways?Whether targeting OPN can prolong survival while attenuating tumor stromal fibrosis and collagen accumulation?These questions have not yet been fully understanding,and further research is needed.Therefore,this study aimed to explore the regulatory mechanism between tumor cell-derived OPN and tumor stromal fibrosis,and to verify the effect of the crosstalk between OPN and CAFs on chemotherapeutic resistance.Provide strategic support for the development of new drugs or treatment options targeting tumor stromal fibrosis.Methods:1.According to the varying degrees of tumor fibrosis,four mouse tumor cell lines are selected for transcriptome sequencing analysis,combined with the analysis of human breast cancer data in the GEPIA database,we confirm the correlation between OPN and tumor fibrosis.2.The CRISPR-Cas9 system is used to construct a stable OPN knockout cell line,and the effects of knockout OPN on the proliferation,apoptosis and migration of 4T1tumor cells are observed in vitro.3.Stably overexpressing OPN is constructed by lentivirus,and the effects of OPN on the proliferation,apoptosis and migration of B16-F0 cells ar evaluated in vitro.4.We have eastblished the 4T1 and B16-F0 mice model,and observe the effects of knockout or overexpression of OPN on the proportion of TAMs and the degree of tumor stromal fibrosis by flow cytometry and immunohistochemical staining,and analyze the proportion of macrophages in spleen and monocytes in peripheral blood of tumorbearing mice.5.In macrophages depletion model,we have used flow cytometry,quantitative-PCR,western blot,immunofluorescence and immunohistochemical staining to investigate the regulation mechanism of tumor cell-derived OPN and TAMs on tumor stromal fibrosis.6.TAMs(CD45~+CD11b~+F4/80~+)and CAFs(CD45~-CD90.2~+)in 4T1 tumors are sorted to explore the mechanism of TAMs on the activation and migration of CAFs.And observe the sensitivity of 4T1-WT and 4T1-SPP1-KO tumors to docetaxel by in vitro and in vivo experiments.Results:1.Among the four types of tumors,the expression of OPN and tumor stroma fibrosis are highest in 4T1 tumor,which is highly consistent with the expression trends of OPN and CAFs biomarkers(ACTA2,S100A4 and FAP)in human TNBC samples,demonstrating that 4T1 model can be used to uncover the relationship between OPN and tumor stromal fibrosis.2.The 4T1-SPP1-KO cell line is successfully constructed by using CRISPR-Cas9.Knockout OPN do not affect the proliferation rate and total apoptosis ratio of 4T1 cells,but the migration ability is impaired.3.The B16-SPP1-OE cell line is constructed by lentivirus.Overexpression of OPN do not increase the proliferation,total apoptosis ratio or migration of B16 cells.4.In 4T1 model,the proportion of TAMs in 4T1-SPP1-KO tumor is significantly reduced compared with 4T1-WT tumor,and the degree of tumor stromal fibrosis is alleviated obviously.In B16 model,overexpressing OPN have an increased proportion of TAMs and aggravated degree of tumor stromal fibrosis compared with control groups.5.Depleting macrophages in 4T1-WT tumorbearing mice with Clodronate-Liposomes can significantly inhibited the tumor growth rate,reduced the proportion of TAMs and alleviated the degree of tumor stromal fibrosis,but it cannot further reduce the proportion of TAMs nor the degree of fibrosis in 4T1-SPP1-KO tumorbearing mice.It suggests that tumor cell-derived OPN is the main factor to promot the infiltration of TAMs in 4T1 tumor,and the regulation of tumor stromal fibrosis by OPN is indirectly mediated by TAMs.6.TAMs can recruit CAFs to migrate through the CCL5-CCR5 signal axis,and TAMs can also stimulate the up-regulation ofα-SMA,FSP1 and PDPN in CAFs through TGF-β1/c-Rel.CAFs are resistant to docetaxel-induced apoptosis after co-culture with tumor cells in vitro.In vivo experiments have showed that knocking out OPN could improve the sensitivity of 4T1 tumor to docetaxel.Conclusion:In this study,it is suggested that tumor cell-derived OPN can significantly increase the growth rate of tumor volume,enhance the infiltration of TAMs and aggravate the degree of tumor stromal fibrosis.Moreover,the depletion of macrophages in tumor-bearing mice confirme that tumor cell-derived OPN is the main factor to promote the infiltration of TAMs.In the conditon of OPN,TAMs can recruit CAFs to migrate through the CCL5-CCR5 signaling axis.Meanwhile,TAMs stimulate CAFs to overexpressα-SMA,FSP1 and PDPN through TGF-β1/c-Rel pathway,and promote the apoptosis resistance of 4T1 to docetaxel.The complicated crosstalk in TME together contribute to TNBC disease progression.