Mouse Hepatoma Research Articles

Cholecystohepatic shunt pathway reduces secondary bile acid accumulation to enhance natural killer T cell-mediated anti-hepatocellular carcinoma immunity.

The impact of cholecystectomy, which blocks the cholecystohepatic shunt pathway (CHSP), on the prognosis of patients with hepatocellular carcinoma (HCC) is unclear. Hepatic secondary bile acids (BAs) inhibit natural killer T (NKT) cell-mediated immunity against HCC, and the regulation of homeostasis of hepatic secondary BAs is controlled by the CHSP. However, the influence of CHSP on NKT cell-mediated immunity against HCC remains unclear. The clinical data of hospitalized patients undergoing HCC resection were collected. Meanwhile, an in situ HCC mouse model was established, and the CHSP was augmented using oleanolic acid (OA). After 1:1 propensity score matching, Cox regression analysis revealed that cholecystectomy was an independent risk factor for HCC recurrence after hepatectomy (P=0.027, hazard ratio: 1.599, 95% confidence interval: 1.055-2.422). Experimentally, when OA enhanced CHSP, a significant decrease was observed in the accumulation of secondary BAs in the livers of mice. Additionally, a significant increase was observed in the levels of C-X-C ligand 16 and interferon γ in the serum and tumor tissues. Further, the percentage of C-X-C receptor 6 (+) NKT cells in the tumor tissues increased significantly, and the growth of liver tumors was inhibited. This clinical study revealed that cholecystectomy promoted the recurrence after radical hepatectomy in patients with HCC. Preserving the normal-functioning gallbladder as much as possible during surgery may be beneficial to the patient's prognosis. Further investigation into the mechanism revealed that CHSP enhanced NKT cell-mediated immunity against HCC by reducing the hepatic accumulation of secondary BAs.

Dihydroartemisinin breaks the positive feedback loop of YAP1 and GLUT1-mediated aerobic glycolysis to boost the CD8+ effector T cells in hepatocellular carcinoma

Aerobic glycolysis is a hallmark of hepatocellular carcinoma (HCC). Dihydroartemisinin (DHA) exhibits antitumor activity towards liver cancer. Our previous studies have shown that DHA inhibits the Warburg effect in HCC cells. However, the mechanism still needs to be clarified. Our study aimed to elucidate the interaction between YAP1 and GLUT1-mediated aerobic glycolysis in HCC cells and focused on the underlying mechanisms of DHA inhibiting aerobic glycolysis in HCC cells. In this study, we confirmed that inhibition of YAP1 expression lowers GLUT1-mediated aerobic glycolysis in HCC cells and enhances the activity of CD8+T cells in the tumor niche. Then, we found that DHA was bound to cellular YAP1 in HCC cells. YAP1 knockdown inhibited GLUT1-mediated aerobic glycolysis, whereas YAP1 overexpression promoted GLUT1-mediated aerobic glycolysis in HCC cells. Notably, liver-specific Yap1 knockout by AAV8-TBG-Cre suppressed HIF-1α and GLUT1 expression in tumors but not para-tumors in DEN/TCPOBOP-induced HCC mice. Even more crucial is that YAP1 forms a positive feedback loop with GLUT1-mediated aerobic glycolysis, which is associated with HIF-1α in HCC cells. Finally, DHA reduced GLUT1-aerobic glycolysis in HCC cells through YAP1 and prevented the binding of YAP1 and HIF-1α. Collectively, our study revealed the mechanism of DHA inhibiting glycolysis in HCC cells from a perspective of a positive feedback loop involving YAP1 and GLUT1 mediated-aerobic glycolysis and provided a feasible therapeutic strategy for targeting enhanced aerobic glycolysis in HCC.

Fluorescence imaging sheds light on the immune evasion mechanisms of hepatic stellate cells mediated by superoxide anion

Whether and how the reactive oxygen species generated by hepatic stellate cells (HSCs) promote immune evasion of hepatocellular carcinoma (HCC) remains mysterious. Therefore, investigating the function of superoxide anion (O2•−), the firstly generated reactive oxygen species, during the immune evasion become necessary. In this work, we establish a novel in situ imaging method for visualization of O2•− changes in HSCs based on a new two-photon fluorescence probe TPH. TPH comprises recognition group for O2•− and HSCs targeting peptides. We observe that O2•− in HSCs gradually rose, impairing the infiltration of CD8+ T cells in HCC mice. Further studies reveal that the cyclin-dependent kinase 4 is deactivated by O2•−, and then cause the up-regulation of PD-L1. Our work provides molecular insights into HSC-mediated immune evasion of HCC, which may represent potential targets for HCC immunotherapy.

Reprogramming tumor-associated macrophages and inhibiting tumor neovascularization by targeting MANF–HSF1–HSP70-1 pathway: An effective treatment for hepatocellular carcinoma

In advanced hepatocellular carcinoma (HCC) tissues, M2-like tumor-associated macrophages (TAMs) are in the majority and promotes HCC progression. Contrary to the pro-tumor effect of M2-like TAMs, M1-like TAMs account for a small proportion and have anti-tumor effects. Since TAMs can switch from one type to another, reprogramming TAMs may be an important treatment for HCC therapy. However, the mechanisms of phenotypic switch and reprogramming TAMs are still obscure. In this study, we analyzed differential genes in normal macrophages and TAMs, and found that loss of MANF in TAMs accompanied by high levels of downstream genes negatively regulated by MANF. MANF reprogrammed TAMs into M1 phenotype. Meanwhile, loss of MANF promoted HCC progression in HCC patients and mice HCC model, especially tumor neovascularization. Additionally, macrophages with MANF supplement suppressed HCC progression in mice, suggesting MANF supplement in macrophage was an effective treatment for HCC. Mechanistically, MANF enhanced the HSF1–HSP70-1 interaction, restricted HSF1 in the cytoplasm of macrophages, and decreased both mRNA and protein levels of HSP70-1, which in turn led to reprogramming TAMs, and suppressing neovascularization of HCC. Our study contributes to the exploration the mechanism of TAMs reprogramming, which may provide insights for future therapeutic exploitation of HCC neovascularization.

Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells

BackgroundDysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC.MethodsThe clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells.ResultsHCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells.ConclusionsIL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

Regulatory effect of Yinchenhao decoction on bile acid metabolism to improve the inflammatory microenvironment of hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is a malignant tumor with extremely high mortality. The tumor microenvironment is the "soil" of its occurrence and development, and the inflammatory microenvironment is an important part of the "soil". Bile acid is closely related to the occurrence of HCC. Bile acid metabolism disorder is not only directly involved in the occurrence and development of HCC but also affects the inflammatory microenvironment of HCC. Yinchenhao decoction, a traditional Chinese medicine formula, can regulate bile acid metabolism and may affect the inflammatory microenvironment of HCC. To determine the effect of Yinchenhao decoction on bile acid metabolism in mice with HCC and to explore the possible mechanism by which Yinchenhao decoction improves the inflammatory microenvironment of HCC by regulating bile acid metabolism, we established mice model of orthotopic transplantation of hepatocellular carcinoma. These mice were treated with three doses of Yinchenhao decoction, then liver samples were collected and tested. Yinchenhao decoction can regulate the disorder of bile acid metabolism in liver cancer mice. Besides, it can improve inflammatory reactions, reduce hepatocyte degeneration and necrosis, and even reduce liver weight and the liver index. Taurochenodeoxycholic acid, hyodeoxycholic acid, and taurohyodeoxycholic acid are important molecules in the regulation of the liver inflammatory microenvironment, laying a foundation for the regulation of the liver tumor inflammatory microenvironment based on bile acids. Yinchenhao decoction may improve the inflammatory microenvironment of mice with HCC by ameliorating hepatic bile acid metabolism.

Allicin regulates Sestrin2 ubiquitination to affect macrophage autophagy and senescence, thus inhibiting the growth of hepatoma cells

BackgroundAllicin regulates macrophage autophagy and senescence, and inhibits hepatoma cell growth. This study investigated the mechanism by which allicin inhibits the growth of hepatoma cells. MethodsHepa1–6 mouse hepatoma cells were subcutaneously injected into C57BL/6 J mice to construct a tumor transplantation model. Macrophages were cultured with the supernatant of hepatoma cells to construct a cell model. The levels of mRNA and proteins and the level of Sestrin2 ubiquitination were measured by RTqPCR, immunofluorescence and Western blotting. The levels of autophagy-related factors and the activity of senescence-associated β-galactosidase were determined by kits, and protein stability was detected by cycloheximide (CHX) tracking. ResultsData analysis of clinical samples revealed that RBX1 was highly expressed in tumor tissues, while Sestrin2 was expressed at low levels in tumor tissues. Allicin can promote the expression of the autophagy-related proteins LC3 and Beclin-1 in tumor macrophages and inhibit the expression of the aging-related proteins p16 and p21, thus promoting autophagy in macrophages and inhibiting cell senescence. Moreover, allicin can inhibit the expression of RBX1, thereby reducing the ubiquitination of Sestrin2, enhancing the stability of Sestrin2, activating autophagy in tumor macrophages and inhibiting senescence. In addition, allicin treatment inhibited the proliferation and migration of hepatoma carcinoma cells cocultured with macrophages and significantly improved the development of liver cancer in mice. ConclusionAllicin can affect the autophagy of macrophages and restrain the growth of hepatoma cells by regulating the ubiquitination of Sestrin2.

Dihydroartemisinin breaks the immunosuppressive tumor niche during cisplatin treatment in Hepatocellular carcinoma

ObjectiveHepatocellular carcinoma, characterized by high mortality rates, often exhibits limited responsiveness to conventional treatments such as surgery, radiotherapy, and chemotherapy. Therefore, identifying a sensitizer for cisplatin has become crucial. Dihydroartemisinin, known for its potent role of tumor treatment, arises as a prospective candidate for cisplatin sensitization in clinical settings. MethodsA mouse model of liver tumor was established through chemical induction of DEN/TCPOBOP. Upon successful model establishment, ultrasound was employed to detect tumors, Hematoxylin and eosin staining was conducted for observation of liver tissue pathology, and ELISA was utilized to assess cytokine changes (IFN-γ, IL-2, IL-4, IL-10, TGF-β, IL-1β, CCL2, and CCL21) in peripheral blood, para-tumor tissues, and tumor tissues. The infiltration of CD8+T cells and macrophages in tumor tissue sections was detected by immunofluorescence. ResultsDihydroartemisinin combined with cisplatin obviously restrained the growth of liver tumors in mice and improved the weight and spleen loss caused by cisplatin. Cisplatin treatment of liver tumor mice increased the content of CCL2 and the number of macrophages in tumor tissues and promoted the formation of an immunosuppressive microenvironment. The combination therapy decreased the content of TGF-β in tumor tissues while increasing CCL2 levels in para-tumor tissues. Both combination therapy and cisplatin alone increased the number of CD8+T cells in tumor tissue, but there was no difference between them. ConclusionDihydroartemisinin combined with cisplatin obviously prevented the deterioration of liver tumor in hepatocellular carcinoma mice and improve the therapeutic effect of cisplatin by improving the immunosuppressive microenvironment induced by cisplatin. Our findings provide a theoretical basis for considering dihydroartemisinin as an adjuvant drug for cisplatin in the treatment of hepatocellular carcinoma in the future.

Targeting OXCT1-mediated ketone metabolism reprograms macrophages to promote antitumor immunity via CD8+ T cells in hepatocellular carcinoma

The liver is the main organ of ketogenesis, while ketones are mainly metabolized in peripheral tissues via the critical enzyme 3-oxoacid CoA-transferase 1 (OXCT1). We previously found that ketolysis is reactivated in hepatocellular carcinoma (HCC) cells through OXCT1 expression to promote tumor progression; however, whether OXCT1 regulates antitumor immunity remains unclear. To investigate the expression pattern of OXCT1 in HCC invivo, we conducted multiplex immunohistochemistry experiments on human HCC specimens. To explore the role of OXCT1 in mouse HCC tumor-associated macrophages (TAMs), we generated LysMcreOXCT1f/f (OXCT1 conditional knockout in macrophages) mice. Here, we found that inhibiting OXCT1 expression in tumor-associated macrophages reduced CD8+ T-cell exhaustion through the succinate-H3K4me3-Arg1 axis. Initially, we found that OXCT1 was highly expressed in liver macrophages under steady state and that OXCT expression was further increased in TAMs. OXCT1 deficiency in macrophages suppressed tumor growth by reprogramming TAMs toward an antitumor phenotype, reducing CD8+ T-cell exhaustion and increasing CD8+ T-cell cytotoxicity. Mechanistically, high OXCT1 expression induced the accumulation of succinate, a byproduct of ketolysis, in TAMs, which promoted Arg1 transcription by increasing the H3K4me3 level in the Arg1 promoter. In addition, pimozide, an inhibitor of OXCT1, suppressed Arg1 expression as well as TAM polarization toward the protumor phenotype, leading to decreased CD8+ T-cell exhaustion and slower tumor growth. Finally, high expression of OXCT1 in macrophages was positively associated with poor survival in patients with HCC. In conclusion, our results demonstrate that OXCT1 epigenetically suppresses antitumor immunity, suggesting that suppressing OXCT1 activity in TAMs could be an effective approach for treating liver cancer. The intricate metabolism of liver macrophages plays a critical role in shaping hepatocellular carcinoma progression and immune modulation. Targeting macrophage metabolism to counteract immune suppression presents a promising avenue for hepatocellular carcinoma treatment. Herein, we found that the ketogenesis gene OXCT1 was highly expressed in tumor-associated macrophages (TAMs) and promoted tumor growth by reprogramming TAMs toward a protumor phenotype. Pharmacological targeting or genetic downregulation of OXCT1 in TAMs enhances antitumor immunity and slows tumor growth. Our results suggest that suppressing OXCT1 activity in TAMs could be an effective approach for treating liver cancer.

Seedless black Vitis vinifera polyphenols suppress hepatocellular carcinoma in vitro and in vivo by targeting apoptosis, cancer stem cells, and proliferation

Hepatocellular carcinoma (HCC) is an aggressive tumor and one of the most challenging cancers to treat. Here, we evaluated the in vitro and in vivo ameliorating impacts of seedless black Vitis vinifera (VV) polyphenols on HCC. Following the preparation of the VV crude extract (VVCE) from seedless VV (pulp and skin), three fractions (VVF1, VVF2, and VVF3) were prepared. The anticancer potencies of the prepared fractions, compared to 5-FU, were assessed against HepG2 and Huh7 cells. In addition, the effects of these fractions on p-dimethylaminoazobenzene-induced HCC in mice were evaluated. The predicted impacts of selected phenolic constituents of VV fractions on the activity of essential HCC-associated enzymes (NADPH oxidase “NADPH-NOX2”, histone deacetylase 1 “HDAC1”, and sepiapterin reductase “SepR”) were analyzed using molecular docking. The results showed that VVCE and its fractions induced apoptosis and collapsed CD133+ stem cells in the studied cancer cell lines with an efficiency greater than 5-FU. VVF1 and VVF2 exhibited the most effective anticancer fractions in vitro; therefore, we evaluated their influences in mice. VVF1 and VVF2 improved liver morphology and function, induced apoptosis, and lowered the fold expression of various crucial genes that regulate cancer stem cells and other vital pathways for HCC progression. For most of the examined parameters, VVF1 and VVF2 had higher potency than 5-FU, and VVF1 showed more efficiency than VVF2. The selected phenolic compounds displayed competitive inhibitory action on NADPH-NOX2, HDAC1, and SepR. In conclusion, these findings declare that VV polyphenolic fractions, particularly VVF1, could be promising safe anti-HCC agents.

Deficiency of betaine-homocysteine methyltransferase activates glucose-6-phosphate dehydrogenase (G6PD) by decreasing arginine methylation of G6PD in hepatocellular carcinogenesis.

Betaine-homocysteine methyltransferase (BHMT) regulates protein methylation and is correlated with tumorigenesis; however, the effects and regulation of BHMT in hepatocarcinogenesis remain largely unexplored. Here, we determined the clinical significance of BHMT in the occurrence and progression of hepatocellular carcinoma (HCC) using tissue samples from 198 patients. BHMT was to be frequently found (86.6%) expressed at relatively low levels in HCC tissues and was positively correlated with the overall survival of patients with HCC. Bhmt overexpression effectively suppressed several malignant phenotypes in hepatoma cells in vitro and in vivo, whereas complete knockout of Bhmt (Bhmt-/-) produced the opposite effect. We combined proteomics, metabolomics, and molecular biological strategies and detected that Bhmt-/- promoted hepatocarcinogenesis and tumor progression by enhancing the activity of glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolism in DEN-induced HCC mouse and subcutaneous tumor-bearing models. In contrast, restoration of Bhmt with an AAV8-Bhmt injection or pharmacological inhibition of G6PD attenuated hepatocarcinogenesis. Additionally, coimmunoprecipitation identified monomethylated modifications of the G6PD, and BHMT regulated the methylation of G6PD. Protein sequence analysis, generation and application of specific antibodies, and site-directed mutagenesis indicated G6PD methylation at the arginine residue 246. Furthermore, we established bidirectionally regulated BHMT cellular models combined with methylation-deficient G6PD mutants to demonstrate that BHMT potentiated arginine methylation of G6PD, thereby inhibiting G6PD activity, which in turn suppressed hepatocarcinogenesis. Taken together, this study reveals a new methylation-regulatory mechanism in hepatocarcinogenesis owing to BHMT deficiency, suggesting a potential therapeutic strategy for HCC treatment.

Chemically engineered mTOR-nanoparticle blockers enhance antitumour efficacy

Hepatocellular carcinoma (HCC) is a highly prevalent and deadly type of cancer, and although pharmacotherapy remains the cornerstone of treatment, therapeutic outcomes are often unsatisfactory. Pharmacological inhibition of mammalian target of rapamycin (mTOR) has been closely associated with HCC regression. Herein, we covalently conjugated AZD8055, a potent mTORC1/2 blocker, with a small panel of unsaturated fatty acids via a dynamically activating linkage to enable aqueous self-assembly of prodrug conjugates to form mTOR nanoblockers. Cell-based experiments were carried out to evaluate the effects of the nanoblocker against hepatocellular carcinoma (HCC) cells. The orthotopic and subcutaneous HCC mouse models were established to examine its antitumour activity. Among several fatty acids as promoieties, linoleic acid-conjugated self-assembling nanoblocker exhibited optimal size distribution and superior physiochemical properties. Compared with free agents, PEGylated AZD8055 nanoblocker (termed AZD NB) was pharmacokinetically optimized after intravenous administration. Invivo investigations confirmed that AZD NB significantly suppressed tumour outgrowth in subcutaneous HCCLM3 xenograft, Hepatoma-22, and orthotopic Hepa1-6 liver tumour models. Strikingly, treatment with AZD NB, but not free agent, increased intratumour infiltration of IFN-γ+CD8+ T cells and CD8+ memory T cells, suggesting a potential role of the mTOR nanoblocker to remodel the tumour microenvironment. Overall, a single conjugation with fatty acid transformed a hydrophobic mTOR blocker into a systemically injectable nanomedicine, representing a facile and generalizable strategy for improving the therapeutic index of mTOR inhibition-based cancer therapy. The mTOR inhibition by chemically engineered nanoblocker presented here had enhanced efficacy against tumours compared with the pristine drug and thus has the potential to improve the survival outcomes of patients with HCC. Additionally, this new nanosystem derived from co-assembling of small-molecule prodrug entities can serve as a delivery platform for the synergistic co-administration of distinct pharmaceutical agents. This work was supported by the National Natural Science Foundation of China (32171368,81721091), the Zhejiang Provincial Natural Science Foundation of China (LZ21H180001), the Jinan Provincial Laboratory Research Project of Microecological Biomedicine (JNL-2022039c and JNL-2022010B), State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (zz202310), and Natural Science Foundation of Shandong Province (ZR2023ZD59).

Sustainable production of extracellular polymeric substances and iron or copper complex from glutinous rice processing wastewater

Essential trace minerals play vital roles in maintaining human and animal health. However, an overdose of the existing inorganic trace minerals is prone to induce detrimental effects that outweigh positive benefits. In this study, an extracellular polymeric substances (EPS)-producing bacterium, identified as Bacillus licheniformis CCTCC M2020298, was isolated from marine using glutinous rice processing wastewater as enrichment medium. The EPS yield of Bacillus licheniformis CCTCC M2020298 could reach 8.62 g/L by using glutinous rice-processing wastewater containing medium. Furthermore, the potential of the EPS as a carrier for synthesizing EPS-iron (Fe) and EPS-copper (Cu) complex was explored. The results showed that the optimum condition for the synthesis EPS-Fe were the reaction temperature 70°C, pH 8.5–9.0 and mass ratio of EPS to trisodium citrate 2:1. The iron content of EPS-Fe reached 77.4 mg/g. Under the same condition, the copper content of EPS-Cu reached 90.7 mg/g. The elemental composition, functional groups and valence state of the mineral elements of EPS-Fe and EPS-Cu were well characterized. The EPS-Fe and EPS-Cu exhibited antioxidant activity in scavenging ·OH, DPPH and ·O2− free radicals, thereby leading to reduced oxidative stress and apoptosis levels in human colonic epithelial cells in vitro. They also inhibited the proliferation of mouse hepatocellular carcinoma H22 and the growth of intestinal pathogens in vitro. This study provided an effective avenue for EPS production from glutinous rice processing wastewater and proved the potential of EPS-Fe and EPS-Cu complexes as a new-type comprehensive essential trace mineral supplement.

Proteomic analysis of DEN and CCl4-induced hepatocellular carcinoma mouse model.

Hepatocellular carcinoma (HCC) seriously threatens human health, mostly developed from liver fibrosis or cirrhosis. Since diethylnitrosamine (DEN) and carbon tetrachloride (CCl4)-induced HCC mouse model almost recapitulates the characteristic of HCC with fibrosis and inflammation, it is taken as an essential tool to investigate the pathogenesis of HCC. However, a comprehensive understanding of the protein expression profile of this model is little. In this study, we performed proteomic analysis of this model to elucidate its proteomic characteristics. Compared with normal liver tissues, 432 differentially expressed proteins (DEPs) were identified in tumor tissues, among which 365 were up-regulated and 67 were down-regulated. Through Gene Ontology (GO) analysis, Ingenuity Pathway Analysis (IPA), protein-protein interaction networks (PPI) analysis and Gene-set enrichment analysis (GSEA) analysis of DEPs, we identified two distinguishing features of DEN and CCl4-induced HCC mouse model in protein expression, the upregulation of actin cytoskeleton and branched-chain amino acids metabolic reprogramming. In addition, matching DEPs from the mouse model to homologous proteins in the human HCC cohort revealed that the DEN and CCl4-induced HCC mouse model was relatively similar to the subtype of HCC with poor prognosis. Finally, combining clinical information from the HCC cohort, we screened seven proteins with prognostic significance, SMAD2, PTPN1, PCNA, MTHFD1L, MBOAT7, FABP5, and AGRN. Overall, we provided proteomic data of the DEN and CCl4-induced HCC mouse model and highlighted the important proteins and pathways in it, contributing to the rational application of this model in HCC research.

Rspo2-LGR4 exacerbates hepatocellular carcinoma progression via activation of Wnt/β-catenin signaling pathway

BackgroundThe leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) plays an important role in stem cell differentiation, organ development and cancer. Whether LGR4 affects the progression of hepatocellular carcinoma (HCC) remains unknown. This study aimed to reveal the role of LGR4 in HCC. MethodsClinical samples of HCC were collected to assess the expression of LGR4 and its correlation with patients’ clinical characteristics. The expression level of LGR4 in HCC cells was altered by pharmacological and genetic methods, and the role of LGR4 in HCC progression was analyzed by in vivo and in vitro assays. HCC was induced by diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) in wild-type and LGR4 deficient mice, the effect of LGR4 on HCC was examined by histopathological evaluation and biochemical assays. ResultsLGR4 expression was up-regulated in HCC samples, and its expression level was positively correlated with tumor size, microvascular invasion (MVI), TNM stage and pathological differentiation grade of HCC patients. In the mouse HCC model induced by DEN+CCl4, knockdown of LGR4 effectively inhibited the progression of HCC. Silencing of LGR4 inhibited the proliferation, migration, invasion, stem cell-like properties and Warburg effect of HCC cells. These phenotypes were promoted by R-spondin2 (Rspo2), an endogenous ligand for LGR4. Rspo2 markedly increased the nuclear translocation of β-catenin, whereas IWR-1, an inhibitor of Wnt/β-catenin signaling, reversed its effect. Deficiency of LGR4 significantly reduced the nuclear translocation of β-catenin and the expression of its downstream target genes cyclinD1 and c-Myc. ConclusionsLGR4 promotes HCC progression via Wnt/β-catenin signaling pathway.

Analysis of the Upregulated Expression Mechanism of Apoptotic Chromatin Condensation Inducer 1 in Hepatocellular Carcinoma Based on Bioinformatics.

A large number of differentially expressed molecules exist in hepatocellular carcinoma (HCC), and the mechanism by which they upregulate or downregulate expression is still unclear. The purpose of this study is to explore the possible mechanism of differential expression of apoptotic chromatin condensation inducer 1 (Acin1) in HCC. A mouse HCC model was constructed, and the expression of Acin1 in HCC was analyzed by whole transcriptome sequencing, bioinformatics analysis, and reverse transcription-quantitative polymerase chain reaction, and differentially expressed Acin1-related genes were screened to construct a protein-protein interaction and competing endogenous RNA (ceRNA) network. The microRNA (miRNAs) targeting Acin1 were further predicted using online databases and finally compared with sequencing data. The expression of Acin1 was significantly up-regulated in HCC compared to the paracancerous and healthy control groups (P<.001). The top 10 upregulated genes closely related to Acin1 (Slc3a2, Wiz, Srrm2, Akt1, Hnrnpu, Sap18b, Pabpn1, Ddx39b, Eif4a3, and Rnps1) were mainly involved in pathways such as messenger RNA (mRNA) surveillance, RNA transport, spliceosome, Janus kinase/ signal transducers and activators of transcription signaling, apoptosis, and ubiquitin-mediated proteolysis. The ceRNA network identified several molecules (2 long noncoding RNAs, 50 miRNAs, and 49 mRNAs) interacting with Acin1, among which miR-674-5p was highly expressed in all sample tissues, and higher than that of other differentially expressed miRNAs, and significantly downregulated in HCC. Multiple online databases such as miRWalk also predicted that miR-674-5p targets Acin1. This shows that miR-674-5p may be an important molecule for targeting Acin1. Acin1 is overexpressed in HCC, and the overexpressed Acin1 is most likely regulated by miR-674-5p and other ceRNA molecules.

Abstract 5335: Immune cell population variations in the livers of commonly used mouse strains

Abstract Previous studies have shown that the distribution of major immune cell populations differ across mouse strains in peripheral blood and hematopoietic organs at baseline. Additionally, variations in immune responses exist between mouse strains in Leishmania spp. and Melanoma models, but these studies in the context of liver diseases including Non Alcoholic Fatty Liver Disease (NAFLD) and Hepatocellular Carcinoma (HCC) have not been carried out. With the effort to state if these variations exist within the liver, we performed comprehensive spectral flow cytometry analysis on the liver and spleens of three female mouse strains commonly used in cancer research (BALB/c, C57BL/6, and FVBN). NAFLD was induced by providing mice with a methionine choline deficient diet. To promote HCC, a single genotype (myc/p53+/-) was induced by administering transposon-based vectors to overexpress Myc-Luc and CRISPR/Cas-9 vectors to knock down p53 via hydrodynamic tail-vein injection. To confirm vector integration and monitor tumor growth (luciferase activity), in vivo imaging was performed at different time points. As a result, we confirm throughout this study that differences exist within immune cell populations of the liver and spleen at baseline. Moreover, we depict differences in the shift of immune cell populations across mouse strains in the pathologic states included. In NAFLD, there is an increase of dendritic cells (DCs), macrophages, NK cells, ILC1s, T regulatory cells (Tregs), TH1, and TH2, and a decrease of CD4+ T cells, B cells, γδ-T cells, and NKT cells in the liver across all mouse strains, when compared to controls. However, the population of neutrophils in BALB/c decreases, and the population of CD8+ T cells increases in C57BL/6 mice, which differs from the patterns of change seen in the other mouse strains. Moreover, in spleens, NAFLD promotes an increase of TH1 and NK cells, and a decrease of TH2 and NKT Cells across the mouse strains included in the study, whereas the patterns of change differ between mouse strains among the remaining immune cell types. When evaluating liver immune cell population shifts on our HCC model, BALB/c mice showed a decrease in macrophages and ILC1s, C57BL/6 mice showed a decrease in γδ-T cells, prominent shifts across Neutrophils, TH1, and NKT cells, and null changes in TH2 and CD4+ T cell shifts, and FVBN mice had a less prominent decrease of B cells, which differed from the shifts observed in the mouse strains used for comparison. Immune cell populations in the spleens of our HCC mouse model also presented with differences across the mouse strains studied. In conclusion, we highlight that key differences in immune profiles exist across commonly used mouse strains for cancer research in the presence of liver pathology including HCC, suggesting that mouse strains could be possible confounders in the study of immune oncology of HCC. Citation Format: Francisco J. Rodriguez-Matos, Patrick Huang, Rajiv Trehan, Benjamin Ruf, Chi Ma, Tim F. Greten. Immune cell population variations in the livers of commonly used mouse strains [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5335.

Abstract 6755: NKT activation enhanced the therapeutic efficacy of neoantigen vaccine in hepatocellular carcinoma

Abstract One sentence introduction: This study indicated the potential of combinational therapy of neoantigen vaccine and iNKT activator α-GalCer in Hepatocellular carcinoma. Hepatocellular carcinoma (HCC) is a leading cause of death around the world. Current immune checkpoint blockade therapies only show effectiveness in some cancer patients, so there is an urgent need for more effective immunotherapy strategies. One alternative is the use of neoantigen-based cancer vaccines, which target tumor-specific antigens (neoantigens) on cancer cells and induce strong T-cell responses. Advances in technology now allow for precise identification of tumor neoantigens using bioinformatic analysis or mass spectrometry. While cancer vaccines have demonstrated strong anti-tumor immune responses and therapeutic effectiveness in some cancer types, their success in HCC remains limited. A contributing factor is the suppressive tumor microenvironment (TME) found in advanced HCC. Some studies suggest that combining neoantigen vaccines with anti-PD1/PD-L1 therapies can significantly enhance HCC treatment outcomes. Overcoming the suppressive immune microenvironment is crucial in cancer treatment, and reactivating intratumoral immunity is essential. In our study, we developed an HCC cancer vaccine using transcriptome sequencing and bioinformatics analysis. Our ELISpot assay results showed that this vaccine induced strong tumor-specific immune responses and significantly inhibited tumor growth in early-stage orthotopic HCC mouse models when administered five days after tumor inoculation. However, the effectiveness decreased in advanced tumors when administered ten days after tumor inoculation. We also discovered a significant relationship between the therapeutic effectiveness of the neoantigen vaccine and the activation of intratumoral NKT cells in HCC. Our study suggested that the type-I NKT cell activator, α-Galactosylceramide (α-GalCer), significantly improved the therapeutic outcomes of the cancer neoantigen vaccine in advanced orthotopic HCC mouse models. In some cases, tumors were almost completely eliminated. This indicates the potential of combining iNKT activators with neoantigen vaccines for HCC treatment. To further understand the role of NKT cells in HCC's TME, we plan to investigate intratumoral NKT cell subtypes following neoantigen vaccine therapy. Additionally, we will develop a new delivery system to deliver type I NKT cell activators and type II NKT inhibitors to tumor sites, aiming to reactivate the suppressive TME in HCC after neoantigen vaccine therapy. Citation Format: Renhao Li, Jing-Chu Hu, Li Rong, Yangfan Wu, Yige He, Jiandong Huang. NKT activation enhanced the therapeutic efficacy of neoantigen vaccine in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6755.

Abstract 5927: ACBP/DBI inhibition suppresses NASH-induced hepatocarcinogenesis

Abstract Background: Non-alcoholic fatty liver disease (NAFLD) or its severe form non-alcoholic steatohepatitis (NASH) is becoming an ever more prevalent risk factor of hepatocellular carcinoma (HCC). Currently, there are no approved drugs for the treatment of NASH. Extracellular ACBP/DBI (Acyl coenzyme A binding protein, also called diazepam-binding inhibitor) acts as an autophagy checkpoint and neutralization of ACBP/DBI prevents NASH. Therefore, we investigated the functional roles, mechanisms, and clinical relevance of ACBP/DBI in HCC. Methods: ACBP/DBI was blocked by four strategies: an inducible Acbp/Dbi knockout (Acbp-/-, control: Acbp+/+), a constitutive Gabrg2F77I mutation that prevents binding of ACBP/DBI to the receptor (control: WT), induction of ACBP/DBI-specific autoantibodies (induced by KLH-DBI, control: KLH), and monoclonal anti-DBI antibody (control: isotype). Three different types of HCC mouse models, including toxins/diet-based NASH-induced models (western diet plus CCl4), hydrodynamic transfection of oncogenes (Myc+Ctnnb1), and orthotopic transplantation (Hep55.1C) were used in combination with the aforementioned ACBP/DBI inhibition strategies to determine the association of ACBP/DBI and HCC. Results: ACBP/DBI inhibition attenuates tumorigenesis in mice with NASH, blunts oncogene induced HCC, and reduces the growth of orthotopically transplanted cancers. Mechanistically, ACBP/DBI inhibition retards cell cycle, suppresses cell proliferation, and favors ferroptosis, as determined by RNA-seq data and mass spectrometric metabolomics. CCK-8 proliferation, colony formation, and cell cycle assays performed on Huh-7, HepG2 and Hep55.1C cells demonstrated that ACBP/DBI knockdown reduced cell proliferation and induced G0/G1 arrest. IHC detection of Ki67 and PCNA in liver sections revealed antiproliferative effects of ACBP/DBI inhibition. qRT-PCR demonstrated the ACBP/DBI inhibition-induced upregulation of cell cycle inhibitor genes and downregulation of genes invoded in G0/G1 phase in liver tissues. qRT-PCR and immunoblot confirmed the upregulation of pro-ferroptotic and the downregulation of anti-ferroptotic genes and proteins in liver tissues upon ACBP/DBI inhibition. Primary HCC cell lines from Acbp+/+ and Acbp-/- mice further confirmed the pro-ferroptotic effects of ACBP/DBI inhibition obtained in liver tissues. Acbp-/- cell lines were more sensitivity to RSL3-induced ferroptosis. Conclusion: ACBP/DBI inhibition constitutes a potential strategy for treating NASH and NASH-induced HCC. Citation Format: Sijing Li, Omar Motiño, Flavia Lambertucci, Maria Chiara Maiuri, Isabelle Martins, Guido Kroemer. ACBP/DBI inhibition suppresses NASH-induced hepatocarcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5927.

Abstract 5447: Microtubule-associated protein MAP2 promotes drug resistance and cancer stemness in hepatocellular carcinoma through integrin dysregulation

Abstract Integrins are adhesion molecules that mediate mechanical and chemical signal transduction pathways to support cell survival and proliferation. Dysregulated integrin signaling empowers tumor cells to drive oncogenic stemness functions, including tumor initiation, epithelial plasticity, metastatic reactivation, and resistance to therapies. However, the interplay between cancer stemness and integrin signaling in hepatocellular carcinoma (HCC) remains poorly understood. HCC cells marked by CD133 represent an important functional subset of HCC tumors, displaying a dedifferentiated status with stem cell traits. In this study, transcriptome profiling reveals specific downregulation of integrin α family genes and integrin signaling in ‘HCC’ CD133+ cells isolated from NRAS+AKT-driven HCC, but not epithelial-specific ‘normal’ CD133+ cells isolated from regenerating liver. Of note, one of the most differentially upregulated genes identified in CD133+ HCC cell profiling, microtubule-associated protein MAP2, demonstrates the ability to suppress integrin expression. MAP2 overexpression is frequently observed in HCC and correlates with aggressive clinical and stemness features, including survival, tumor stage, and stemness signatures. Epigenetic modifications by H3K27Ac contribute to MAP2 upregulation. Functionally, MAP2 promotes cancer stemness and cell invasion, and confers resistance to the targeted therapy sorafenib. Mechanistically, the inhibition of collagen-binding integrin α subunits by MAP2 subsequently ablates integrin β1-mediated cell adhesion and FAK signaling. Estramustine Phosphate (EMP), previously reported to inhibit the interaction of MAP2 with actin filaments, attains a synergistic effect in suppressing tumor initiation and growth of HCC cell lines, HCC patient-derived organoids and NRAS+AKT HCC mouse model when used in combination with sorafenib. In summary, MAP2 inhibition may represent a potential novel therapy for HCC by targeting its cancer stemness roots and altering integrin signaling. Ongoing work is focused on the study of MAP2 regulation of integrin signaling and cell behaviours in the maintenance of a more stemness state in HCC. Citation Format: Ut Kei Lou, Ka-Hei Lam, Huajian Yu, Jia Jian Loh, Ki-Fong Man, Lei Zhou, Yuan Gao, Tin-Lok Wong, Cheng-Han Yu, Stephanie Ma. Microtubule-associated protein MAP2 promotes drug resistance and cancer stemness in hepatocellular carcinoma through integrin dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5447.