Effect of fusobacterium nucleatum on NF-κB/HIF-1α/CCL20 pathway and M2 macrophages infiltration in esophageal squamous cell carcinoma.

Abstract

2554 Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common digestive malignant tumor with the highest mortality rate in China.Recent studies have shown that Fusobacterium nucleatum (F. nucleatum) can attenuate the efficacy of immunotherapy in ESCC patients through various mechanisms.One of them is recriuting more M2-like macrophages through tumor-derived cytokines such as CCL20. Methods: From January 2022 to June 2023, a total of 30 patients with ESCC from 4th Hospital of HeBei Medical University were enrolled.All of the pateints did not apply any neoadjuvant therapy before and received radical resection.Real-time reverse transcriptase - PCR(RT-PCR) were performed to examine the expressions of F. nucleatum in tumor tissues.According to the CT values,the level of the F. nucleatum infection could be seperated in two groups - positive group and negative group. Immunohistochemistry (IHC) staining were used to examine the expressions of CCL20、CD206 and HIF-1α in both groups. Immunofluorescence(IF) was characterised CD206 on CD68 + macrophages.In addition,transwell assay was carried on to quantify macrophages migration ability in vitro.At last,Western blot was used to determine the expression level of CCL20, HIF-1α and p65/p-p65 on ESCC cells. Results: F. nucleatum DNA positivity was significantly associated with higher expression of CCL20, HIF-1α and accumulation of CD68 + CD206 + macrophages.IHC showed that the expression of CCL20 and HIF-1α were higher in F. nucleatum positive tumor tissue. After coculture with F. nucleatum and Eca109 cells in vitro, CCL20 and HIF-1α production by ESCC cells were accelerated.Transwell assay showed using siCCL20, CCL20-Nab or siCCR6 could decline the macrophages invasion level caused by CCL20. Otherwise, siHIF-1α could lowered the CCL20 expression level caused by F. nucleatum.In addition,Western blot revealed NF-κB pathway was highly activated in Fn educated Eca109 cells.Using BAY11-7082 could decline both CCL20 and HIF-1α level triggered by F. nucleatum. Conclusions: Fusobacterium nucleatum promotes esophageal squamous cell carcinoma progression via NF-κB/HIF-1α/CCL20 pathway-mediated migration of M2-like macrophages into the tumour micro-environment and deteriorates the suppression of the local immune micro-environment within the tumor.Such bacteria may be a biomarker to predict the efficacy of immunotherapy in ESCC.