Mouse Model Of Hepatocellular Carcinoma Research Articles

Personalized neoantigen vaccine combined with PD-1 blockade increases CD8+ tissue-resident memory T-cell infiltration in preclinical hepatocellular carcinoma models

BackgroundPersonalized neoantigen vaccine could induce a robust antitumor immune response in multiple cancers, whose efficacy could be further enhanced by combining with programmed cell death 1 blockade (α-PD-1). However, the...

756P Glypican-3 (GPC3) and NKp46 directed FLEX-NK engager antibody (CYT-303) recruits natural killer (NK) cells to tumors in a preclinical hepatocellular carcinoma (HCC) mouse model

GPC3 is an oncofetal antigen that is highly expressed in HCC while minimally expressed in adult normal tissues except placenta. CYT-303 is a multifunctional bispecific NK engager targeting NK cell activating receptor NKp46 and GPC3 expressed on tumor cells constructed with Cytovia’s Flex-NK scaffold with a fully functional Fc. Previously, we reported CYT-303 in vitro treatment of Hep3B tumor cells demonstrated cytolysis and cytokine production as well as in vivo tumor growth inhibition when combined with both PBNK and iNK cells. Here we further characterized the CYT-303 dose-response for tumor growth inhibition in vivo and its influence on NK cell distribution. CYT -303 pharmacokinetics was evaluated in tumor-bearing mice. Anti-tumor efficacy and dose response of CYT-303 in the presence of human PBNKs or iNKs was evaluated in NSG-IL15 mice bearing subcutaneous Hep3B tumors. Biodistribution of the NK cells were analyzed by flow cytometry. CYT-303 pharmacokinetics in tumor bearing mice was similar to normal mice suggesting no target mediated clearance. In the Hep3B tumor model in PBNK or iNK injected NSG-hIL-15 mice, CYT-303 showed dose dependent tumor growth inhibition compared to control hIgG1 treated mice. Consistent with CYT-303 tumor growth inhibition, increased NK cells were detected in the tumor compared to control animals. Blood NK cells in CYT-303 treated animals were significantly lower compared to IgG1 isotype control treated mice suggesting CYT-303 could facilitate NK cell trafficking from blood into the tumor. Blood AFP (alpha fetoprotein), a biomarker in HCC, decreased with CYT-303 tumor growth inhibition. Pharmacologically active CYT-303 doses were identified. CYT-303 can help recruit NK cells to the tumor site to achieve tumor growth inhibition.

Non-invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Non-invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Non-invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma.

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1α expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Immune modulation by molecularly targeted photothermal ablation in a mouse model of advanced hepatocellular carcinoma and cirrhosis

Immunotherapy is a promising new treatment approach for hepatocellular carcinoma (HCC), but there are numerous barriers to immunotherapy in HCC, including an immunosuppressive microenvironment and the “immunotolerance” of the liver. Hyperthermia treatment modalities are standard of care for early stage HCC, and hyperthermia is known to have immunomodulatory effects. We have developed a molecularly targeted photothermal ablation (MTPA) technology that provides thermally tunable, tumor-specific heat generation. The purpose of this study was to evaluate the morphologic and immunologic effects of MTPA in an immunotherapy-resistant syngeneic mouse model of HCC in a background of toxin-induced cirrhosis. We found that the anatomic, cellular, and molecular features of this model recapitulate the characteristics of advanced human HCC. MTPA as a monotherapy and in combination with immune checkpoint therapy significantly increased intratumoral CD3+ and activated CD8+ T cells while decreasing regulatory T cells relative to control or immune checkpoint therapy alone based on immunohistochemistry, flow cytometry, and single cell RNA sequencing data. Furthermore, we identified evidence of MTPA’s influence on systemic tumor immunity, with suppression of remote tumor growth following treatment of orthotopic tumors. The results of this study suggest that tumor-specific hyperthermia may help overcome resistance mechanisms to immunotherapy in advanced HCC.

N-acetyl-galactosamine modified metal-organic frameworks to inhibit the growth and pulmonary metastasis of liver cancer stem cells through targeted chemotherapy and starvation therapy

Hepatocellular carcinoma (HCC) is a common high-mortality malignancy which still needs efficient treatments. HCC patients undergoing extrahepatic metastases are mostly with unsatisfactory prognosis. Therefore, specific attention has been paid to extrahepatic HCC metastasis. We integrated Sorafenib (Sor) and glucose oxidase (GOx) into a N-acetyl-galactosamine (GalNAc) modified zeolitic imidazolate framework (ZIF-8), designated as SG@GR-ZIF-8, for targeted bimodal therapies of chemotherapy and starvation therapy against HCC. The hepatic delivery of SG@GR-ZIF was mediated by the specific recognition of GalNAc residues with asialoglycoprotein (ASGPR) on the liver cell surface. Sor is a clinically approved anti-proliferation and anti-angiogenesis drug for advanced HCC treatment. GOx can efficiently induce cell death and disturb malignant progression by suppressing glucose supply of cancer cells, which is highly associated with metabolic rewiring in metastasis. The nano-formulation exhibit significant anti-metastatic HCC activity against C5WN1 cells, a liver cancer stem cell-like cell line with tumorigenicity and pulmonary metastasis activity. In a subcutaneous C5WN1-tumor carrying mouse model, SG@GR-ZIF exhibits potent synergistic anti-tumor activity with a tumor inhibition rate of 89% and a prolonged survival status. The growth and pulmonary metastasis of HCC in an orthotopic mouse model of HCC was remarkably suppressed in SG@GR-ZIF treated group. The therapeutic strategy targeting energy supply combined with first-line treatment holds great promise for the future treatment of metastatic HCC. Statement of significanceSG@GR-ZIF, a N-acetyl-galactosamine modified metal-organic framework carrying Sorafenib and glucose oxidase, was fabricated for treating metastatic hepatocellular carcinoma (HCC). Sorafenib is an anti-proliferation and anti-angiogenesis drug for advanced HCC treatment. Glucose oxidase blocks energy demand in HCC metastasis by depleting glucose. C5WN1 was used for therapeutic evaluations as a liver cancer stem cell-like cell line with tumorigenicity and pulmonary metastasis activity. In subcutaneous C5WN1-tumor bearing mice, SG@GR-ZIF exhibited a tumor inhibition rate of 89% and prolonged survival period. In orthotopic C5WN1-tumor carrying mice, the growth and pulmonary metastasis of hepatocarcinoma was remarkably suppressed by SG@GR-ZIF. Together, this study suggests the great potential of synergistic chemo/starvation therapy mediated by SG@GR-ZIF for treating metastatic HCC.

Multimodal Imaging Response after the Singular or Combination Treatments of Vascular Endothelial Growth Factor Inhibitor and Immune Checkpoint Inhibitor.

This study aims to dynamically assess tumor changes after variable treatments with vascular endothelial growth factor (VEGF) inhibitor and/or immune checkpoint inhibitor (ICI) using multimodal imaging of MRI and 18F-FDG PET/CT in a hepatocellular carcinoma (HCC) mice model. Based on different treatments, 24 mice were randomly divided into four groups: control (isotype-matched IgG antibody 10 mg/kg), VEGF inhibitor (sorafenib 50 mg/kg), ICI (anti-PD-L1 antibody 10 mg/kg), and combination groups (sorafenib 50 mg/kg + anti-PD-L1 antibody 10 mg/kg). Quantitative imaging assessments, including volume transfer constant (Ktrans), apparent diffusion coefficient (ADC), lactate/choline ratio, and the maximum standardized 18F-FDG uptake value ratio of tumor to muscle (SUVtumor/SUVmuscle ratio), were acquired at different time points (before treatment and 7, 14, and 21 days after treatment). Quantitative data were presented as the mean ± standard errors and two-way repeated-measure ANOVA tests were performed for intergroup and intertime point comparisons. After 21 days from the initiation of therapies, combination group showed the lowest tumor volume and weight, followed by ICI, VEGF inhibitor, and control group, with no significance between the VEGF inhibitor and control groups. In addition, Ktrans values significantly decreased, and the lactate/choline ratio and SUVtumor/SUVmuscle ratio were significantly elevated in the VEGF inhibitor group. ADC significantly increased in the ICI and combination groups, with no significant differences in ADC observed between the control and VEGF inhibitor groups, which showed a similar dynamic change to the tumor volume. Furthermore, Ktrans, lactate/choline ratio, and ADC were significantly correlated with CD31+ area, hypoxyprobe+ area, and apoptosis, respectively. Our results suggest that the singular treatment and combination of the VEGF inhibitor and ICI treatments for HCC present different multimodal imaging changes in accordance with the specific histopathological features. These findings might facilitate the formulation of better treatment response criteria; besides, we find ADC is probably an indicator easily to obtain for treatment response evaluation.

Intraperitoneal administration of thermosensitive hydrogel Co-loaded with norcantharidin nanoparticles and oxaliplatin inhibits malignant ascites of hepatocellular carcinoma

Malignant ascites is a common complication of some advanced cancers. Although intraperitoneal (IP) administration of chemotherapy drugs is routinely used to treat cancerous ascites, conventional drugs have poor retention and therefore need to be administered frequently to maintain a sustained anti-tumor effect. In this study, a thermosensitive hydrogel composite loaded with norethindrone nanoparticles (NPs) and oxaliplatin (N/O/Hydrogel) was developed to inhibit ascites of hepatocellular carcinoma (HCC) through IP injection. N/O/Hydrogel induced apoptosis in the H22 cells in vitro, and significantly inhibited ascites formation, tumor cell proliferation and micro-angiogenesis in a mouse model of advanced HCC with ascites, and prolonged the survival of tumor-bearing mice. Histological examination of the major organs indicated that the hydrogel system is safe. Taken together, the N/O/Hydrogel system is a promising platform for in-situ chemotherapy of malignant ascites.

Berberine inhibits carcinogenesis through antagonizing the ATX-LPA-LPAR2-p38-leptin axis in a mouse hepatoma model

Chemoprevention of hepatocellular carcinoma (HCC) is highly desirable in clinic. Berberine (BBR) is reported to play potential roles in cancer treatment and prevention. We studied the chemopreventive effect of BBR on hepatocellular carcinogenesis in an inflammation-driven mouse model, as it was enriched in liver after oral administration. Oral BBR significantly decreased the number and volume of visible nodular tumors, and prolonged the median overall survival by 9 and 8 weeks in the diethylnitrosamine (DEN)-injected male and female mice respectively. The nodular tumors were induced through activation of the lysophosphatidic acid (LPA) pathway in liver. LPA stimulated the abnormal leptin transcription through interacting with LPA receptor-2 (LPAR2) followed by p38 activation, and BBR inhibited carcinogenesis by suppressing the bioactivity of LPA. Specifically, BBR significantly reduced the expression of the LPA synthetase autotaxin (ATX) and LPAR2 in the nodular tumors of DEN-injected mice. Subsequently, BBR repressed the abnormal transcription of leptin stimulated by LPA-induced phosphorylation of p38 in hepatoma cells. In fact, BBR reduced the abnormal expression of leptin in livers of DEN-injected male mice throughout the course of an 8-month experiment. BBR might be a preventive agent for HCC, working at least partially through antagonizing the ATX-LPA-LPAR2-p38-leptin axis in liver.

Modulation of EphA7 and pEphA7 Protein Expression: Potential Biomarkers for Early Detection of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading drivers of cancer-related mortality in the world. As a result, researchers are constantly looking for ways to optimize the screening and diagnosis of the said malignancy. To establish the mice model of hepatocellular carcinoma with the administration of a suitable dose of diethylnitrosamine (DEN) and examine the utility of EphA7 and pEphA7 as ideal diagnostic markers in HCC. Swiss Albino (BALB/c) mice of around 10-12 weeks old were exposed to a known hepatocarcinogen-diethylnitrosamine at a dose of 20 mg/kg body weight at weekly intervals for a period of 4, 8, 12, & 16 weeks. Blood was collected from mice of different experimental groups, and age-matched control and serum were separated from whole blood samples. The liver homogenate was prepared after completion of treatment, and the resulting supernatant was used for enzyme assays. A range of liver biomarker enzyme assays such as Gamma-glutamyl transpeptidase (GGT), Acetylcholine esterase (AChE), GPx activity and GSH level, Heme oxygenase-1 (HO-1), GPC3 and alpha-fetoprotein (AFP) level along with the expression of Caspase-3, EphA7 and pEphA7 were evaluated. An elevation in body weight and relative liver weight across the treatment period (4, 8, 12, 16 weeks) was observed in DEN-treated mice. Significant differences in GGT levels between control and DEN treated mice were noted in the present study (P < 0.005). In the 16th week of the treatment period, a significant difference in AchE level was noted between the treated and control group (P < 0.001). However, there was no statistically significant difference in the levels of SGOT and SGPT levels between the control and DEN treated groups (P > 0.001). Lower GSH and GPx levels were demonstrated in the treated mice as compared to control over all the treatment period. Loss of Caspase-3 expression and significant differences in expression of HO-1 activity in treated vs. non-treated group of mice were observed. Significant differences in EphA7 and pEphA7 protein expression levels were noted in the DEN-treated vs. control groups across all the treatment periods (4 weeks: P < 0.05; 8 weeks: P < 0.05; 12 weeks: P < 0.005; 16 weeks: P < 0.05). The present study indicated that EphA7 and phosphoEphA7 over-expression might contribute to the malignancy transition, invasion development, and metastasis of HCC. As a result, along with the known markers such as AFP and others, EphA7 and pEphA7 could be a very putative biomarkers of HCC, particularly at a very early stage of cancer development.

Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) has been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like “off-target” toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment.MethodsThe peripheral blood was acquired from healthy donors and T cells were separated by density-gradient centrifugation. We used an electroporation system to deliver anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures as target genes into the T cells. The cell membrane was opened by the momentary electric current effect, and the target gene was delivered into the cell by non-viral minicircle DNA (mcDNA) vector. The flow cytometry and western blot assays were used to detect whether the two scFv were simultaneously transfected and the transfection efficacy of this bispecific CAR T cell generation method. We respectively detected the in vitro and in vivo tumor-suppression efficacy of CAR T cells through the CCK-8 assays and the HCC xenograft mice models. The CoG133-CAR T cells containing both CD133 and GPC3 antigen recognition sites were the effector cells. CD133-CAR T cells and GPC3-CAR T cells were defined as single-targeted control groups, normal T and mock T cells were defined as blank control groups.ResultsThe mcDNA vector accommodated two target gene structures successfully transfected to generate bispecific CAR T cells. The detection methods on gene level and protein level confirmed that CoG133-CAR T cells had considerable transfection efficiency and exhibited both antigen-binding capacity of CD133 and GPC3. Compared to single-targeted CAR T cells or control T cells, CoG133-CAR T cells performed enhanced eliminated efficacy against CD133 and GPC3 double-positive HCC cell line in vitro and HCC xenograft mice in vivo. Hematoxylin and eosin (H&E) staining indicated no fatal “off-target” combination existed on CoG133-CAR T cells and major organs.ConclusionOur study suggests that it is with higher efficiency and more safety to prepare bispecific CAR T cells through non-viral mcDNA vectors. CoG133-CAR T cells have enhanced tumor-suppression capacity through dual antigen recognition and internal activation. It provides an innovative strategy for CAR T therapy of HCC, even solid tumors.

CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis.

BackgroundThe shortening of 3’ untranslated regions (3’UTRs) of messenger RNAs(mRNAs) by alternative polyadenylation (APA) is an important mechanism for oncogene activation. Cleavage stimulation factor 2 (CSTF2), an important regulator of APA, has been reported to have a tumorigenic function in urothelial carcinoma of the bladder and lung cancers. However, the tumor-promoting role of CSTF2 in hepatocellular carcinoma (HCC) and its underlying molecular mechanism remains unclear.MethodsMultiple databases were used to analyze the expression level and prognostic value of CSTF2 in HCC. Function enrichment analysis was used to investigate the molecular mechanism of CSTF2 for the occurrence and development of HCC. The biological function in HCC cell lines in vitro was determined by CCK8, colony formation, Transwell migration, and invasion assay. Moreover, the tumorigenic function of CSTF2 in vivo was measured by a subcutaneous tumor formation or injecting four plasmids into a mouse tail vein within 5–7 s in an immunocompetent HCC mouse model. In addition, aerobic glycolysis in HCC cells was determined by measuring the extracellular acid rate (ECAR) and extracellular glucose and lactate levels.ResultsBioinformatics analysis revealed that CSTF2 was overexpressed in HCC tissues. The high expression of CSTF2 was correlated with a poor prognosis and high histological grades. CSTF2 knockout inhibited the proliferation, migration, and invasion of HCC cells. In addition, CSTF2 knockout HCC cells failed to form tumors by a subcutaneous graft experiment. Furthermore, endogenous CSTF2 knockout attenuated hepatocarcinogenesis in an immunocompetent HCC mouse model. Function enrichment analysis suggested that the high expression of CSTF2 was associated with enhanced glycolysis. Moreover, we found that CSTF2 knockout reduced the level of the short 3’ UTR isoform of hexokinase 2 and increased its level of long 3’UTR. Furthermore, CSTF2 knockout inhibited ECAR levels, glucose uptake, and lactate production.ConclusionOur results indicated that CSTF2 is highly expressed in HCC and is correlated with a poor prognosis and high histological grade. The knockout of CSTF2 inhibits the tumorigenesis and procession of HCC both in vitro and in vivo. Moreover, CSTF2 is associated with enhanced glycolysis. Therefore, this study suggests that CSTF2 might be a new prognostic biomarker and therapeutic target for HCC.

Investigation of Functional Synergism of CENPF and FOXM1 Identifies POLD1 as Downstream Target in Hepatocellular Carcinoma.

BackgroundLines of evidence implicate CENPF and FOXM1 may have novel co-operative roles in driving hepatocellular carcinoma (HCC).ObjectiveWe investigated the clinicopathological correlation, functional characterization, molecular mechanism and translational significance of CENPF and FOXM1.MethodsWe carried out integrative studies investigating functional synergism of CENPF and FOXM1 in HCC and its metastasis. Human HCC samples, HCC cell lines and mouse model were used in the studies. Stable knockdown, q-PCR, Western blotting, whole-transcriptomic sequencing (RNA-seq), as well as cell and mouse assays were performed.ResultsUpon clinicopathological correlation, we found that co-overexpression of CENPF and FOXM1 in human HCCs was associated with more aggressive tumor behavior including presence of venous invasion, tumor microsatellite formation, and absence of tumor encapsulation. Moreover, co-silencing FOXM1 and CENPF using shRNA approach in HCC cell lines resulted in significantly reduced cell proliferation. Furthermore, our RNA-seq and differential gene expression analysis delineated that CENPF and FOXM1 co-regulated a specific set of target genes in various metabolic processes and oncogenic signaling pathways. Among them, POLD1, which encodes the catalytic subunit of DNA polymerase δ, was ranked as the top downstream target co-regulated by CENPF and FOXM1. POLD1 expression was positively correlated with that of FOXM1 and CENPF in HCCs. In addition, POLD1 expression was significantly upregulated in HCC tumors. Functionally, in vivo orthotopic injection model showed that stable knockdown of POLD1 in HCC cells suppressed tumor incidence and tumorigenicity and had a trend of diminished lung metastasis.ConclusionTaken together, our data suggest that CENPF and FOXM1 could synergistically support hepatocarcinogenesis via the regulation of POLD1. CENPF and FOXM1 may represent new vulnerabilities to novel drug-based therapy in HCC.

Growth hormone receptor antagonism downregulates ATP-binding cassette transporters contributing to improved drug efficacy against melanoma and hepatocarcinoma in vivo

Knockdown of GH receptor (GHR) in melanoma cells in vitro downregulates ATP-binding cassette-containing (ABC) transporters and sensitizes them to anti-cancer drug treatments. Here we aimed to determine whether a GHR antagonist (GHRA) could control cancer growth by sensitizing tumors to therapy through downregulation of ABC transporters in vivo. We intradermally inoculated Fluc-B16-F10 mouse melanoma cells into GHA mice, transgenic for a GHR antagonist (GHRA), and observed a marked reduction in tumor size, mass and tumoral GH signaling. Moreover, constitutive GHRA production in the transgenic mice significantly improved the response to cisplatin treatment by suppressing expression of multiple ABC transporters and sensitizing the tumors to the drug. We confirmed that presence of a GHRA and not a mere absence of GH is essential for this chemo-sensitizing effect using Fluc-B16-F10 allografts in GH knockout (GHKO) mice, where tumor growth was reduced relative to that in GH-sufficient controls but did not sensitize the tumor to cisplatin. We extended our investigation to hepatocellular carcinoma (HCC) using human HCC cells in vitro and a syngeneic mouse model of HCC with Hepa1-6 allografts in GHA mice. Gene expression analyses and drug-efflux assays confirm that blocking GH significantly suppresses the levels of ABC transporters and improves the efficacy of sorafenib towards almost complete tumor clearance. Human patient data for melanoma and HCC show that GHR RNA levels correlate with ABC transporter expression. Collectively, our results validate in vivo that combination of a GHRA with currently available anti-cancer therapies can be effective in attacking cancer drug resistance.

Mouse Models of Hepatocellular Carcinoma: Classification, Advancement, and Application.

Hepatocellular carcinoma (HCC) is the subtype of liver cancer with the highest incidence, which is a heterogeneous malignancy with increasing incidence rate and high mortality. For ethical reasons, it is essential to validate medical clinical trials for HCC in animal models before further consideration on humans. Therefore, appropriate models for the study of the pathogenesis of the disease and related treatment methods are necessary. For tumor research, mouse models are the most commonly used and effective in vivo model, which is closer to the real-life environment, and the repeated experiments performed on it are closer to the real situation. Several mouse models of HCC have been developed with different mouse strains, cell lines, tumor sites, and tumor formation methods. In this review, we mainly introduce some mouse HCC models, including induced model, gene-edited model, HCC transplantation model, and other mouse HCC models, and discuss how to choose the appropriate model according to the purpose of the experiments.

Opposing roles of HDAC6 in liver regeneration and hepatocarcinogenesis

Histone deacetylase 6 (HDAC6), a deacetylase of p53, has emerged as a privileged inhibitory target for cancer therapy because of its deacetylating activity for p53 at K120 and K373/382. However, intricate roles of HDAC6 in hepatocellular carcinogenesis have been suggested by recent evidence, namely that HDAC6 ablation suppresses innate immunity, which plays critical roles in tumor immunosurveillance and antitumor immune responses. Therefore, it is valuable to determine whether HDAC6 ablation inhibits hepatocellular carcinogenesis using in vivo animal models. Here, we firstly showed that HDAC6 ablation increased K320 acetylation of p53, known as pro‐survival acetylation, in all tested animal models but did not always increase K120 and K373/382 acetylation of p53, known as pro‐apoptotic acetylation. HDAC6 ablation induced cellular senescence in primary MEFs and inhibited cell proliferation in HepG2 cells and liver regeneration after two‐thirds partial hepatectomy. However, the genetic ablation of HDAC6 did not inhibit hepatocarcinogenesis, but instead slightly enhanced it in two independent mouse models (DEN + HFD and DEN + TAA). Notably, HDAC6 ablation significantly promoted hepatocarcinogenesis in a multiple DEN treatment hepatocellular carcinoma (HCC) mouse model, mimicking chronic DNA damage in the liver, which correlated with hyperacetylation at K320 of p53 and a decrease in inflammatory cytokines and chemokines. Our data from three independent in vivo animal HCC models emphasize the importance of the complex roles of HDAC6 ablation in hepatocellular carcinogenesis, highlighting its immunosuppressive effects.

Caspase-3–Induced Activation of SREBP2 Drives Drug Resistance via Promotion of Cholesterol Biosynthesis in Hepatocellular Carcinoma

This study finds that cholesterol biosynthesis supports the expansion of cancer stem cell populations to drive resistance to tyrosine kinase inhibitor therapy in hepatocellular carcinoma, identifying potential therapeutic approaches for improving cancer treatment.

Gasdermin D-mediated release of IL-33 from senescent hepatic stellate cells promotes obesity-associated hepatocellular carcinoma.

Long-term senescent cells exhibit a secretome termed the senescence-associated secretory phenotype (SASP). Although the mechanisms of SASP factor induction have been intensively studied, the release mechanism and how SASP factors influence tumorigenesis in the biological context remain unclear. In this study, using a mouse model of obesity-induced hepatocellular carcinoma (HCC), we identified the release mechanism of SASP factors, which include interleukin-1β (IL-1β)- and IL-1β-dependent IL-33, from senescent hepatic stellate cells (HSCs) via gasdermin D (GSDMD) amino-terminal-mediated pore. We found that IL-33 was highly induced in senescent HSCs in an IL-1β-dependent manner in the tumor microenvironment. The release of both IL-33 and IL-1β was triggered by lipoteichoic acid (LTA), a cell wall component of gut microbiota that was transferred and accumulated in the liver tissue of high-fat diet-fed mice, and the release of these factors was mediated through cell membrane pores formed by the GSDMD amino terminus, which was cleaved by LTA-induced caspase-11. We demonstrated that IL-33 release from HSCs promoted HCC development via the activation of ST2-positive Treg cells in the liver tumor microenvironment. The accumulation of GSDMD amino terminus was also detected in HSCs from human NASH-associated HCC patients, suggesting that similar mechanism could be involved in a certain type of human HCC. These results uncover a release mechanism for SASP factors from sensitized senescent HSCs in the tumor microenvironment, thereby facilitating obesity-associated HCC progression. Furthermore, our findings highlight the therapeutic potential of inhibitors of GSDMD-mediated pore formation for HCC treatment.

Inhibition of anti-inflammatory pathway through suppressors of cytokine signalling (Socs2/Socs3) in the initiation of hepatocellular carcinoma

Hepatocellular carcinoma (HCC), a leading cause of cancer related deaths is predominantly driven by chronic inflammatory responses. Due to asymptomatic nature and lack of early patient biopsies, precise involvement of inflammation in hepatic injury initiation remains unidentified. Aim of the study was to elucidate the regulation patterns of inflammatory signalling from initiation of hepatic injury to development of HCC. HCC mice model was established using DEN followed by repeated doses of CCl4 and sacrificed at three different stages of disease comprising 7, 14 and 21 weeks. Serum biochemical tests, hepatic lipids quantification, histopathology and qPCR analyses were conducted to characterize the initiation and progression of liver injury and inflammatory signalling. Notably, at 7 weeks, we observed hepatocyte damage and periportal necrotic bodies coupled with induction of Socs2/Socs3 and anti-inflammatory cytokine Il-10. At 14 weeks, mice liver showed advancement of liver injury with micro-vesicular steatosis and moderate collagen deposition around portal zone. With progression of injury, the expression of Socs3 was declined with further reduction of Il-10 and Tgf-β indicating the disturbance of anti-inflammatory mechanism. In contrast, pro-inflammatory cytokines Il1-β, Il6 and Tnf-α were upregulated contributing inflammation. Subsequently, at 21 weeks severe liver damage was estimated as characterized by macro-vesicular steatosis, perisinusoidal collagen bridging, immune cell recruitment and significant upregulation of Col-1α and α-Sma. In parallel, there was significant upregulation of pro/anti-inflammatory cytokines highlighting the commencement of chronic inflammation.Findings of the study suggest that differential regulation of cytokine suppressors and inflammatory cytokines might play role in the initiation and progression of hepatic injury leading towards HCC.

Abstract 1654: Mouse models of hepatocellular carcinoma: A comprehensive and functional preclinical platform for immunotherapy research

Abstract Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and accounts for over 80% of primary liver cancer worldwide. Early stage HCC can be treated by local ablation, surgical resection or liver transplantation. Systemic pharmacological options are limited (kinase and immune-checkpoint inhibitors). Most cases of liver cancer occur in the setting of chronic liver diseases. Risk factors include chronic Hepatitis B and C, alcohol addiction and metabolic diseases. HCC is a multistep process comprising chronic liver injury, inflammation, fibrosis/cirrhosis and cancer formation. Therefore, providing palliative and curative options remains a high medical need. And with the recent success of immunotherapies in HCC, mouse models that better recapitulate the human disease and antitumor immune response are needed. In order to better evaluate new preventive and curative treatments of liver cancers we developed complementary and integrated strategies to mimic the liver cancer initiation and progression steps in mouse models. These models involve chemotoxic agents, diet-induced disorders and syngeneic or xenogeneic tumor implantation strategies. We established an orthotopic syngeneic model using Hepa1.6 mouse liver hepatoma cells, characterized through liver index, alpha-fetoprotein measurement in serum and liver, and MRI. The response to chemotherapy (sorafenib) and immunotherapy (anti PD-1, anti-TLR) was also assessed and show moderate to high efficiency. Moreover, a panel of xenograft models including Patient-Derived Xenograft models (PDXs) are available to assess new treatment options in human HCC with regards of the genetic mutations and the variety of etiologies seen in human. But as xenograft models are not completely mimicking the human situation of both immune and liver microenvironment, we have recently initiated the development of a double humanized (immune and liver) transgenic mouse as a better host for human tumor engraftment. During the course of HCC formation, the liver undergoes cycles of inflammation, necrosis with regeneration, fibrosis, cell dysplasia and ultimately HCC. Thus, we developed a model induced by a low dose of streptozotocin and high fat diet regimen. In this model, mice develop metabolic syndrome NASH and fibrosis within 12 weeks and HCC within 16 weeks. Of interest, 100% of male mice develop HCC within 16 weeks, in accordance with studies showing that men had a 2-to 7-fold higher risk of developing HCC in human. Treatment of mice with lenvatinib alone or in combination with anti-PD1 increases survival and reduces tumor burden as shown with reduced liver weight/body weight ratio at 16 weeks. Altogether, these results demonstrate the usefulness of this comprehensive platform of preclinical in vivo HCC models to discover and identify novel therapeutic strategies that could circumvent the progression of liver cancers. Citation Format: Olivier Duchamp, Gael Krysa, Loic Morgand, Hugo Quillery, Robin Artus, Maxime Ramelet, Jeremy Odillard, Peggy Provent, Sylvie Maubant, Caroline Mignard, Fabrice Viviani, Samira Benhamouche-Trouillet. Mouse models of hepatocellular carcinoma: A comprehensive and functional preclinical platform for immunotherapy research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1654.