Background Different experimental techniques were used to search for evidence of bacteria in HCC. LPS/TLR4/CCL2/CCR2/M-MDSCs hypothesis was proposed using TCGA and transcriptome sequencing data. Combined with cells, animal models and clinical samples confirmed that Gram’s negative bacteria and endotoxins affect the immune microenvironment of HCC and promote the development of HCC through this mechanism.

Methods (1) The presence of bacteria was verified in HCC by different experimental techniques. (2) LPS^Low and LPS^High groups were distinguished according to the strength of LPS staining. Kaplan-Meier was used to analyze the survival difference between the two groups, and 16S rDNA sequencing was used to analyze the difference in bacterial community. (3) Differences of CD33 and CD8 expression between LPS^Low and LPS^High groups were verified by WB and IF in 30 fresh HCC tissues. FCM was used to verify the difference of MDSCs between LPS^Low and LPS^High. (5)

Results (1) LPS were found in 93 HCC pathological sections (IDDF2024-ABS-0281 Figure 1) and were located in the cytoplasm of immune cells and non-immune cells. 16S rRNA signal was found in HCC (IDDF2024-ABS-0281 Figure 2). 40956 OTUs were obtained by 16S rDNA sequencing for HCC and paracancer. Culture omics found that 57% of the HCC samples were positive (IDDF2024-ABS-0281 Figure 3). (2) The prognosis of HCC patients in the LPS^High group was poor. The EUB338 probe signal of the LPS^High group was strong. Combined with TCGA data, TLR4 was positively correlated with CCL2, CCR2, and CD33 expression (IDDF2024-ABS-0281 Figure 4). IHC (IDDF2024-ABS-0281 Figure 5) and WB(Fig. 6) verified the relation of CD33 and CD8. The abundance of Proteobacteria in the LPS^High group increased, while the abundance of Lactobacillus decreased. FCM showed that MDSCs in the LPSHigh group were higher (IDDF2024-ABS-0281 Figure 6).

Conclusions Gram-negative bacteria and endotoxins in HCC affect the growth and prognosis of HCC through LPS/TLR4/CCL2/CCR2/M-MDSCs. The relative abundance of Proteobacteria in the LPSHigh group increased, and Lactobacillus decreased in human HCC. In the HCC model constructed by DEN combined with CCL₄, neomycin treatment decreased the relative abundance of Proteobacteria and increased the relative abundance of Lactobacillus. Targeting this path becomes a new method for HCC.