Murine Hepatoma Cells Research Articles

AAV-mediated genome editing is influenced by the formation of R-loops

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3’ end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

Oncogenic accumulation of cysteine promotes cancer cell proliferation by regulating the translation of D-type cyclins

Malignant cells exhibit a high demand for amino acids to sustain their abnormal proliferation. Particularly, the intracellular accumulation of cysteine is often observed in cancer cells. Previous studies have shown that deprivation of intracellular cysteine in cancer cells results in the accumulation of lipid peroxides in the plasma membrane and induction of ferroptotic cell death, indicating that cysteine plays a critical role in the suppression of ferroptosis. Herein, we found that the oncogenic accumulation of cysteine also contributes to cancer cell proliferation by promoting the cell cycle progression, which is independent of its suppressive effect on ferroptosis. The growth ability of four types of cancer cells, including murine hepatocarcinoma cells, but not of primary hepatocytes, were dependent on the exogenous supply of cysteine. Deprivation of intracellular cysteine in cancer cells induced cell cycle arrest at the G0/G1 phase, accompanied by a decrease in the expression of cyclin D1 and D2 proteins. The cysteine deprivation-induced decrease in D-type cyclin expression was associated with the upregulation of eukaryotic translation initiation factor 4E binding protein (4E-BP1), which represses the translation of cyclin D1 and D2 proteins by binding to eukaryotic translation initiation factor 4E (eIF4E). Similar results were observed in hepatocarcinoma cells treated with erastin, an xCT inhibitor. These findings reveal an unappreciated role of cysteine in regulating the growth of malignant cancer cells and deepen our understanding of the cytotoxic effect of xCT inhibitor to prevent cancer cell proliferation.

Epigenetic repression of de novo cysteine synthetases induces intra-cellular accumulation of cysteine in hepatocarcinoma by up-regulating the cystine uptake transporter xCT

BackgroundThe metabolic reprogramming of amino acids is critical for cancer cell growth and survival. Notably, intracellular accumulation of cysteine is often observed in various cancers, suggesting its potential role in alleviating the oxidative stress associated with rapid proliferation. The liver is the primary organ for cysteine biosynthesis, but much remains unknown about the metabolic alterations of cysteine and their mechanisms in hepatocellular carcinoma cells.MethodsRNA-seq data from patients with hepatocarcinoma were analyzed using the TNMplot database. The underlying mechanism of the oncogenic alteration of cysteine metabolism was studied in mice implanted with BNL 1ME A.7 R.1 hepatocarcinoma.ResultsDatabase analysis of patients with hepatocellular carcinoma revealed that the expression of enzymes involved in de novo cysteine synthesis was down-regulated accompanying with increased expression of the cystine uptake transporter xCT. Similar alterations in gene expression have also been observed in a syngeneic mouse model of hepatocarcinoma. The enhanced expression of DNA methyltransferase in murine hepatocarcinoma cells caused methylation of the upstream regions of cysteine synthesis genes, thereby repressing their expression. Conversely, suppression of de novo cysteine synthesis in healthy liver cells induced xCT expression by up-regulating the oxidative-stress response factor NRF2, indicating that reduced de novo cysteine synthesis repulsively increases cystine uptake via enhanced xCT expression, leading to intracellular cysteine accumulation. Furthermore, the pharmacological inhibition of xCT activity decreased intracellular cysteine levels and suppressed hepatocarcinoma tumor growth in mice.ConclusionsOur findings indicate an underlying mechanism of the oncogenic alteration of cysteine metabolism in hepatocarcinoma and highlight the efficacy of alteration of cysteine metabolism as a viable therapeutic target in cancer.

Reprogramming of Glutamine Amino Acid Transporters Expression and Prognostic Significance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver malignancy and is a major cause of cancer-related mortality in the world. This study aimed to characterize glutamine amino acid transporter expression profiles in HCC compared to those of normal liver cells. In vitro and in vivo models of HCC were studied using qPCR, whereas the prognostic significance of glutamine transporter expression levels within patient tumors was analyzed through RNAseq. Solute carrier (SLC) 1A5 and SLC38A2 were targeted through siRNA or gamma-p-nitroanilide (GPNA). HCC cells depended on exogenous glutamine for optimal survival and growth. Murine HCC cells showed superior glutamine uptake rate than normal hepatocytes (p < 0.0001). HCC manifested a global reprogramming of glutamine transporters compared to normal liver: SLC38A3 levels decreased, whereas SLC38A1, SLC7A6, and SLC1A5 levels increased. Also, decreased SLC6A14 and SLC38A3 levels or increased SLC38A1, SLC7A6, and SLC1A5 levels predicted worse survival outcomes (all p < 0.05). Knockdown of SLC1A5 and/or SLC38A2 expression in human Huh7 and Hep3B HCC cells, as well as GPNA-mediated inhibition, significantly decreased the uptake of glutamine; combined SLC1A5 and SLC38A2 targeting had the most considerable impact (all p < 0.05). This study revealed glutamine transporter reprogramming as a novel hallmark of HCC and that such expression profiles are clinically significant.

Arsenic trioxide augments immunogenic cell death and induces cGAS-STING-IFN pathway activation in hepatocellular carcinoma

The treatment of hepatocellular carcinoma (HCC) is particularly challenging due to the inherent tumoral heterogeneity and easy resistance towards chemotherapy and immunotherapy. Arsenic trioxide (ATO) has emerged as a cytotoxic agent effective for treating solid tumors, including advanced HCC. However, its effectiveness in HCC treatment remains limited, and the underlying mechanisms are still uncertain. Therefore, this study aimed to characterize the effects and mechanisms of ATO in HCC. By evaluating the susceptibilities of human and murine HCC cell lines to ATO treatment, we discovered that HCC cells exhibited a range of sensitivity to ATO treatment, highlighting their inherent heterogeneity. A gene signature comprising 265 genes was identified to distinguish ATO-sensitive from ATO-insensitive cells. According to this signature, HCC patients have also been classified and exhibited differential features of ATO response. Our results showed that ATO treatment induced reactive oxygen species (ROS) accumulation and the activation of multiple cell death modalities, including necroptosis and ferroptosis, in ATO-sensitive HCC cells. Meanwhile, elevated tumoral immunogenicity was also observed in ATO-sensitive HCC cells. Similar effects were not observed in ATO-insensitive cells. We reported that ATO treatment induced mitochondrial injury and mtDNA release into the cytoplasm in ATO-sensitive HCC tumors. This subsequently activated the cGAS-STING-IFN axis, facilitating CD8+ T cell infiltration and activation. However, we found that the IFN pathway also induced tumoral PD-L1 expression, potentially antagonizing ATO-mediated immune attack. Additional anti-PD1 therapy promoted the anti-tumor response of ATO in ATO-sensitive HCC tumors. In summary, our data indicate that heterogeneous ATO responses exist in HCC tumors, and ATO treatment significantly induces immunogenic cell death (ICD) and activates the tumor-derived mtDNA-STING-IFN axis. These findings may offer a new perspective on the clinical treatment of HCC and warrant further study.

Abstract 5545: FADD activation renders hepatocellular carcinoma sensitive to immune checkpoint blockade by turning cold tumors hot

Abstract The heterogenicity of hepatocellular carcinoma (HCC) remains a key obstacle in turning the majority of ‘immune-cold’ tumors ‘hot’ for effective immune checkpoint blockade (ICB) therapy. Through studying the naturally-existed ‘hot’ HCC variant lymphoepithelioma-like (LEL)-HCC by RNA and whole exome sequencing analysis, we identified fas-associated death domain (FADD) as one of the top differentially expressed genes that associated with high tumoral CD8+T cell abundance. Of note, FADD upregulation was also observed in HCC patients with better ICB responsiveness. As FADD was mainly expressed in tumor cells from HCC by scRNAseq analysis, we constructed Fadd knockout stable cells using murine HCC cell lines. Interestingly, CRISPR knockout of tumoral Fadd led to increased tumor weights in immunocompetent but not immunodeficient mice, accompanied with decreased tumoral CD8+T cells. Mechanistically, FADD phosphorylation promoted NF-κB transcriptional activity to increase CCL5 secretion from HCC cells via interacting with its nuclear binding partners Src-associated-substrate-during-mitosis-of-68kDa/KH domain containing, RNA binding, signal transduction associated 1 (Sam68/KHDRBS1) and lysine (K)-specific methyltransferase 2D (KMT2D), thus directing CD8+T cell tumor infiltration. In addition, anti-PD1 triggered FADD phosphorylation via IFN-γ and TNF-ɑ release from CD8+T cells in ICB-sensitive but not resistant tumors. Most importantly, sequential FADD activation via genetic and pharmacologic approaches could re-sensitize the therapeutic efficacy of anti-programmed death 1 (PD1) antibody in ICB-resistant orthotopic and spontaneous HCC models by turning cold tumors hot. Taken together, our findings reveal a key molecular feature of ICB-responsive hot tumors that may guide the development of sequential combinatory immunotherapies for the majority of HCC patients with low responsiveness to ICB monotherapy. This project is supported by the University Grants Committee through General Research Fund (24110323, 14107622, 14104820) and the Health and Medical Research Fund (07180556). Citation Format: Jingying Zhou, Jiahuan Lu. FADD activation renders hepatocellular carcinoma sensitive to immune checkpoint blockade by turning cold tumors hot [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 5545.

Combined inhibition of surface CD51 and γ-secretase-mediated CD51 cleavage improves therapeutic efficacy in experimental metastatic hepatocellular carcinoma

Integrin αv (ITGAV, CD51) is regarded as a key component in multiple stages of tumor progression. However, the clinical failure of cilengitide, a specific inhibitor targeting surface CD51, suggests the importance of yet-unknown mechanisms by which CD51 promotes tumor progression. In this study, we used several hepatocellular carcinoma (HCC) cell lines and murine hepatoma cell lines. To investigate the role of CD51 on HCC progression, we used a 3D invasion assay and invivo bioluminescence imaging. We used periostin-knockout transgenic mice to uncover the role of the tumor microenvironment on CD51 cleavage. Moreover, we used several clinically relevant HCC models, including patient-derived organoids and patient-derived xenografts, to evaluate the therapeutic efficacy of cilengitide in combination with the γ-secretase inhibitor LY3039478. We found that CD51 could undergo transmembrane cleavage by γ-secretase to produce a functional intracellular domain (CD51-ICD). The cleaved CD51-ICD facilitated HCC invasion and metastasis by promoting the transcription of oxidative phosphorylation-related genes. Furthermore, we identified cancer-associated fibroblast-derived periostin as the major driver of CD51 cleavage. Lastly, we showed that cilengitide-based therapy led to a dramatic therapeutic effect when supplemented with LY3039478 in both patient-derived organoid and xenograft models. In summary, we revealed previously unrecognized mechanisms by which CD51 is involved in HCC progression and uncovered the underlying cause of cilengitide treatment failure, as well as providing evidence supporting the translational prospects of combined CD51-targeted therapy in the clinic. Integrin αv (CD51) is a widely recognized pro-tumoral molecule that plays a crucial role in various stages of tumor progression, making it a promising therapeutic target. However, despite early promising results, cilengitide, a specific antagonist of CD51, failed in a phase III clinical trial. This prompted further investigation into the underlying mechanisms of CD51's effects. This study reveals that the γ-secretase complex directly cleaves CD51 to produce an intracellular domain (CD51-ICD), which functions as a pro-tumoral transcriptional regulator and can bypass the inhibitory effects of cilengitide by entering the nucleus. Furthermore, the localization of CD51 in the nucleus is significantly associated with the prognosis of patients with HCC. These findings provide a theoretical basis for re-evaluating cilengitide in clinical settings and highlight the importance of identifying a more precise patient subpopulation for future clinical trials targeting CD51.

Boosting intracellular sodium selectively kills hepatocarcinoma cells and induces hepatocellular carcinoma tumor shrinkage in mice

Pharmacological treatments for advanced hepatocellular carcinoma (HCC) have a partial efficacy. Augmented Na+ content and water retention are observed in human cancers and offer unexplored targets for anticancer therapies. Na+ levels are evaluated upon treatments with the antibiotic cation ionophore Monensin by fluorimetry, ICP-MS, 23Na-MRI, NMR relaxometry, confocal or time-lapse analysis related to energy production, water fluxes and cell death, employing both murine and human HCC cell lines, primary murine hepatocytes, or HCC allografts in NSG mice. Na+ levels of HCC cells and tissue are 8-10 times higher than that of healthy hepatocytes and livers. Monensin further increases Na+ levels in HCC cells and in HCC allografts but not in primary hepatocytes and in normal hepatic and extrahepatic tissue. The Na+ increase is associated with energy depletion, mitochondrial Na+ load and inhibition of O2 consumption. The Na+ increase causes an enhancement of the intracellular water lifetime and death of HCC cells, and a regression and necrosis of allograft tumors, without affecting the proliferating activity of either HCCs or healthy tissues. These observations indicate that HCC cells are, unlike healthy cells, energetically incapable of compensating and surviving a pharmacologically induced Na+ load, highlighting Na+ homeostasis as druggable target for HCC therapy.

Alteration of the tumor microenvironment by pharmacological inhibition of EZH2 in hepatocellular carcinoma.

Enhancer of zeste homolog 2 (EZH2), a core component of polycomb repressive component 2 is overexpressed in a variety of cancers and recognized as a therapeutic target molecule. However, EZH2 possesses immunomodulatory functions in the tumor microenvironment (TME). The impact of EZH2 on TME of hepatocellular carcinoma (HCC) using immunocompetent mouse model was evaluated in the present study. UNC1999, an EZH2 inhibitor, impaired growth of the murine HCC cells (H22 cells) and induced apoptosis in a dose-dependent manner. Although UNC1999 significantly inhibited the growth of H22 cells-derived and Hepa1-6 cells-derived tumors in nonobese diabetic/severe combined immunodeficiency mice, its antitumor effect was diminished in allogenic BALB/c and C57BL/6 mice. Flow cytometric analyses of TME cells in BALB/c mice demonstrated a significant decrease in the number of interferon‑γ+ CD8+ T cells and regulatory T cells and a significant increase in the number of myeloid-derived suppressor cells (MDSCs). Administration of Gr-1 neutralizing antibody concomitant with UNC1999 restored antitumor effect accompanied by an increase in the number of CD8+ T cells followed by a decrease in the number of MDSCs. Chemokine antibody array demonstrated an enhanced expression of chemokines responsible for MDSCs recruitment such as C5a, CCL8, and CCL9. In conclusion, the study results demonstrated that EZH2 inhibitor contributed to attenuation of tumor immunity caused by TME arrangement. Combination therapy with EZH2 inhibitors and agents that reduce MDSCs might represent a novel therapeutic strategy for HCC.

Synthesis of New Shogaol Analogues as NRF2 Activators and Evaluation of Their Anti-Inflammatory Activity, Modes of Action and Metabolic Stability

6-shogaol is a natural and the most potent bioactive vanilloid in dried Zingiber officinale rhizomes. Many scientific studies have reported the diverse biological activities of 6-shogaol. However, the major drawback of 6-shogaol is its instability at room temperature. We synthesised new shogaol thiophene compounds (STCs) by replacing the pentyl group in the sidechain with thiophene derivatives. The STCs were tested for their nuclear factor erythroid 2-related factor 2 (NRF2) activation ability in murine hepatoma cells (Hepa1c1c-7) by determining their NAD(P)H quinone oxidoreductase 1 (NQO1) inducing ability and expression of NRF2-associated antioxidant genes. The anti-inflammatory activity of STCs was determined in Escherichia coli lipopolysaccharide (LPSEc)-stimulated NR2-proficient and -silenced mouse microglial cells (BV-2) by measuring the inflammatory markers, cytokines, and mediators. The modes of action (interacting with the Kelch domain of KEAP1, covalent bonding with cysteines of KEAP1, and inhibition of GSK-3β enzyme activity) of NRF2 activation by STCs were determined using commercially available kits. The in vitro metabolic stability of the STCs in liver microsomes (humans, rats, and mice) was also investigated. The molecular docking and molecular dynamics studies were conducted to identify the binding poses, stability, and molecular interactions of the STCs in the binding pockets of Kelch and BTB domains of KEAP1 and GSK-3β enzyme. The new STCs were synthesised in good yields of > 85%, with a purity of about 95%, using a novel synthesis method by employing a reusable proline-proline dipeptide catalyst. The STCs are more potent than 6-shogaol in activating NRF2 and reducing inflammation. The nature of substituents on thiophene has a profound influence on the bioactivity of the STCs. Phenylthiophene STC (STC5) is the most potent, while thiophenes containing electron-withdrawing groups showed weaker bioactivity. The bioactivity of 6-shogaol is in the micromolar range, whereas STC5 showed bioactivity in the sub micromolar range. The STCs showed anti-inflammatory effects via NRF2-dependent and NRF2-independent mechanisms. The STCs improved NRF2 activity through multiple (KEAP1-independent and -dependent) mechanisms. The STCs showed decreased reactivity with thiols than 6-shogaol and thus may possess fewer side-effects than 6-shogaol. The STCs were more metabolically stable than 6-shogaol.

Treatment with a Cholecystokinin Receptor Antagonist, Proglumide, Improves Efficacy of Immune Checkpoint Antibodies in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated deaths worldwide. Treatment with immune checkpoint antibodies has shown promise in advanced HCC, but the response is only 15-20%. We discovered a potential target for the treatment of HCC, the cholecystokinin-B receptor (CCK-BR). This receptor is overexpressed in murine and human HCC and not in normal liver tissue. Mice bearing syngeneic RIL-175 HCC tumors were treated with phosphate buffer saline (PBS; control), proglumide (a CCK-receptor antagonist), an antibody to programmed cell death protein 1 (PD-1Ab), or the combination of proglumide and the PD-1Ab. In vitro, RNA was extracted from untreated or proglumide-treated murine Dt81Hepa1-6 HCC cells and analyzed for expression of fibrosis-associated genes. RNA was also extracted from human HepG2 HCC cells or HepG2 cells treated with proglumide and subjected to RNA sequencing. Results showed that proglumide decreased fibrosis in the tumor microenvironment and increased the number of intratumoral CD8+ T cells in RIL-175 tumors. When proglumide was given in combination with the PD-1Ab, there was a further significant increase in intratumoral CD8+ T cells, improved survival, and alterations in genes regulating tumoral fibrosis and epithelial-to-mesenchymal transition. RNAseq results from human HepG2 HCC cells treated with proglumide showed significant changes in differentially expressed genes involved in tumorigenesis, fibrosis, and the tumor microenvironment. The use of the CCK receptor antagonist may improve efficacy of immune checkpoint antibodies and survival in those with advanced HCC.

Immunization of Mice with Gold Nanoparticles Conjugated to Thermostable Cancer Antigens Prevents the Development of Xenografted Tumors.

Gold nanoparticles as part of vaccines greatly increase antigen stability, antigen accumulation in the lymph nodes, and antigen uptake by antigen-presenting cells. The use of such particles as part of anticancer vaccines based on heat shock proteins to increase vaccine effectiveness is timely. We prepared and characterized nanoconjugates based on 15-nm gold nanoparticles and thermostable tumor antigens isolated from MH22a murine hepatoma cells. The whole-cell lysate of MH22a cells contained the main heat shock proteins. BALB/c mice were injected with the conjugates and then received transplants of MH22a cells. The highest titer was produced in mice immunized with the complex of gold nanoparticles + antigen with complete Freund's adjuvant. The immunized mice showed no signs of tumor growth for 24 days. They also showed a decreased production of the INF-γ, IL-6, and IL-1 proinflammatory cytokines compared to the mice immunized through other schemes. This study is the first to show that it is possible in principle to use gold nanoparticles in combination with thermostable tumor antigens for antitumor vaccination. Antitumor vaccines based on thermostable tumor antigens can be largely improved by including gold nanoparticles as additional adjuvants.

Alizarin as a New Activator of the Aryl Hydrocarbon Receptor Signaling Pathway

Alizarin (1,2-dihydroxyanthraquinone) is a natural red dye extracted from the roots of Rubia cordifolia L. (family Rubiaceae). Alizarin has been used as a biological red stain for calcium. The aryl hydrocarbon receptor (AhR) has critical roles in multiple physiological pathways. This study aimed to determine whether alizarin is an unreported ligand of AhR. In the present study, we investigated the effects on cytochrome P450 (CYP) 1A1 mRNA, protein expression, AhR nuclear translocation, aryl hydrocarbon response element (AHRE) reporter activity, and AhR-specific antagonist following alizarin treatment of cells of the human hepatoma cell line, HepG2, and murine hepatoma cell line, Hepa-1c1c7. Alizarin induced CYP1A1 mRNA and protein expression in HepG2 and Hep-1c1c7 cells. Such induction was not present in C4 (B13NBii1) cells, which are AhR signal deficient, C12 (B15ECiii2) cells, which reduce AhR protein levels. The alizarin-induced responses were blocked by CH-223191, which is an AhR antagonist. Alizarin, the same as with the AhR ligand, induced the nuclear localization of AhR, as well as stimulated the transcriptional activity of AHRE. The results of this study suggest that alizarin is an AhR agonist.

Irradiation Induced Activation of cGAS/STING Signaling Promotes Macrophage Anti-Tumor Activity via CXCL9, CXCL10-CXCR3 Axis

<h3>Purpose/Objective(s)</h3> Irradiation induced abundant dsDNA fragments, which can stimulate tumor immune responses by DNA sensing pathway. However, the underlying mechanism, mainly involved immune cells remains unknown. Herein we discovered that macrophage was the main immune cell involved in cGAS/STING DNA sensing pathway induced anti-tumor immune response, and macrophage derived CXCL9, CXCL10 played an important role in infiltration of T cells in tumor via CXCL9, CXCL10-CXCR3 axis. <h3>Materials/Methods</h3> cGAS knockout mice (cGAS-/- mice) and STING-deficient mice were used in the study. Xenograft tumor mouse model was established for in vivo study, using murine hepatoma cell line H22 or ovalbumin (OVA)-expressing H22. Irradiation locally delivered to the tumor area at 8 Gy/day for 3 days using a micro-CT/micro irradiator. <h3>Results</h3> Macrophage was found recruitment to the irradiated tumor site after irradiation. Using C57BL/6J mice bearing xenograft H22 tumors, we found cGAS-/- mice showed higher growth rate after irradiation comparing to tumors growing on wild type mice (WT mice). Using a both sides subcutaneous H22-OVA tumor mouse model, we found a substantial increased OVA-specific CD8+ T cells within the DLN(draining lymph node) of right irradiated tumor of WT mice, but low level within the right DLN of the irradiated tumor of STING-deficient mice and the no-irradiated right tumors of STING-deficient mice or WT mice, which confirmed that cGAS/STING pathway was involved in the radiation-mediated immunomodulation and promoted the radiation-induced adaptive anti-tumor immune activation. We also noted macrophage depletion partial reverse antitumor effect of irradiation in WT tumors, while no significant difference is observed in cGAS^−/− tumor, which revealed macrophage was the main immune cell involved in cGAS/STING DNA sensing pathway induced anti-tumor immune response. Further mechanism study revealed macrophage derived CXCL9, CXCL10 was induced by radiation in a cGAS/STING pathway dependent manner. Intraperitoneal injection of anti-CXCL9, CXCL10 significantly reduce the anti-tumor effect of irradiation in WT mice, but not in STING-deficient mice, these observations suggested macrophage derived CXCL9, CXCL10 were involved in infiltration of T cells in tumor via CXCL9, CXCL10-CXCR3 axis. <h3>Conclusion</h3> cGAS/STING pathway is crucial in the radiation-induced adaptive anti-tumor immune activation. macrophage derived CXCL9, CXCL10, which induced by radiation in a cGAS/STING pathway dependent manner, mediated infiltration of T cells in tumor via CXCL9, CXCL10- CXCR3 axis.

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...

Anti-Inflammatory Effects of Auranamide and Patriscabratine-Mechanisms and In Silico Studies.

Auranamide and patriscabratine are amides from Melastoma malabathricum (L.) Smith. Their anti-inflammatory activity and nuclear factor erythroid 2-related factor 2 (NRF2) activation ability were evaluated using Escherichia coli lipopolysaccharide (LPSEc)-stimulated murine macrophages (RAW264.7) and murine hepatoma (Hepa-1c1c7) cells, respectively. The cytotoxicity of the compounds was assessed using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The anti-inflammatory activity was determined by measuring the nitric oxide (NO) production and pro-inflammatory cytokines (Interleukin (IL)-1β, Interferon (IFN)-γ, tumour necrosis factor (TNF)-α, and IL-6) and mediators (NF-κB and COX-2). NRF2 activation was determined by measuring the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) quinone oxidoreductase 1 (NQO1), nuclear NRF2 and hemeoxygenase (HO)-1. In vitro metabolic stability was assessed using the mouse, rat, and human liver microsomes. The compounds were non-toxic to the cells at 10 μM. Both compounds showed dose-dependent effects in downregulating NO production and pro-inflammatory cytokines and mediators. The compounds also showed upregulation of NQO1 activity and nuclear NRF2 and HO-1 levels. The compounds were metabolically stable in mouse, rat and human liver microsomes. The possible molecular targets of NRF2 activation by these two compounds were predicted using molecular docking studies and it was found that the compounds might inhibit the Kelch domain of KEAP1 and GSK-3β activity. The physicochemical and drug-like properties of the test compounds were predicted using Schrodinger small molecule drug discovery suite (v.2022-2).

Dysregulated glucuronic acid metabolism exacerbates hepatocellular carcinoma progression and metastasis through the TGFβ signalling pathway.

BackgroundGlucuronic acid metabolism participates in cellular detoxification, extracellular matrix remodeling and cell adhesion and migration. Here, we aimed to explore the crosstalk between dysregulated glucuronic acid metabolism and crucial metastatic signalling in glutathione S‐transferase zeta 1 (GSTZ1)‐deficient hepatocellular carcinoma (HCC).MethodsTranswell, HCC xenograft and Gstz1 −/‐ mouse models were used to examine the role of GSTZ1 in HCC metastasis. Non‐targeted and targeted metabolomics and global transcriptomic analyses were performed to screen significantly altered metabolic and signalling pathways in GSTZ1 overexpressing hepatoma cells. Further, RNA‐binding protein immunoprecipitation, Biotin‐RNA pull‐down, mRNA decay assays and luciferase reporter assays were used to explore the interaction between RNA and RNA‐binding proteins.ResultsGSTZ1 was universally silenced in both human and murine HCC cells, and its deficiency contributed to HCC metastasis in vitro and in vivo. UDP‐glucose 6‐dehydrogenase (UGDH)‐mediated UDP‐glucuronic acid (UDP‐GlcUA) accumulation promoted hepatoma cell migration upon GSTZ1 loss. UDP‐GlcUA stabilized TGFβR1 mRNA by enhancing its binding to polypyrimidine tract binding protein 3, contributing to the activation of TGFβ/Smad signalling. UGDH or TGFβR1 blockade impaired HCC metastasis. In addition, UGDH up‐regulation and UDP‐GlcUA accumulation correlated with increased metastatic potential and decreased patient survival in GSTZ1‐deficient HCC.ConclusionsGSTZ1 deficiency and subsequent up‐regulation of the glucuronic acid metabolic pathway promotes HCC metastasis by increasing the stability of TGFβR1 mRNA and activating TGFβ/Smad signalling. UGDH and a key metabolite, UDP‐GlcUA, may serve as prognostic markers. Targeting UGDH might be a promising strategy for HCC therapy.

Establishment and evaluation of cell and animal models expressing BORIS subfamily 2 variant.

BackgroundThe possibility of a cancer vaccine aimed at stimulating or mobilizing the body’s immune system to control and kill tumor cells is emerging as a potential new strategy for tumor immunological therapy. CCCTC-binding Factor Like (CTCFL)/Brother of the Regulator of Imprinted Sites (BORIS), a cancer-testis antigen (CTA), has 23 mRNA splice variants classified into six subfamilies (sfs) and potentially encodes 17 distinct polypeptides. Based on our previous long-term research on hepatocellular carcinoma (HCC), we were particularly interested in whether BORIS sf2 could be a promising candidate for immunotherapy targeting liver cancer cells. Therefore, in this study, we aimed to construct an animal model to study the immunogenicity of human BORIS sf2 in murine hepatoma cells.MethodsWe established a hepatoma cell line expressing human BORIS sf2/C68 by inserting the sequences into a lentiviral vector pLVX-EF1α-IRES-Puro carrying the puromycin resistance gene. We achieved the stabilized expression of BORIS sf2/C68 in the oncogenic Hepa1c1c7 cells through lentivirus-mediated approach. The Hepa1c1c7 cells expressing the BORIS sf2/C68 (5×106/mouse) were inoculated subcutaneously into 6-week-old C57BL/6 mice to induce the formation of tumors.ResultsIn the tumor formation experiment, the murine hepatoma cells expressing human BORIS sf2/C68 showed progressive growth in C57BL/6 mice. The animal model we constructed could be used to study the in vivo immunogenicity of the human BORIS sf2 in murine hepatoma cells.ConclusionsThe animals bearing BORIS sf2/C68-positive tumors may serve as an animal model for studying the therapeutic potency and safety of HCC vaccine directed at the CT-antigen BORIS sf2.

HepG2-Based Designer Cells with Heat-Inducible Enhanced Liver Functions

Functional human hepatocytes have been a pivotal tool in pharmacological studies such as those investigating drug metabolism and hepatotoxicity. However, primary human hepatocytes are difficult to obtain in large quantities and may cause ethical problems, necessitating the development of a new cell source to replace human primary hepatocytes. We previously developed genetically modified murine hepatoma cell lines with inducible enhanced liver functions, in which eight liver-enriched transcription factor (LETF) genes were introduced into hepatoma cells as inducible transgene expression cassettes. Here, we establish a human hepatoma cell line with heat-inducible liver functions using HepG2 cells. The genetically modified hepatoma cells, designated HepG2/8F_HS, actively proliferated under normal culture conditions and, therefore, can be easily prepared in large quantities. When the expression of LETFs was induced by heat treatment at 43 °C for 30 min, cells ceased proliferation and demonstrated enhanced liver functions. Furthermore, three-dimensional spheroid cultures of HepG2/8F_HS cells showed a further increase in liver functions upon heat treatment. Comprehensive transcriptome analysis using DNA microarrays revealed that HepG2/8F_HS cells had enhanced overall expression of many liver function-related genes following heat treatment. HepG2/8F_HS cells could be useful as a new cell source for pharmacological studies and for constructing bioartificial liver systems.

Tumor endothelial cell-induced CD8+ T-cell exhaustion via GPNMB in hepatocellular carcinoma.

Tumor endothelial cells (TECs) promote tumor angiogenesis and regulate cytotoxic T cells in the tumor microenvironment. However, the roles of TECs for tumor‐infiltrating T‐cell in hepatocellular carcinoma (HCC) is still unknown. Here, we aimed to investigate how TECs influenced tumor growth and immune responses of HCC focusing on CD8+ T‐cell infiltration and exhaustion. First, TECs were isolated from subcutaneous HCC tumors with murine HCC cell lines (BNL‐T) with magnetic selection of CD31+ cells, and normal endothelial cells (NECs) were isolated from normal liver. Second, immunocompetent mice were injected with BNL‐T alone, BNL‐T + NECs, or BNL‐T + TECs for tumor formation, and the functions and exhaustion of tumor‐infiltrating CD8+ T cells were evaluated. The mice injected with BNL‐T + TEC showed rapid tumorigenesis and a decrease in the number of infiltrating CD8+ T cells. In addition, the percentage of CD8+ T‐cell exhaustion was significantly higher in tumors from the administration of BNL‐T + TEC. Third, the next‐generation sequencing on TECs was performed to identify mRNAs that might be a novel treatment target. The molecule of glycoprotein nonmetastatic melanoma protein B (GPNMB) was identified and the functions of GPNMB was analyzed by silencing of GPNMB expression using small interfering RNAs. The silencing of GPNMB expression in TECs induced the suppression of tumor growth and T‐cell exhaustion. In conclusion, TECs induced tumor‐infiltrating T‐cell exhaustion via GPNMB expression and GPNMB might be a novel therapeutic target in HCC.