Gram-negative bacteria and endotoxins promote hepatocellular carcinoma progression through the recruitment of myeloid-derived suppressor cells

Figure 1 Lipopolysaccharide was found present in both tumor and non-tumor tissues in paraffin sections of hepatocellular carcinoma. A: Hematoxylin-eosin staining and immunohistochemistry (50 μm) revealed the presence of lipopolysaccharide (LPS) in both tumor tissue regions and non-tumor tissue regions; B: LPS immunohistochemistry in different batches of HepG2 cells (100 μm). T: Tumor tissue region; N: Non-tumor tissue region; HE: Hematoxylin-eosin; PBS: Phosphate-buffered saline; LPS: Lipopolysaccharide; LTA: Lipoteichoic acid.

Figure 2 Bacteria and their products are components of human hepatocellular carcinoma. A: Through fluorescence in situ hybridization detection, bacterial 16S rRNA was found adjacent to the cell nucleus (25 μm), and its localization was more clearly observed under oil immersion microscopy (10 μm); B: The presence of bacterial DNA in human hepatocellular carcinoma (HCC) was assessed by reverse transcription-quantitative PCR; C: Comparison of peripheral blood leukocyte count between HCC patients and healthy controls (no-template control, n = 8; environmental blank control, n = 9; sample solution, n = 30; normal, n = 7; HCC, n = 30); D: Schematic representation of the analysis pipeline applied to 16S rDNA sequencing data; E: Major genus average distribution in cancer and para-cancer groups; F: Alpha diversity assessed using the Simpson index and β diversity evaluated by principal coordinate analysis comparing cancerous and paracancerous tissue groups (n = 30); G: Sample homogenates and HCC tissues were cultured on LB agar without antibiotics. ^aP < 0.0001; ^bP < 0.01; NS: Not significant. NTC: No-template control; EBC: Environmental blank control; HCC: Hepatocellular carcinoma; PCoA: Principal coordinate analysis; OTU: Operational taxonomic unit; G⁺: Gram-positive; G^-: Gram-negative.

Figure 3 Gram-negative bacteria and endotoxins affected the survival of hepatocellular carcinoma patients through recruitment of monocytic myeloid-derived suppressor cells. A: Relative expression of lipopolysaccharide (LPS) in LPS^low and LPS^high groups; B: Kaplan-Meier survival analysis of LPS^low and LPS^high groups; C: EUB338 fluorescence in situ hybridization of LPS^low and LPS^high groups (LPS^low, n = 47; LPS^high, n = 46); D and E: The relative expression levels of CD33 and CD8 in LPS^low and LPS^high groups (LPS^low, n = 16; LPS^high, n = 14); F: Representative images of IF staining with CD33⁺CD14⁺ cells in group of LPS^low and LPS^high (LPS^low, n = 16; LPS^high, n = 14); G: The flow cytometry data of MDSCs in LPS^low and LPS^high groups (LPS^low, n = 8, LPS^high, n = 6). ^aP < 0.05, ^bP < 0.01; ^cP < 0.0001. LPS: Lipopolysaccharide.

Figure 4 An impaired intestinal mucosal barrier renders the liver susceptible to exposure to gut microbiota and promotes hepatocellular carcinoma growth. A: Occludin relative expression in ileum in control (Ctrl) and CCL₄ groups; B: Interleukin-1β and interleukin-17 mRNA expression in ileum in Ctrl and CCL₄ groups (n = 5 for each group); C: Hepatocellular carcinoma (HCC) tissues from Ctr and CCL₄ mice were homogenized and cultured on a BHI medium without antibiotics. The clone formation units were calculated; D: The flow cytometry-based proportional changes of monocytic myeloid-derived suppressor cells and polymorphonuclear-myeloid-derived suppressor cells in the tumor microenvironment (TME) between Ctrl and CCl₄ groups; E: The Ly6C relative expression in TME in Ctrl and CCL₄ groups; F: Establishment of murine HCC models and monitoring tumor growth; G: Weekly in vivo imaging was performed to monitor tumor growth in Ctrl and CCL₄ groups; H: Following the schematic diagram of animal interventions, tumor size and liver weight changes were compared between the Ctrl and CCl4 groups post-intervention. ^aP < 0.05, ^bP < 0.01; ^bP < 0.001; ^cP < 0.0001; NS: Not significant. IL: Interleukin; CFU: Clone formation unit; PMN: Polymorphonuclear; MDSC: Myeloid-derived suppressor cell; MMDSC: Monocytic myeloid-derived suppressor cell; Ctrl: Control.

Figure 5 Gram-negative bacteria and endotoxins mediate monocytic myeloid-derived suppressor cells aggregation through lipopolysaccharide/TLR4/CCL2. A: The mRNA expression levels of TLR4 and CCL2 in human HepG2, MHCC-97H and LX-2 cells were detected by phosphate-buffered saline (PBS), lipopolysaccharide (LPS), and lipoteichoic acid (LTA) after stimulation, respectively (n = 3 for each group); B: After the intervention with PBS, LPS, and LTA, the mRNA expression level of CCL2 was detected by reverse transcription-quantitative PCR (RT-qPCR); C: The aggregation of monocytic myeloid-derived suppressor cells (MMDSCs) was detected by flow cytometry (n = 4 for each group); D: Weekly in vivo imaging was performed to monitor tumor growth in control (Ctrl), LPS, and LTA groups; E: The changes in tumor size and liver weight in Ctrl, LPS, and LTA groups were assessed in animals that received different interventions; F: Volcano map of differential gene expression in Ctrl and LPS groups (n = 3); G: Kyoto Encyclopedia of Genes and Genomes enrichment pathway; H: Gene Set Enrichment Analyses enrichment pathway; I: Transcriptome sequencing showed the expression levels of interest genes. The mRNA expression levels of CCR2 and Ly6C in Ctrl and LPS group were detected by RT-qPCR (n = 4 for each group); J: The expression of CCL2 mRNA was detected by RT-qPCR in Ctrl and TAK-242 groups; K: The aggregation of MMDSCs was detected by flow cytometry in Ctrl and TAK-242 groups; L: Weekly in vivo imaging was performed to monitor tumor growth in Ctrl and TAK-242 groups; M: The changes in tumor size in Ctrl and TAK-242 groups were assessed in animals that received different interventions. ^aP < 0.05, ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001; NS: Not significant. LPS: Lipopolysaccharide; LTA: Lipoteichoic acid; MMDSC: Monocytic myeloid-derived suppressor cell; GSEA: Gene Set Enrichment Analyses; Ctrl: Control.

Figure 6 Targeting CCL2/CCR2/monocytic myeloid-derived suppressor cell inhibits tumor growth. A: The aggregation of monocytic myeloid-derived suppressor cells was detected by flow cytometry in control (Ctrl), CCL2 Ab and CCR2 antag groups; B: Weekly in vivo imaging was performed to monitor tumor growth in Ctrl, CCL2 Ab and CCR2 antag groups; C: The Change of tumor size in Ctrl, CCL2 Ab and CCR2 antag groups by giving animals different interventions (n = 5 for Ctrl and CCR2 antag, 4 for CCL2 Ab); D: Weekly in vivo imaging was performed to monitor tumor growth in Ctrl and Ly6C Ab groups; E: The change of tumor size in Ctrl and Ly6C Ab groups by giving animals different interventions; F: The aggregation of IFN-γ⁺CD8⁺T was detected by flow cytometry in Ctrl and Ly6C Ab groups (n = 4 for each group); G: Molding diagram of DEN combination with CCL₄ (n = 5 for each group); H: Major phylum average distribution in Ctrl and neomycin groups; I: The ratio of Bacteroidetes/Firmicutes in Ctrl and neomycin groups; J: Major genus average distribution in Ctrl and neomycin groups; K and L: Alpha diversity was evaluated using the Simpson and Chao1 indices, while beta diversity was assessed by principal coordinate analysis, revealing distinct microbial community structures between the Ctrl and neomycin groups. ^aP < 0.05, ^bP < 0.01. PBS: Phosphate-buffered saline; PCoA: Principal coordinate analysis; MMDSC: Monocytic myeloid-derived suppressor cell; Ctrl: Control.

Figure 7 Neomycin sensitive Gram-negative bacteria promote hepatocellular carcinoma growth. A: Portal endotoxin levels in control (Ctrl) and neomycin groups; B: Hepatocellular carcinoma tissues from Ctrl and neomycin mice were homogenized and cultured on a medium without antibiotics. The clone formation units were calculated; C: Major phylum average distribution in Ctrl and neomycin groups; D: Major genus average distribution in Ctrl and neomycin groups; E: The differential bacteria at genus level was detected in Ctrl and neomycin groups by t-test; F: The LEfSe analysis revealed genus-level differences in bacterial composition between the Ctrl and neomycin groups; G: The mRNA expression levels of TLR4, CCL2, and CCR2 were detected by reverse transcription-quantitative PCR in Ctrl and neomycin groups; H: The aggregation of monocytic myeloid-derived suppressor cells was detected by flow cytometry in Ctrl and neomycin groups; I: The relative expression of Ly6C was detected by immunohistochemical in Ctrl and neomycin groups. Tumor size, maximum tumor diameter, and liver weight were assessed in in Ctrl and neomycin groups (n = 5 for each group). ^aP < 0.05, ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. CFU: Clone formation unit; Ctrl: Control; Neo: Neomycin.