CRISPR/CAS99-MEDIATED TLR4 KNOCKOUT ATTENUATES HEPATOCELLULAR CARCINOMA PROGRESSION IN MICE VIA THE KINETICS OF MYELOID-DERIVED SUPPRESSOR CELL SUBSETS DYNAMICS

Hepatocellular carcinoma (HCC) significantly contributes to global cancer mortality, highlighting the urgent need for new treatments. Toll-like receptor 4 (TLR4) is linked to metabolic dysfunction associated steatotic liver disease (MASLD)-related HCC, while MDSCs (myeloid-derived suppressor cell) suppress the immune system in the tumor microenvironment. This study investigates how TLR4 ablation using CRISPR/Cas9 technology influences HCC progression in mice with MASLD-associated HCC by examining MDSC subset dynamics. Method: CRISPR/Cas9 TLR4 mutant mice (CCT #712 to CAT) (CTLR4), global TLR4 knockout (TLR4), and wild-type (WT) HCC models with or without HFD were used. HDI was used to transfect C3H/HeN mice with surrogate tumor antigens, NRAS, ShP53, and ORFs. Mice were examined at two, four, six, and eight weeks after HCC induction. Mouse gut microbiota (GM) was explored through germ-free (GF) to specific pathogen-free (SPF) environment shift mice and GF mice. Comprehensive studies include metabolic profile, serum analysis, hepatotoxicity assessment, and immune cell flow cytometry. The GM composition was analyzed by TTGE and 16SrDNA NGS. HCC was verified by histology and immunohistochemistry. Result: Histological evaluation of NAFLD features indicated that HFD diet in combination with addition to hydrodynamic injections of oncogenes created a new MASLD-HCC model. Both CTLR4 and TLR4 mice demonstrated a substantial decrease in the progression of HCC in this model when compared to WT mice. CTLR4 and TLR4 mice exhibited decreased levels of AST and ALT. Flow cytometry analysis of CTLR4 and TLR4 mice reveals fewer pan-keratin Ki67 cells, indicating a deceleration in tumor growth. The livers of WT mice contained a greater quantity of monocytic MDSCs (mMDSC) compared to mice lacking CTLR4 and TLR4. In contrast to WT mice, CTLR4 and TLR4 mice exhibit a reduction in granulocytic MDSCs (gMDSCs) during the final phases (6-8 weeks) of HCC progression. Suggesting that mMDSCs may foster immunological tolerance in the initial microenvironment of HCC tumors, whereas gMDSCs become more abundant during the intermediate and terminal phases. Hepatic tumor MDSC infiltration appears to be TLR4-regulated. SPF mice were outlived by GF mice subsequent to GM modifications. Significantly, GF mice that were transplanted into SPF conditions exhibited a greater abundance of myeloid cell TLR4s than both GF and WT mice. Conclusion: This study provides an HFD-induced MASLD-HCC model with accelerated carcinogenesis. CRISPR/Cas9-mediated TLR4 deletion prevents mouse HCC progression. Early mMDSCs decrease and late gMDSCs decrease was observed in both CTLR4 and TLR4 mice, along with attenuated HCC progression. The findings suggest TLR4 regulation could reverse HCC and highlight immune cell-cancer interactions.