Murine Hepatoma Research Articles

GPER1 signaling restricts macrophage proliferation and accumulation in human hepatocellular carcinoma

BackgroundSex hormones and their related receptors have been reported to impact the development and progression of tumors. However, their influence on the composition and function of the tumor microenvironment is not well understood. We aimed to investigate the influence of sex disparities on the proliferation and accumulation of macrophages, one of the major components of the tumor microenvironment, in hepatocellular carcinoma (HCC).MethodsImmunohistochemistry was applied to assess the density of immune cells in HCC tissues. The role of sex hormone related signaling in macrophage proliferation was determined by immunofluorescence and flow cytometry. The underlying regulatory mechanisms were examined with both in vitro experiments and murine HCC models.ResultsWe found higher levels of macrophage proliferation and density in tumor tissues from male patients compared to females. The expression of G protein–coupled estrogen receptor 1 (GPER1), a non-classical estrogen receptor, was significantly decreased in proliferating macrophages, and was inversely correlated with macrophage proliferation in HCC tumors. Activation of GPER1 signaling with a selective agonist G-1 suppressed macrophage proliferation by downregulating the MEK/ERK pathway. Additionally, G-1 treatment reduced PD-L1 expression on macrophages and delayed tumor growth in mice. Moreover, patients with a higher percentage of GPER1+ macrophages exhibited longer overall survival and recurrence-free survival compared to those with a lower level.ConclusionsThese findings reveal a novel role of GPER1 signaling in regulating macrophage proliferation and function in HCC tumors and may offer a potential strategy for designing therapies based on understanding sex-related disparities of patients.

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.

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.

Assessment of the In Vitro Phosphatidylinositol Glycan Class A (PIG-A) Gene Mutation Assay Using Human TK6 and Mouse Hepa1c1c7 Cell Lines.

Gene mutations linked to diseases like cancer may be caused by exposure to environmental chemicals. The X-linked phosphatidylinositol glycan class A (PIG-A) gene, required for glycosylphosphatidylinositol (GPI) anchor biosynthesis, is a key target locus for in vitro genetic toxicity assays. Various organisms and cell lines may respond differently to genotoxic agents. Here, we compared the mutagenic potential of directly genotoxic ethyl methane sulfonate (EMS) to metabolically activated pro-mutagenic polycyclic aromatic hydrocarbons (PAHs). The two classes of mutagens were compared in an in vitro PIG-A gene mutation test using the metabolically active murine hepatoma Hepa1c1c7 cell line and the human TK6 cell line, which has limited metabolic capability. Determination of cell viability is required for quantifying mutagenicity. Two common cell viability tests, the MTT assay and propidium iodide (PI) staining measured by flow cytometry, were evaluated. The MTT assay overestimated cell viability in adherent cells at high benzo[a]pyrene (B[a]P) exposure concentrations, so PI-based cytotoxicity was used in calculations. The spontaneous mutation rates for TK6 and Hepa1c1c7 cells were 1.87 and 1.57 per million cells per cell cycle, respectively. TK6 cells exposed to 600 µM and 800 µM EMS showed significantly higher mutation frequencies (36 and 47 per million cells per cell cycle, respectively). Exposure to the pro-mutagen benzo[a]pyrene (B[a]P, 10 µM) did not increase mutation frequency in TK6 cells. In Hepa1c1c7 cells, mutation frequencies varied across exposure groups (50, 50, 29, and 81 per million cells per cell cycle when exposed to 10 µM B[a]P, 5-methylcholanthrene (5-MC), chrysene, or 16,000 µM EMS, respectively). We demonstrate that the choice of cytotoxicity assay and cell line can determine the outcome of the Pig-A mutagenesis assay when assessing a specific mutagen.

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.

FOXM1/DEPDC1 feedback loop promotes hepatocarcinogenesis and represents promising targets for cancer therapy.

Forkhead box M1 (FOXM1) is a key regulator of mitosis and is identified as an oncogene involved in several kinds of human malignancies. However, how it induces carcinogenesis and related therapeutic approaches remains not fully understood. In this study, we aimed to identify a regulatory axis involving FOXM1 and its target gene DEP domain containing 1 (DEPDC1) and investigate their biological functions. FOXM1 bound to the promoter and transcriptionally induced DEPDC1 expression, in turn, DEPDC1 physically interacted with FOXM1, promoted its nuclear translocation, and reinforced its transcriptional activities. The FOXM1/DEPDC1 axis was indispensable for cancer cells, as evidenced by the fact that DEPDC1 rescued cell growth inhibition caused by FOXM1 knockdown, and silencing DEPDC1 efficiently attenuated tumor growth in a murine hepatocellular carcinoma model. Furthermore, strong positive associations between FOXM1/DEPDC1 axis and poor clinical outcome were observed in human hepatocellular carcinoma samples, further indicating their significance for hepatocarcinogenesis. Finally, we attempted to exploit immunotherapy approaches to target the FOXM1/DEPDC1 axis. Several HLA-A24:02-restricted T-cell epitopes targeting FOXM1 or DEPDC1 were identified through bioinformatic analysis. Then, T cell receptor (TCR)-engineered T cells targeting FOXM1262-270 or DEPDC1294-302 were successfully established and proved to efficiently eradicate tumor cells. Our findings highlight the significance of the FOXM1/DEPDC1 axis in the process of oncogenesis and indicate their potential as immunotherapy targets.

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.

IMmuneCite: an integrated workflow for analysis of immune enriched spatial proteomic data.

Spatial proteomics enable detailed analysis of tissue at single cell resolution. However, creating reliable segmentation masks and assigning accurate cell phenotypes to discrete cellular phenotypes can be challenging. We introduce IMmuneCite, a computational framework for comprehensive image pre-processing and single-cell dataset creation, focused on defining complex immune landscapes when using spatial proteomics platforms. We demonstrate that IMmuneCite facilitates the identification of 32 discrete immune cell phenotypes using data from human liver samples while substantially reducing nonbiological cell clusters arising from co-localization of markers for different cell lineages. We established its versatility and ability to accommodate any antibody panel and different species by applying IMmuneCite to data from murine liver tissue. This approach enabled deep characterization of different functional states in each immune compartment, uncovering key features of the immune microenvironment in clinical liver transplantation and murine hepatocellular carcinoma. In conclusion, we demonstrated that IMmuneCite is a user-friendly, integrated computational platform that facilitates investigation of the immune microenvironment across species, while ensuring the creation of an immune focused, spatially resolved single-cell proteomic dataset to provide high fidelity, biologically relevant analyses.

Tumor cell-intrinsic MELK enhanced CCL2-dependent immunosuppression to exacerbate hepatocarcinogenesis and confer resistance of HCC to radiotherapy

BackgroundThe outcome of hepatocellular carcinoma (HCC) is limited by its complex molecular characteristics and changeable tumor microenvironment (TME). Here we focused on elucidating the functional consequences of Maternal embryonic leucine zipper kinase (MELK) in the tumorigenesis, progression and metastasis of HCC, and exploring the effect of MELK on immune cell regulation in the TME, meanwhile clarifying the corresponding signaling networks.MethodsBioinformatic analysis was used to validate the prognostic value of MELK for HCC. Murine xenograft assays and HCC lung metastasis mouse model confirmed the role of MELK in tumorigenesis and metastasis in HCC. Luciferase assays, RNA sequencing, immunopurification–mass spectrometry (IP-MS) and coimmunoprecipitation (CoIP) were applied to explore the upstream regulators, downstream essential molecules and corresponding mechanisms of MELK in HCC.ResultsWe confirmed MELK to be a reliable prognostic factor of HCC and identified MELK as an effective candidate in facilitating the tumorigenesis, progression, and metastasis of HCC; the effects of MELK depended on the targeted regulation of the upstream factor miR-505-3p and interaction with STAT3, which induced STAT3 phosphorylation and increased the expression of its target gene CCL2 in HCC. In addition, we confirmed that tumor cell-intrinsic MELK inhibition is beneficial in stimulating M1 macrophage polarization, hindering M2 macrophage polarization and inducing CD8 + T-cell recruitment, which are dependent on the alteration of CCL2 expression. Importantly, MELK inhibition amplified RT-related immune effects, thereby synergizing with RT to exert substantial antitumor effects. OTS167, an inhibitor of MELK, was also proven to effectively impair the growth and progression of HCC and exert a superior antitumor effect in combination with radiotherapy (RT).ConclusionsAltogether, our findings highlight the functional role of MELK as a promising target in molecular therapy and in the combination of RT therapy to improve antitumor effect for HCC.

Manipulating the Crosstalk between Cancer and Immunosuppressive Cells with Phototherapeutic Gold-Nanohut for Reprogramming Tumor Microenvironment.

Photoimmunotherapy faces challenges due to insufficient intratumoral accumulation of photothermal agents and the reversion of the cancer-immunity cycle during treatment. In this study, an anti-PD-L1-immobilized magnetic gold nanohut, AuNH-2-Ab, with photoresponsive, thermosensitive, and immunomodulatory properties to effectively suppress the growth of primary tumors, elevate immunogenic cell death (ICD) levels, reverse the tumor immune microenvironment (TIME), and consequently inhibit metastases are developed. AuNH-2-Ab achieves high tumor accumulation (9.54% injected dose) following systemic administration, allowing the modulation of hyperthermia dose of over 50°C in the tumor. By optimizing the hyperthermia dose, AuNH-2-Ab simultaneously target and eliminate cancer cells and tumor-associated macrophages, thereby activating potent antitumor immunity without being compromised by immunosuppressive elements. Hyperthermia/pH induced morphological transformation of AuNH-2-Ab involving the detachment of the surface antibody for in situ PD-L1 inhibition, and exposure of the inner fucoidan layer for natural killer (NK) cell activation. This precision photoimmunotherapy approach reprograms the TIME, significantly prolongs survival in a murine hepatocellular carcinoma model (Hep55.1c), and harnesses the synergistic effects of ICD production and checkpoint inhibitors by utilizing a single nanoplatform.

The Loss of an Orphan Nuclear Receptor NR2E3 Augments Wnt/β-catenin Signaling via Epigenetic Dysregulation that Enhances Sp1-β catenin-p300 Interactions in Hepatocellular Carcinoma.

The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3) is an epigenetic player that modulates chromatin accessibility to activate p53 during liver injury. Nonetheless, a precise tumor suppressive and epigenetic role of NR2E3 in hepatocellular carcinoma (HCC) development remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/β-catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice consistently exhibits accelerated liver tumor formation accompanied by enhanced activation of Wnt/β-catenin signaling pathway and inactivation of p53 signaling. At cellular level, the loss of NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates key genes in the WNT/β-catenin pathway with increased chromatin accessibility. This event is mediated through increased formation of active transcription complex involving Sp1, β-catenin, and p300, a histone acetyltransferase, on the promoters of target genes. These findings demonstrate that the loss of NR2E3 activates Wnt/β-catenin signaling at cellular and organism levels and this dysregulation is associated with aggressive HCC development and poor clinical outcomes. In summary, NR2E3 is a novel tumor suppressor with a significant prognostic value, maintaining epigenetic homeostasis to suppress the Wnt/β-catenin signaling pathway that promotes HCC development.

Pantoprazole suppresses carcinogenesis and growth of hepatocellular carcinoma by inhibiting glycolysis and Na+/H+ exchange.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers. The prevention and therapy for this deadly disease remain a global medical challenge. In this study, we investigated the effect of pantoprazole (PPZ) on the carcinogenesis and growth of HCC. Both diethylnitrosamine (DEN) plus CCl4-induced and DEN plus high fat diet (HFD)-induced HCC models in mice were established. Cytokines and cell proliferation-associated gene in the liver tissues of mice and HCC cells were analyzed. Cellular glycolysis and Na+/H+ exchange activity were measured. The preventive administration of pantoprazole (PPZ) at a clinically relevant low dose markedly suppressed HCC carcinogenesis in both DEN plus CCl4-induced and HFD-induced murine HCC models, whereas the therapeutic administration of PPZ at the dose suppressed the growth of HCC. In the liver tissues of PPZ-treated mice, inflammatory cytokines, IL1, CXCL1, CXCL5, CXCL9, CXCL10, CCL2, CCL5, CCL6, CCL7, CCL20, and CCL22, were reduced. The administration of CXCL1, CXCL5, CCL2, or CCL20 all reversed PPZ-suppressed DEN plus CCL4-induced HCC carcinogenesis in mice. PPZ inhibited the expressions of CCNA2, CCNB2, CCNE2, CDC25C, CDCA5, CDK1, CDK2, TOP2A, TTK, AURKA, and BIRC5 in HCC cells. Further results showed that PPZ reduced the production of these inflammatory cytokines and the expression of these cell proliferation-associated genes through the inhibition of glycolysis and Na+/H+ exchange. In conclusion, PPZ suppresses the carcinogenesis and growth of HCC, which is related to inhibiting the production of inflammatory cytokines and the expression of cell proliferation-associated genes in the liver through the inhibition of glycolysis and Na+/H+ exchange.

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.

Induction of the Inflammasome Pathway by Tyrosine Kinase Inhibitors Provides an Actionable Therapeutic Target for Hepatocellular Carcinoma.

During the last decade, tyrosine kinase inhibitors (TKIs) sorafenib and regorafenib have been standard systemic treatments for advanced hepatocellular carcinoma (HCC). Previous data associated sorafenib with inflammasome activation. However, the role of the inflammasome in sorafenib and regorafenib signaling has not been described in liver cancer patients. For this purpose, we analyzed inflammasome-related transcriptomic changes in a murine HCC model. Our data confirmed inflammasome activation after both TKI treatments, sharing a similar pattern of increased gene expression. According to human database results, transcriptional increase of inflammasome genes is associated with poorer prognosis for male liver cancer patients, suggesting a sex-dependent role for inflammasome activation in HCC therapy. In biopsies of HCC and its surrounding tissue, we detected durable increases in the inflammasome activation pattern after sorafenib or regorafenib treatment in male patients. Further supporting its involvement in sorafenib action, inflammasome inhibition (MCC950) enhanced sorafenib anticancer activity in experimental HCC models, while no direct in vitro effect was observed in HCC cell lines. Moreover, activated human THP-1 macrophages released IL-1β after sorafenib administration, while 3D Hep3B spheres displayed increased tumor growth after IL-1β addition, pointing to the liver microenvironment as a key player in inflammasome action. In summary, our results unveil the inflammasome pathway as an actionable target in sorafenib or regorafenib therapy and associate an inflammasome signature in HCC and surrounding tissue with TKI administration. Therefore, targeting inflammasome activation, principally in male patients, could help to overcome sorafenib or regorafenib resistance and enhance the efficacy of TKI treatments in HCC.

In situ chemoimmunotherapy hydrogel elicits immunogenic cell death and evokes efficient antitumor immune response

BackgroundChemoimmunotherapy has shown promising advantages of eliciting immunogenic cell death and activating anti-tumor immune responses. However, the systemic toxicity of chemotherapy and tumor immunosuppressive microenvironment limit the clinical application.MethodsHere, an injectable sodium alginate hydrogel (ALG) loaded with nanoparticle albumin-bound-paclitaxel (Nab-PTX) and an immunostimulating agent R837 was developed for local administration. Two murine hepatocellular carcinoma and breast cancer models were established. The tumor-bearing mice received the peritumoral injection of R837/Nab-PTX/ALG once a week for two weeks. The antitumor efficacy, the immune response, and the tumor microenvironment were investigated.ResultsThis chemoimmunotherapy hydrogel with sustained-release character was proven to have significant effects on killing tumor cells and inhibiting tumor growth. Peritumoral injection of our hydrogel caused little harm to normal organs and triggered a potent antitumor immune response against both hepatocellular carcinoma and breast cancer. In the tumor microenvironment, enhanced immunogenic cell death induced by the combination of Nab-PTX and R837 resulted in 3.30-fold infiltration of effector memory T cells and upregulation of 20 biological processes related to immune responses.ConclusionsOur strategy provides a novel insight into the combination of chemotherapy and immunotherapy and has the potential for clinical translation.

Sorafenib and Piperine co-loaded PLGA nanoparticles: Development, characterization, and anti-cancer activity against hepatocellular carcinoma cell line

Hepatocellular carcinoma (HCC) exhibits high mortality rates in the advanced stage (>90 %). Sorafenib (SORA) is a targeted therapy approved for the treatment of advanced HCC; however, the reported response rate to such a therapeutic is suboptimal (<3%). Piperine (PIP) is an alkaloid demonstrated to exert a direct tumoricidal activity in HCC and improve the pharmacokinetic profiles of anticancer drugs including SORA. In this study, we developed a strategy to improve efficacy outcomes in HCC using PIP as an add-on treatment to support the first-line therapy SORA using biodegradable Poly (D, L-Lactide-co-glycolide, PLGA) nanoparticles (NPs). SORA and PIP (both exhibit low aqueous solubility) were co-loaded into PLGA NPs (PNPs) and stabilized with various concentrations of polyvinyl alcohol (PVA). The SORA and PIP-loaded PNPs (SP-PNPs) were characterized using Fourier Transform Infrared (FTIR) Spectroscopy, X-ray Powder Diffraction (XRD), Dynamic Light Scattering (DLS), and Scanning Electron Microscopy (SEM), Release of these drugs from SP-PNPs was investigated in vitro at both physiological and acidic pH, and kinetic models were employed to assess the mechanism of drug release. The in vitro efficacy of SP-PNPs against HCC cells (HepG2) was also evaluated. FTIR and XRD analyses revealed that the drugs encapsulated in PNPs were in an amorphous state, with no observed chemical interactions among the drugs or excipients. Assessment of drug release in vitro at pH 5 and 7.4 showed that SORA and PIP loaded in PNPs with 0.5 % PVA were released in a sustained manner, unlike pure drugs, which exhibited relatively fast release. SP-PNPs with 0.5 % PVA were spherical, had an average size of 224 nm, and had a high encapsulation efficiency (SORA ∼ 82 %, PIP ∼ 79 %), as well as superior cytotoxicity compared to SORA monotherapy in vitro. These results suggest that combining PIP with SORA using PNPs may be an effective strategy for the treatment of HCC and may set the stage for a comprehensive in vivo study to evaluate the efficacy and safety of this novel formulation using a murine HCC model.

Abstract 1520: A novel cluster of cytolytic alpha/beta TCR+CD4−CD8−T cells originating from CD8+T cells with potent innate-like T cell killing ability: implicating adoptive T cell transfer therapy for hepatocellular carcinoma

Abstract Tumor immunotherapies represent promising strategies for treating various cancers, including hepatocellular carcinoma. In this context, immune cells, including the adoptive T cell transfer treatment, become the focus of interests. Notably, alpha/beta TCR+CD4−CD8−T (DNT) cells is a subgroup of T cells that is ignored for a long time but was re-focused recently. These DNT cells are a group of cells with opposite functions in different condition, including pro-inflammatory or suppressive functions and possibly originated from over-stimulation of peripheral CD4+T cells/CD8+T cells or a special group of T cells formed in thymus. In our research results, we have found these DNT cells increased in murine hepatoma model, especially in the tumor microenvironment. In addition, these DNT cells from tumor microenvironment of murine hepatoma possessed potent tumor-killing ability. Furthermore, we analyzed the transcriptome profile of T cells in tumor microenvironment by single cell RNAseq approach (CITE-seq). We found DNT cells could be categorized into two main clusters, one is cytolytic DNT cells, resemble the potent CD8+T cells, and another is the CD4+T cell-like DNT cells. These cytolytic DNT cells are not exhausted but with high expression of cytolytic molecule and low hypoxia-inducible factor-1alpha (HIF-1a) expression. By trajectory analysis, cytolytic DNT cells come from the conventional CD8+T cells but following a unique developmental pathway that is different from the exhausted CD8+T cells. Interesting, these cytolytic DNT cells also expressed high level of Fcer1g molecule, a marker of innate CD8+T cells. In addition, these DNT cells could be generated and expanded in-vitro by IL-15 without TCR activation. In conclusion, we identified a cluster of cytolytic DNT cells, originated from CD8+T cells, exhibits innate T cells behavior and potent killing ability. In addition, these cytolytic DNT cells could be generated and expanded in-vitro for the adoptive T cell transfer treatment of hepatoma. Citation Format: Cheng-Heng Wu, Te-Wei Tseng, Chen-Yang Huang, Ping-Hsun Ou, Yi-Chen Lin, Pin-Jung Chen, Po-Ting Lin, Chan-Keng Yang, Wei Teng, Tsung-Han Wu, Yung-Chang Lin, Chun-Yen Lin. A novel cluster of cytolytic alpha/beta TCR+CD4−CD8−T cells originating from CD8+T cells with potent innate-like T cell killing ability: implicating adoptive T cell transfer therapy for 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 1520.

Abstract 2703: Targeting STING pathway as a therapeutic strategy against β-catenin driven immune escape and resistance to anti-PD1 therapy in hepatocellular carcinoma

Abstract β-catenin mutation occurs in around 40% of hepatocellular carcinoma(HCC); yet no precision therapies are available to treat this tumor subset. Immune checkpoint inhibitors (ICIs) are now widely used for the treatment of patients with advanced HCC. Unfortunately, preliminary evidence showed that β-catenin mutation drives resistance to anti-PD1 immunotherapy in HCC. In this study, we studied the molecular mechanisms underlying β-catenin mutation during HCC pathogenesis to develop novel therapeutics targeting β-Catenin-driven HCC. Firstly, three HCC cohorts were examined, including 70 untreated, 20 treated patients with nivolumab, and patients from a public TCGA database. By single-cell RNA sequencing analysis, we identified that anti-PD-1 treatment displayed effectiveness with the increase of CD27+CD8+ T-cell infiltration, correlating to improved prognosis with longer progression-free survival (PFS) and OS. Importantly, multiplex IHC staining showed that β-catenin activation is associated with significantly reduced CD8+CD27+ T cell infiltration in tumors (n=70), demonstrating that β-catenin promoted immune escape and resistance to anti-PD-1 treatment involved defective cytotoxic T-cell activity. Subsequently, we found that the interaction of β-Catenin with sirtuin 1 (SIRT1) mediated suppression of IRF3/IRF7 activity, resulting in inhibition of Type I interferon (IFN-I) production. However, by CRISPRi screening, we found that disruption of β-catenin-SIRT1 interaction via STING caused persistent high-level IFN-I response which increased the transcription of T-cell costimulatory molecule CD27 via IL10/STAT3/BATF signaling on infiltrated CD8+ T cells. This effect was also confirmed in the STING null mice, emphasizing that STING signaling is a therapeutic target to overcome β-catenin driven immune escape. Further, using hydrodynamic tail-vein injection murine HCC models, we found that the STING agonist inhibited β-catenin-SIRT1 activity, restored IFN signaling to rescue T-cell cytotoxic capacities, and enhanced the anti-PD-1 therapy efficacy. In conclusion, we demonstrated that STING reprogrammed β-Catenin mutated the HCC tumor microenvironment and a combination of STING and anti-PD-1 treatment represents an effective novel therapeutic strategy for HCC patients with β-Catenin mutation. Citation Format: Qingmei Zhang, Jiacheng Bi, Oscar Wai-Ho Yeung, Tao Ding, Wing-Lim Chan, Yueqin Zhu, Jiang Liu, Kevin Tak-Pan Ng, Kwan Man. Targeting STING pathway as a therapeutic strategy against β-catenin driven immune escape and resistance to anti-PD1 therapy 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 2703.

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.

Abstract 5629: MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas

Abstract Background: Immune escape remains a challenge in the treatment of hepatocellular carcinoma (HCC), one of the most common and deadly cancers in the world. The transcription factor, myocyte enhancer factor 2D (MEF2D), is important in the regulation of differentiation and adaptive responses in many cancers, such that we tested its role in HCC. Methods: We knocked down MEF2D in HCC cell lines and analyzed HCC tissues and cell lines by RNA sequencing, Western blot and immunohistochemistry. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation assay. Chromatin immunoprecipitation was used to analyze the regulation of CD70 transcription by MEF2D. H22 cells, with MEF2D knockout or without (controls), were injected into the livers of syngeneic BALB/c mice. Flow cytometry was used to analyze the function of T cells in tumors, spleens, and lymph nodes. Results: Through database searches, we noted that MEF2D and CD70 were upregulated in HCC and associated with shorter patient survival times. Compared to WT tumors, injection of MEF2D-knockdown HCC cells into immunocompetent mice led to smaller tumors with increased T cell infiltration, activation, and impaired T-regulatory (Treg) cell suppressive function. Mechanistically, we found that MEF2D binds to the promoter region of CD70 gene and activates its transcription. Moreover, acetylation of MEF2D enhances the binding of MEF2D to the CD70 promoter and further promotes its transcription. CD70 blocking antibody inhibited activation of the CD70-CD27 signaling axis in murine HCC tumors, leading to impaired immunosuppressive function of Tregs and enhanced T cell-mediated anti-tumor immune responses. Thus, regulation of the CD70-CD27 signaling axis by MEF2D affects the numbers and functions of Treg cells and thereby controls T cell infiltration. Conclusions: MEF2D in HCC cells increases the expression of CD70 and blocks T cell-mediated anti-tumor immunity via the CD70-CD27 signaling axis. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC. Citation Format: Fanhua Kong, Liqing Wang, Qifa Ye, Yan Xiong, Wayne W. Hancock. MEF2D regulates T cell function via the CD70-CD27 signaling axis and promotes immune escape in hepatocellular carcinomas [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 5629.