Abstract: 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.

Key words: bacterial biofilm(BF), Fusobacterium nucleatum(F.n), tumor-associated macrophages(TAMs), colon cancer, chemotherapy, resistance