具核梭杆菌相关细菌生物膜促进巨噬细胞M2型极化和结肠癌化疗耐药的研究

Study of Fusobacterium nucleatum-related bacterial biofilm promoting M2 polarization of macrophages and chemoresistance in colon cancer

目的·探索具核梭杆菌(Fusobacterium nucleatum,F.n)相关细菌生物膜对结肠癌浸润的肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)极化和化疗耐药的影响。方法·培养并收集液基和生物膜来源的F.n上清干预肠癌细胞或巨噬细胞。采用实时荧光定量PCR检测各组肠癌细胞耐药相关基因表达水平,采用CCK8试剂盒检测化疗药物对各组肠癌细胞生长抑制率,采用实时荧光定量PCR和免疫细胞化学染色检测巨噬细胞极化分型。采用荧光原位杂交和免疫化学染色检测结肠癌患者组织标本生物膜状态和TAMs浸润分型情况。结果·生物膜来源的F.n培养上清上调肠癌细胞耐药基因(如MDR1/Abcb1a/Abcb1b)表达更为明显,化疗药物处理后细胞的生长抑制率更低。生物膜来源的培养上清更能促进巨噬细胞M2型极化。术后化疗复发的结肠癌组织中细菌生物膜阳性率和M2型巨噬细胞比例更高。结论·F.n相关细菌生物膜促进肿瘤相关巨噬细胞M2型极化和结肠癌化疗耐药,影响结肠癌患者预后。

Objective·To investigate whether and how Fusobacterium nucleatum-related bacterial biofilm modulates the infiltration of tumor-associated macrophages into tumor microenvironment and the response to chemotherapy in colon cancer patients.Methods·Both biofilm-based F.n-culture medium(BF-CM)and planktonic F.n-culture medium,(P-CM)Fusobacterium nucleatum was cultured,and the culture-medium was collected to co-culture with CRC cell lines and macrophages.Quantified real time PCR(qRT-PCR)was used to measure genes related to chemoresistance,CCK8 assay was conducted to measure proliferation inhibition rate of chemicals to cancer cells,qRT-PCR was used to measure the expression of genes related to macrophage polarization.Fluorescence in situ hybridization(FISH)and immunohistochemistry(IHC)were conducted to evaluate existence of bacterial biofilm and infiltration of macrophages in tumor tissues of colon cancer.Results·Expression of chemoresistance-related genes(e.g.MDR1/Abcb1a/Abcb1b)were higher in BF-CM treated CRC cells than those in P-CM treated cells.CRC cell inhibition level via LOHP/5-FU were reduced with F.n culture medium(metabolites)co-culture,and much lower in BF-CM group.Expression of M2-polarization markers were also higher after BF-CM treated macrophages than P-CM.Biofilm positivity was higher in recurrent(confirmed by PET-CT,CT,or colonoscopy)colon cancer patients with post-resection adjuvant chemotherapy,than that in non-recurrent ones;correlated with infiltration rate of M2 macrophages.Conclusion·Fusobacterium nucleatum-related bacterial biofilm can induce M2-polarization of intratumor macrophages and could promote chemoresistance to chemicals in CRC cells,which may contribute to prognosis of colon cancer patients.