| Background and objective:Neutrophils are involved in immunomodulatory processes and tissue repair,as well as in tumorigenesis and development.Neutrophils have been found to be associated with lung metastasis of breast cancer,and the increase of neutrophils in the lung facilitates the formation of lung pre-metastatic microenvironment of breast cancer,but the mechanism by which neutrophils promote the formation of lung pre-metastatic microenvironment has not been fully clarified.Caveolin-1(Cav-1)is an oncogenic membrane protein that is associated with tumor invasion,metastasis and treatment resistance.Based on the above research status,this project will investigate the breast cancer cell-derived Cav-1-containing exosomes to promote breast cancer lung metastasis by affecting the recruitment and polarization of lung neutrophils and lung angiogenesis,so as to provide new ideas and theoretical basis for the prevention and treatment of breast cancer.Methods:1.Construction of cell models and acquisition of Cav-1-containing exosomes for physicochemical characterization.2.To investigate the function of breast cancer cell-derived Cav-1-containing exosomes in promoting neutrophil recruitment and polarization in the breast cancer pre-metastatic microenvironment.HL-60 was induced into neutrophil-like dHL-60 using ATRA,and HL-60-induced differentiation was examined by Hoechst,Richter-Kimsa staining and RT-qPCR.In vivo mouse breast cancer in situ tumor model was constructed and HE staining was performed to observe the recruitment of neutrophils around lung metastases;immunofluorescence was used to detect the polarization of neutrophils around lung metastases;immunohistochemistry was used to detect the effect of Cav-1-containing exosomes on CD34 expression in lung tissue.The biological effects of Cav-1-containing exosomes on neutrophils were observed by Transwell in vitro,chemokines secreted by neutrophils were detected by ELISA,the expression of neutrophil N1-type NOS2 and N2-type CD206 markers were detected by flow cytometry,and the effects of Cav-1-containing exosomes on neutrophil VEGF-A and MMP9 expression were detected by Western Blot,MMP9 expression by Western Blot.3.To investigate the mechanism by which breast cancer cell-derived Cav-1-containing exosomes promote neutrophil recruitment in the breast cancer pre-metastatic microenvironment through the TLR4-NF-κB-IL-6/CCL2 signaling axis and neutrophil polarization through the TLR4/NF-κB/NLRP3 signaling pathway.The effects of Cav-1-containing exosomes on neutrophil TLR4 receptor expression in each subgroup were detected by Western Blot,the effects of Cav-1-containing exosomes on neutrophil TLR4,NF-κB,NLRP3 expression in each subgroup were detected by ELISA,the effects of Cav-1-containing exosomes on neutrophil chemokine IL-6,CCL2 expression in each subgroup were detected by flow cytometry,and the effects of Cav-1-containing exosomes on neutrophil chemokine IL-6,CCL2 expression in each subgroup were detected by flow cytometry.The effect of Cav-1-containing exosomes on neutrophil N2-type polarization was detected by flow cytometry.Results:1.In vivo observation of exosome-derived Cav-1 in breast cancer cells promotes breast cancer lung metastasis:HE results showed that breast cancer-derived Cav-1-containing exosomes promoted neutrophil recruitment around lung metastases;IF results showed that breast cancer-derived Cav-1-containing exosomes promoted N2-type polarization of neutrophils;IHC results showed that breast cancer-derived Cav-1-containing exosomes promoted CD34 expression in vivo and facilitated angiogenesis.2.Breast Cancer Cell-Derived Cav-1-Containing Exosomes Promote Neutrophil Recruitment and Polarization and Angiogenesis:(1)2 μM ATRA induction for 5 days and 3 μM ATRA induction for 4 days can induce HL-60 into neutrophils;(2)Transwell and ELISA results show that breast cancer cell-derived Cav-1-containing exosomes attract more neutrophils by promoting the secretion of chemokines IL-6 and CCL2 by neutrophils;(3)Flow-through results showed that breast cancer cell-derived Cav-1containing exosomes stimulated increased CD206 expression and decreased NOS2 expression in neutrophils dHL-60,promoting their N2-type polarization;(4)Western Blot results show that breast cancer-derived Cav-1-containing exosomes promote the expression of VEGF-A,MMP9.3.Breast cancer cell-derived Cav-1-containing exosomes promote neutrophil recruitment through TLR4/NF-κB/IL-6,CCL2 signaling pathway and neutrophil polarization through TLR4/NF-κB/NLRP3 signaling pathway:(1)Knockdown of TLR4 gene and inhibition of NF-κB gene expression in neutrophils and breast cancer cell-derived Cav-1-containing exosomes promote the expression of TLR4,NF-κB,IL-6,and CCL2;(2)Knockdown of TLR4 gene and inhibition of NLRP3 gene expression in neutrophils and breast cancer cell-derived Cav-1-containing exosomes promote TLR4,NF-κB,NLRP3,and CD206 expression.Conclusion:1.Breast cancer cell-derived Cav-1-containing exosomes promote recruitment and N2-type polarization of neutrophils in the lung pre-metastatic microenvironment,promote angiogenesis,and accelerate the development of breast cancer lung metastases.2.Breast cancer cell-derived Cav-1-containing exosomes may promote neutrophil recruitment through the TLR4-NF-κB-IL-6/CCL2 signaling axis and neutrophil polarization through the TLR4/NF-κB/NLRP3 signaling pathway. |
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