2-己基-4-戊炔酸重塑肿瘤免疫微环境抑制乳腺癌肺转移的作用及机制OA

OBJECTIVE To investigate the inhibitory effect of 2-hexyl-4-pentynoic acid(HPTA)on breast tumor growth and lung metastasis and to explore its immunoregulatory mechanisms.METHODS① The animal experiment:Female BALB/c mice were inoculated with 4T1-Luc cells to establish an orthotopic breast tumor model before being randomly assigned to a PBS control or an HPTA group(n=7 per group).The control group received PBS while the HPTA group received HPTA(20 mg·kg-1)by intraperitoneal injection twice daily for 6 consecutive days.Tumor volume and pulmonary biolumi-nescence were dynamically monitored.The onset of lung metastasis was tracked to confirm the estab-lishment of an orthotopic breast cancer model with spontaneous lung metastasis.After euthanasia,lung metastases were evaluated by gross inspection,Bouin's staining,and HE staining.Based on this model,the observation period was extended,and survival was compared between the control and HPTA-treated groups by survival analysis.Immunohistochemistry(IHC)was performed for Ki67,cluster of differentiation 34(CD34),F4/80,integrin alpha M(CD11b),CD80,CD206,inducible nitric oxide synthase(iNOS),arginase-1(Arg-1),CD8,and Granzyme B.Western blotting was used to detect iNOS and Arg-1 protein expressions.RT-qPCR was employed to measure mRNA levels of CD206,interleukin-10(IL-10),IL-12,tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),CD80,Arg-1,iNOS,C-X-C motif chemo-kine ligand 9/10/11(CXCL9/10/11).② The cell experiment:RAW264.7 macrophages were cultured in tumor-conditioned medium(TCM)and divided into a control group and an HPTA group(15 μmol·L-1).After 24 h of culture,cells and supernatants were collected separately.The supernatants were desig-nated as the macrophage-conditioned medium in TCM(M-TCM)and M-TCM+HPTA.RT-qPCR was used to measure the mRNA expression levels of CD163,CD206,IL-10,CD80,CD86,IL-12,and CXCL9/10/11 in RAW264.7 cells.Western blotting was used to detect the protein expression of CXCL9/10/11.RAW264.7 macrophages were also cultured in DMEM and divided into a control group and an HPTA group(15 μmol·L-1).After 24 h of culture,the supernatants were collected and designated as the macrophage-conditioned medium in DMEM(M-DMEM)and M-DMEM+HPTA.MCF-7 cells were seeded into 6-well plates and divided into four groups:M-TCM,M-TCM+HPTA,M-DMEM,and M-DMEM+HPTA.Each group was incubated with the corresponding supernatant for 24 h or 48 h.A wound-healing assay was then performed to evaluate scratch closure in MCF-7 cells.RESULTS ① The animal experiments:compared with the control group,mice in the HPTA group showed significantly lower bioluminescence signals in the primary tumor and lungs,as well as significantly lower tumor weight and lung weight.Expressions of Ki67 and CD34 were significantly decreased in both primary tumors and pulmonary metastatic lesions.The number of pulmonary metastatic nodules and the metastatic area ratio were also significantly reduced.In addition,survival was significantly prolonged.Compared with the control group,primary tumors from HPTA-treated mice showed significantly increased expressions of F4/80,CD11b,CD80,CD8,iNOS,and Granzyme B whiile CD206 and Arg-1 expressions were significantly decreased.Pulmonary metastatic lesions from HPTA-treated mice showed significantly increased expressions of F4/80,CD11b,iNOS,CD8,and Granzyme B along with significantly decreased Arg-1 expression.In both primary tumors and pulmonary metastatic lesions,the mRNA levels of CD206,IL-10,and Arg-1 were significantly decreased,whereas those of CD80,TNF-α,IL-12,CXCL9,CXCL10,and CXCL11 were significantly increased.② The cell experiments:compared with the control group,HPTA-treated macrophages showed significantly increased mRNA expressions of CD80,CD86,IL-12,CXCL9,CXCL10,and CXCL11.The protein levels of CXCL9,CXCL10,and CXCL11 were also signifi-cantly increased while the mRNA levels of CD163,CD206,and IL-10 were significantly decreased.In the wound-healing assay,MCF-7 cells cultured with conditioned medium from the M-TCM+HPTA group showed significantly slower wound closure than those cultured with conditioned medium from the M-TCM group.CONCLUSION HPTA may inhibit primary breast tumor growth and pulmonary metastasis by activating M1 macrophage-mediated immune responses.