Gram-negative intratumoral bacteria mediate lymph node metastasis through LPS-TLR4/MAPK signaling pathway in cervical cancer

BACKGROUND: Intratumoral bacteria have been identified as prevalent in various solid tumors, playing a significant role in tumor progression. Lymph node metastasis is a major clinical feature and the primary cause of mortality in cervical cancer (CC). However, the effect of intratumoral bacteria on lymphatic node metastasis in CC remains unclear. METHOD: This study employed 16S rDNA sequencing and targeted bacterial culture to investigate the distribution of intratumoral bacteria in human CC tissues. The identified Gram-negative bacteria, including Escherichia coli (E. coli), Prevotella bivia (P. bivia), and Fusobacterium nucleatum (F. nucleatum), were isolated, and their roles in metastasis were examined using in vitro transwell and capillary tube formation assays on human lymphatic endothelial cells (HLEC). The signaling pathways involved in metastasis were assessed by examining TLR4/MAPK activation and the expression of prometastatic factors EFNA1 and EDN2. In vivo studies using a mouse footpad tumorigenesis model were also conducted to observe the effect of LPS, which was extracted from these three gram-negative intratumoral bacteria and E. coli on lymph node metastasis. RESULT: A higher abundance of Gram-negative bacteria, especially in metastatic CC tissues, was observed. E. coli, P. bivia, and F. nucleatum enhanced capillary tube formation in lymphatic endothelial cells and facilitated metastasis of uninfected tumor cells through paracrine signaling. These bacteria activated the TLR4/MAPK signaling pathway via lipopolysaccharide (LPS), leading to the upregulation of prometastatic factors EFNA1 and EDN2. Knockdown of EFNA1 and EDN2 attenuated the bacteria-induced metastasis, whereas overexpression of these factors mimicked the effects of bacterial infection. In vivo, LPS, which was extracted from E. coli, P. bivia, and F. nucleatum and live E. coli promoted lymph node metastasis, with elevated LPS levels and MAPK-EFNA1/EDN2 expression observed in infected mice compared to controls. CONCLUSION: The study suggests that Gram-negative bacteria, particularly E. coli, P. bivia, and F. nucleatum, play a causal role in exacerbating lymph node metastasis in CC. These findings highlight the potential of targeting these bacteria and their associated signaling pathways as therapeutic strategies to improve clinical outcomes in CC patients.