Orthotopic Hepatocellular Carcinoma Mouse Model Research Articles

WWOX tuning of oleic acid signaling orchestrates immunosuppressive macrophage polarization and sensitizes hepatocellular carcinoma to immunotherapy

BackgroundImmune checkpoint inhibitors (ICIs) are therapeutically effective for hepatocellular carcinoma (HCC) but are individually selective. This study examined the role of specific common fragile sites (CFSs) related gene in HCC...

Anlotinib potentiates anti-PD1 immunotherapy via transferrin receptor-dependent CD8+ T-cell infiltration in hepatocellular carcinoma.

The therapeutic potential of immune checkpoint blockade (ICB) extends across various cancers; however, its effectiveness in treating hepatocellular carcinoma (HCC) is frequently curtailed by both inherent and developed resistance. This research explored the effectiveness of integrating anlotinib (a broad-spectrum tyrosine kinase inhibitor) with programmed death-1 (PD-1) blockade and offers mechanistic insights into more effective strategies for treating HCC. Using patient-derived organotypic tissue spheroids and orthotopic HCC mouse models, we assessed the effectiveness of anlotinib combined with PD-1 blockade. The impact on the tumour immune microenvironment and underlying mechanisms were assessed using time-of-flight mass cytometry, RNA sequencing, and proteomics across cell lines, mouse models, and HCC patient samples. The combination of anlotinib with an anti-PD-1 antibody enhanced the immune response against HCC in preclinical models. Anlotinib remarkably suppressed the expression of transferrin receptor (TFRC) via the VEGFR2/AKT/HIF-1α signaling axis. CD8+ T-cell infiltration into the tumour microenvironment correlated with low expression of TFRC. Anlotinib additionally increased the levels of the chemokine CXCL14, crucial for attracting CD8+ T cells. CXCL14 emerged as a downstream effector of TFRC, exhibiting elevated expression following the silencing of TFRC. Importantly, low TFRC expression was also associated with a better prognosis, enhanced sensitivity to combination therapy, and a favourable response to anti-PD-1 therapy in patients with HCC. Our findings highlight anlotinib's potential to augment the efficacy of anti-PD-1 immunotherapy in HCC by targeting TFRC and enhancing CXCL14-mediated CD8+ T-cell infiltration. This study contributes to developing novel therapeutic strategies for HCC, emphasizing the role of precision medicine in oncology. Synergistic effects of anlotinib and anti-PD-1 immunotherapy demonstrated in HCC preclinical models. Anlotinib inhibits TFRC expression via the VEGFR2/AKT/HIF-1α pathway. CXCL14 upregulation via TFRC suppression boosts CD8+ T-cell recruitment. TFRC emerges as a potential biomarker for evaluating prognosis and predicting response to anti-PD-1-based therapies in advanced HCC patients.

Pilot study of humanized glypican-3-targeted zirconium-89 immuno-positron emission tomography for hepatocellular carcinoma.

Glypican-3 (GPC3)-targeted radioisotope immuno-positron emission tomography (immunoPET) may lead to earlier and more accurate diagnosis of hepatocellular carcinoma (HCC), thus facilitating curative treatment, decreasing early recurrence, and enhancing patient survival. We previously demonstrated reliable HCC detection using a zirconium-89-labeled murine anti-GPC3 antibody (89Zr-αGPC3M) for immunoPET. This study evaluated the efficacy of the humanized antibody successor (αGPC3H) to further clinical translation of a GPC3-based theranostic for HCC. In vitro αGPC3 binding to HepG2 cells was assessed by flow cytometry. In vivo 89Zr-αGPC3H and 89Zr-αGPC3M tumor uptake was evaluated by PET/CT and biodistribution studies in an orthotopic xenograft mouse model of HCC. αGPC3H maintained binding to GPC3 in vitro and 89Zr-αGPC3H immunoPET identified liver tumors in vivo. PET/CT and biodistribution analyses demonstrated high 89Zr-αGPC3H tumor uptake and tumor-to-liver ratios, with no difference between groups. Humanized αGPC3 successfully targeted GPC3 in vitro and in vivo. 89Zr-αGPC3H immunoPET had comparable tumor detection to 89Zr-αGPC3M, with highly specific tumor uptake, making it a promising strategy to improve HCC detection.

Chemically engineered mTOR-nanoparticle blockers enhance antitumour efficacy

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

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

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

Repolarization of Immunosuppressive Macrophages by Targeting SLAMF7-Regulated CCL2 Signaling Sensitizes Hepatocellular Carcinoma to Immunotherapy.

CCL2 upregulation caused by SLAMF7 deficiency in hepatocellular carcinoma cells induces immunosuppressive macrophage polarization and confers resistance to immune checkpoint blockade, providing potential biomarkers and targets to improve immunotherapy response in patients.

Knockdown of circZMIZ1 enhances the anti-tumor activity of CD8+ T cells to alleviate hepatocellular carcinoma.

ZMIZ1 acts as an oncogene in hepatocellular carcinoma (HCC). circZMIZ1 (hsa_circ_0018964) derives from ZMIZ1; its underlying mechanism in HCC has not been reported. Peripheral blood and peripheral blood mononuclear cells (PBMCs) were obtained from HCC patients and healthy volunteers. CD8+ T cells were sorted from PBMCs of HCC patients. Applying flow cytometry, cell apoptosis and the proportion of KCNJ2/CD8+ T cells were examined. The cytotoxicity of CD8+ T cells against HCC cells was evaluated. The interaction among circZMIZ1, miR-15a-5p, and KCNJ2 was investigated by dual luciferase assay, RNA immunoprecipitation, and RNA pull-down assay. An orthotopic mouse model of HCC was constructed by intrahepatic injection of H22 cells. Upregulation of circZMIZ1 and KCNJ2 and downregulation of miR-15a-5p were observed in peripheral blood and PBMCs of HCC patients. The proportion of KCNJ2/CD8+ T cells was also increased in HCC patients. circZMIZ1 knockdown restrained apoptosis of CD8+ T cells and elevated cytotoxicity of CD8+ T cells. Mechanically speaking, circZMIZ1 elevated KCNJ2 expression by sponging miR-15a-5p. miR-15a-5p inhibitor reversed circZMIZ1 silencing-mediated inhibition of apoptosis and promotion of cytotoxicity in CD8+ T cells. In vivo, orthotopic mice of HCC exhibited increased expression of circZMIZ1 and KCNJ2, elevated proportion of KCNJ2/CD8+ T cells, and decreased expression of miR-15a-5p. This work demonstrated that circZMIZ1 inhibited the anti-tumor activity of CD8+ T cells in HCC by regulating the miR-15a-5p/KCNJ2 axis. This provides a theoretical basis for the development of effective circZMIZ1 in tumor immunotherapy.

Bufalin suppresses hepatocellular carcinogenesis by targeting M2 macrophage-governed Wnt1/β-catenin signaling

BackgroundThe interplay of tumor-associated macrophages (TAMs) and tumor cells plays a key role in the development of hepatocellular carcinoma (HCC) and provides an important target for HCC therapy. The communication between them is still on the investigation. Bufalin, the active component derived from the traditional Chinese medicine (TCM) Chansu, has been evidenced to possess anti-HCC activity by directly suppressing tumor cells, while its immunomodulatory effect on the tumor microenvironment (TME) is unclear. PurposeTo explore the mechanism of M2 TAM-governed tumor cell proliferation and the inhibitory effect of bufalin on HCC growth by targeting M2 macrophages. MethodsMorphology and marker proteins were detected to evaluate macrophage polarization via microscopy and flow cytometry. Cellular proliferation and malignant transformation of HCC cells cultured with macrophage conditioned medium (CM) or bufalin-primed M2-CM, were assessed by cell viability, colony formation and soft agar assays. Regulations of gene transcription and protein expression and release were determined by RT-qPCR, immunoblotting, immunoprecipitation, ELISA and immunofluorescence. Tumorigenicity upon bufalin treatment was verified in orthotopic and diethylnitrosamine-induced HCC mouse model. ResultsIn this study, we first verified that M2 macrophages secreted Wnt1, which acted as a mediator to trigger β-catenin activation in HCC cells, leading to cellular proliferation. Bufalin suppressed HCC cell proliferation and malignant transformation by inhibiting Wnt1 release in M2 macrophages, and dose-dependently inhibited HCC progression in mice. Mechanistically, bufalin specially targeted to block Wnt1 transcription, thus inactivating β-catenin signaling cascade in HCC cells and leading to tumor regression in HCC mouse model. ConclusionThese results clearly reveal a novel potential of bufalin to suppress HCC through immunomodulation, and shed light on a new M2 macrophage-based modality of HCC immunotherapy, which additively enhances direct tumor-inhibitory efficacy of bufalin.

Fibroblast growth factor inhibition by molecular-targeted agents mitigates immunosuppressive tissue microenvironment in hepatocellular carcinoma

Background & aimsCombination immunotherapy refers to the use of immune checkpoint inhibitors (ICI) and molecular-targeted agents (MTA), which have recently been approved for the treatment of advanced hepatocellular carcinoma (HCC). Owing to its relatively low antitumor effect (up to 30%), sequential therapy following ICIs treatment is required in patients with HCC. This study aimed to determine the impact of MTAs on the tumor immune microenvironment (TIME).MethodsWe established immune syngeneic orthotopic HCC mouse models using Hep-55.1C and Hep-53.4, and treated them with MTAs (lenvatinib, sorafenib, regorafenib, cabozantinib, and DC101 as anti-vascular endothelial growth factor receptor-2 antibodies, and AZD4547 as a fibroblast growth factor receptor (FGFR)-1/2/3/4 inhibitor) for 2 weeks. Subsequently, alterations in the TIME caused by MTAs were evaluated using immunohistochemistry (antibodies for CD3, CD8, Foxp3, Granzyme B, Arginase-1, NK1.1, F4/80, CD11c, PD-1, and PD-L1). We conducted RNA-seq analysis using lenvatinib- and AZD4547-treated tumors. To confirm the clinical relevance of these findings, we analyzed the transcriptome data of human HCC cells (MHCC-97H) treated with various concentrations of lenvatinib for 24 h using RNA-seq data from the Gene Expression Omnibus database.ResultsThe number of Foxp3- and F4/80-positive cells in the TIME was decreased in many MTAs. Cabozantinib increased the numbers in NK1.1-, Granzyme B, and CD11c-positive cells. Lenvatinib and AZD4547 increased the number of CD8, Granzyme B, and PD-L1-positive cells. Gene ontology enrichment analysis revealed that lipid metabolism-related genes were downregulated by lenvatinib and AZD4547. In total, 161 genes downregulated by FGFR inhibition in rodent models overlapped with those downregulated by lenvatinib in human HCC cells.ConclusionsIn this study, we showed that cabozantinib activated the innate immune system, and lenvatinib and AZD4547, which commonly inhibit FGFR signaling, altered TIME to a hot immune state by downregulating lipid metabolism-related genes. These findings support the therapeutic use of combination immunotherapies.

Establishment of image-guided radiotherapy of orthotopic hepatocellular carcinoma mouse model.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Recently, developments in radiotherapy technology have led to radiotherapy becoming one of the main therapeutics of HCC. Therefore, a suitable animal model for radiotherapy of the orthotopic HCC mouse model is urgently needed. In the present study, Hepa1-6 cells were injected into the liver of C57BL/6 mice in situ to mimic the pathological characteristics of the original HCC. Tumor formation was monitored by applying magnetic resonance imaging techniques and verified by H&E histopathological staining, AFP staining, and Ki67 staining. A single dose of 10 Gy X-ray was applied to simulate clinical radiotherapy plans using image-guided radiotherapy (IGRT) equipment. The efficiency of radiotherapy was then assessed by examining tumor size and weight one week after radiation. Cleaved-caspase3 staining and TUNEL were used to assess apoptosis in tumor tissues. Intrahepatic tumor development was detected in the liver according using MRI. A high-density shadow could be seen 10 days after cell injection, which indicated the formation of HCC in vivo. The tumors grew steadily bigger, and underwent precision radiotherapy 20 days after injection. The typical pathological characteristics of HCC, such as large, deeply stained nuclei and irregular cell size, were visible with H&E staining. After radiotherapy, significantly higher expression of the immunohistochemical markers Ki67 and AFP were detected in tumor tissue than in the nearby normal tissue. Compared with the control group, the tumor volume (p = 0.05) and weight (p < 0.05) of the irradiated group were significantly reduced. In addition, a higher frequency of apoptosis was identified in irradiated HCC tumor tissue using the TUNEL and cleaved-caspase3 staining assay. In a well-established orthotopic HCC model, MRI was utilized to monitor the formation of tumors, and IGRT was used to simulate clinical radiotherapy. The present study could provide a suitable preclinical system for HCC radiotherapy-related studies.

Nanodelivery of scutellarin induces immunogenic cell death for treating hepatocellular carcinoma

Hepatocellular carcinoma (HCC) causes the immunosuppressive tumor microenvironment (TME) resistant to current immunotherapy. The immunogenic apoptosis (currently termed immunogenic cell death, ICD) of cancer cells may induce the adaptive immunity against tumors, thereby providing great potential for treating HCC. In this study, we have confirmed the potential of scutellarin (SCU, a flavonoid found in Erigeron breviscapus) for triggering ICD in HCC cells. To facilitate in vivo application of SCU for HCC immunotherapy, an aminoethyl anisamide-targeted polyethylene glycol-modified poly(lactide-co-glycolide) (PLGA-PEG-AEAA) was produced to facilitate SCU delivery in this study. The resultant nanoformulation (PLGA-PEG-AEAA.SCU) remarkably promoted blood circulation and tumor delivery in the orthotopic HCC mouse model. Consequently, PLGA-PEG-AEAA.SCU reversed the immune suppressive TME and achieved the immunotherapeutic efficacy, resulting in significantly longer survival of mice, without inducing toxicity. These findings uncover the ICD potential of SCU and provide a promising strategy for HCC immunotherapy.

Lenalidomide Promotes Melarsoprol-Activated cGAS-STING-Mediated Immunotherapy for Hepatocellular Carcinoma via Attenuating TNF-α Activity

Current immunotherapy has limited efficacy in hepatocellular carcinoma (HCC) due to the immunosuppressive tumor microenvironment (TME). The activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of the interferon genes (STING) pathway demonstrates great potential to unleash the immunosuppressive TME. The capacity of melarsoprol (MEL; an arsenic-containing drug) on activating the cGAS-STING pathway in HCC cells for antitumor immunotherapy was confirmed for the first time in this study. When MEL (particularly at high doses) activated the cGAS-STING pathway, however, HCC growth was not fully inhibited, mainly due to the hyperactivation of tumor necrosis factor α (TNF-α; a cytokine associated with the cGAS-STING pathway). Accordingly, lenalidomide (LEN; a clinically approved TNF-α inhibitor) was used to alleviate pro-tumorigenic effects of the MEL-activated cGAS-STING pathway while maintaining immunotherapeutic effects. To modulate the cGAS-STING pathway in vivo, a poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticle (NP) was used for co-delivery of MEL and LEN, and the co-loaded NP was coated with aminoethyl anisamide (AEAA, a ligand for Sigma-1 receptor)-modified erythrocyte membrane, forming a co-formulation. In this study, co-formulation was able to modulate the cGAS-STING-mediated efficacy and reverse the immunosuppressive TME in allograft and carcinogen-induced orthotopic HCC mouse models, respectively. Our study reveals an intrinsic hurdle to DNA damaging drug-mediated cGAS-STING monotherapy and provides a promising combination strategy to tackle immunosuppression in HCC.

A nanotherapeutic strategy that engages cytotoxic and immunosuppressive activities for the treatment of cancer recurrence following organ transplantation.

Long-term treatment with immunosuppressants is necessary to attenuate allograft rejection following organ transplantation (OT). Consequently, the overall survival of OT recipients with malignancies has been substantially compromised by tumour recurrence. Rapamycin (RAPA) is a clinically approved immunosuppressive agent with antitumour activity that is considered beneficial in preventing posttransplant tumour recurrence. However, the clinical outcome of RAPA is impeded by acquired drug resistance and its poor oral bioavailability. A nanotherapeutic strategy was developed by supramolecular assembly of RAPA into a polymer cytotoxic 7-ethyl-10-hydroxycamptothecin (SN38) prodrug nanoparticle (termed SRNP) for simultaneous codelivery of cytotoxic/immunosuppressive agents. Cell-based experiments were used to evaluate the cytotoxicity of SRNPs against hepatocellular carcinoma (HCC). The therapeutic efficacy of SRNPs was evaluated in multiple preclinical models including an orthotopic HCC mouse model, an orthotopic liver transplantation (OLT) rat model and a clinically relevant cancer-transplant model to examine its antitumour and immunosuppressive activity. The combination of SN38 with RAPA resulted in synergetic effects against HCC cells and alleviated RAPA resistance by abrogating Akt/mTOR signalling activation. SRNPs exhibited potent antitumour efficiency in the orthotopic HCC model while substantially prolonging the survival of allografts in the OLT model. In the cancer-transplant model that simultaneously bears tumour xenografts and skin allografts, SRNPs not only effectively inhibited tumour growth but also attenuated allograft damage. The nanotherapy presented here had enhanced efficacy against tumours and maintained satisfactory immunosuppressive activity and thus has great potential to improve the survival outcomes of patients with a high risk of tumour recurrence following OT. This work was supported by the National Natural Science Foundation of China (32171368 and 31671019), the Zhejiang Provincial Natural Science Foundation of China (LZ21H180001), the Zhejiang Province Preeminence Youth Fund (LR19H160002), and the Jinan Provincial Laboratory Research Project of Microecological Biomedicine (JNL-2022039c).

Navigating the immunometabolic heterogeneity of B cells in murine hepatocellular carcinoma at single cell resolution

Induction of antitumor immunity is critical for the therapeutic efficacy of hepatocellular carcinoma (HCC) immunotherapy. The cellular metabolic state underpins the effector function of immune cells, yet our understanding of the phenotypic and metabolic heterogeneity of B cells within HCC microenvironment is poorly developed. Herein, we investigated the composition, distribution, phenotype, function and metabolic profiles of B-cell subsets in HCC and adjacent liver tissues from an orthotopic HCC mouse model using single-cell RNA sequencing (scRNA-seq). Our results identified six B-cell clusters, which can be classified into plasma cells and activated and exhausted B cells according to marker expression, functional and temporal distribution. Exhausted B cells exhibited low metabolic activities and impaired effector functions. Activated B and plasma cells showed higher metabolic activity than exhausted B cells, but there were clear differences in their metabolic profiles. In addition, we found that the effector function of exhausted B cells was further diminished in HCC tissues compared with adjacent liver tissues, but their metabolic activity was significantly enhanced. Collectively, we comprehensively characterized the metabolic profile and alterations in B-cell subsets in HCC, which contributes to the understanding of B-cell immunology in HCC and lays the foundation for exploring novel targets in HCC immunotherapy.

Abstract 4452: FASN inhibition overcomes immune evasion by upregulating MHC-I in hepatocellular carcinoma

Abstract Immune evasion is one of the important factors leading to the progression of hepatocellular carcinoma(HCC). Restricted antigen presentation by decrease of MHC-I remains a huge barrier to triggering an immune attack. However, knowledge is limited on how to enhance antigen presentation of cancer cells. Here we identify that FASN inhibition by shRNA and inhibitors could boost MHC-I level on HCC cell surface. FASN deficiency also promotes antigen presentation and augments CD8+ T cell cytotoxicity. Mechanistically, FASN knock-down decreases the degradation of MHC-I in lysosomes. Moreover, ZDHHC3-mediated palmitoylation participates in this process. In vivo, orthotopic HCC mouse model shows that Fasn knock-down greatly improves the cytotoxicity of tumor-infiltrating CD8+ T cells and suppresses HCC tumor growth, indicating that inhibiting FASN is a promising way to enhance the efficiency of immunotherapy in HCC. Citation Format: Jiao Huang, Wai Ying Tsang, Xiaona Fang, Yu Zhang, Jie Luo, Lanqi Gong, Baifeng Zhang, Ching Ngar Wong, Beilei Liu, Jinlin Huang, Yuma Yang, Shan Liu, Zhihong Li, Xinyuan Guan. FASN inhibition overcomes immune evasion by upregulating MHC-I in hepatocellular carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4452.

Disruption of SLFN11 Deficiency–Induced CCL2 Signaling and Macrophage M2 Polarization Potentiates Anti–PD-1 Therapy Efficacy in Hepatocellular Carcinoma

The therapeutic effect of immune checkpoint inhibitors (ICIs) is poor in hepatocellular carcinoma (HCC) and varies greatly among individuals. Schlafen (SLFN) family members have important functions in immunity and oncology, but their roles in cancer immunobiology remain unclear. We aimed to investigate the role of the SLFN family in immune responses against HCC. Transcriptome analysis was performed in human HCC tissues with or without response to ICIs. A humanized orthotopic HCC mouse model and a co-culture system were constructed, and cytometry by time-of-flight technology was used to explore the function andmechanism of SLFN11 in the immune context of HCC. SLFN11 was significantly up-regulated in tumors that responded to ICIs. Tumor-specific SLFN11 deficiency increased the infiltration of immunosuppressive macrophages and aggravated HCC progression. HCC cells with SLFN11 knockdown promoted macrophage migration and M2-like polarization in a C-C motif chemokine ligand 2-dependent manner, which in turn elevated their own PD-L1 expression by activating the nuclear factor-κB pathway. Mechanistically, SLFN11 suppressed the Notch pathway and C-C motif chemokine ligand 2 transcription by binding competitively with tripartite motif containing 21 to the RNA recognition motif 2 domain of RBM10, thereby inhibiting tripartite motif containing 21-mediated RBM10 degradation to stabilize RBM10 and promote NUMB exon 9 skipping. Pharmacologic antagonism of C-C motif chemokine receptor 2 potentiated the antitumor effect of anti-PD-1 in humanized mice bearing SLFN11 knockdown tumors. ICIs were more effective in patients with HCC with high serum SLFN11 levels. SLFN11 serves as a critical regulator of microenvironmental immune properties and an effective predictive biomarker of ICIs response in HCC. Blockade of C-C motif chemokine ligand 2/C-C motif chemokine receptor 2 signaling sensitized SLFN11low HCC patients to ICI treatment.

HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

BackgroundHepatic inflammation is a common initiator of liver diseases and considered as the primary driver of hepatocellular carcinoma (HCC). However, the precise mechanism of inflammation-induced HCC development and immune evasion remains elusive and requires extensive investigation. This study sought to identify the new target that is involved in inflammation-related liver tumorigenesis.MethodsRNA-sequencing (RNA-seq) analysis was performed to identify the differential gene expression signature in primary human hepatocytes treated with or without inflammatory stimulus. A giant E3 ubiquitin protein ligase, HECT domain and RCC1-like domain 2 (HERC2), was identified in the analysis. Prognostic performance in the TCGA validation dataset was illustrated by Kaplan–Meier plot. The functional role of HERC2 in HCC progression was determined by knocking out and over-expressing HERC2 in various HCC cells. The precise molecular mechanism and signaling pathway networks associated with HERC2 in HCC stemness and immune evasion were determined by quantitative real-time PCR, immunofluorescence, western blot, and transcriptomic profiling analyses. To investigate the role of HERC2 in the etiology of HCC in vivo, we applied the chemical carcinogen diethylnitrosamine (DEN) to hepatocyte-specific HERC2-knockout mice. Additionally, the orthotopic transplantation mouse model of HCC was established to determine the effect of HERC2 during HCC development.ResultsWe found that increased HERC2 expression was correlated with poor prognosis in HCC patients. HERC2 enhanced the stemness and PD-L1-mediated immune evasion of HCC cells, which is associated with the activation of signal transducer and activator of transcription 3 (STAT3) pathway during the inflammation-cancer transition. Mechanically, HERC2 coupled with the endoplasmic reticulum (ER)-resident protein tyrosine phosphatase 1B (PTP1B) and limited PTP1B translocation from ER to ER-plasma membrane junction, which ameliorated the inhibitory role of PTP1B in Janus kinase 2 (JAK2) phosphorylation. Furthermore, HERC2 knockout in hepatocytes limited hepatic PD-L1 expression and ameliorated HCC progression in DEN-induced mouse liver carcinogenesis. In contrast, HERC2 overexpression promoted tumor development and progression in the orthotopic transplantation HCC model.ConclusionOur data identified HERC2 functions as a previously unknown modulator of the JAK2/STAT3 pathway, thereby promoting inflammation-induced stemness and immune evasion in HCC.

Caveolin-mediated cytosolic delivery of spike nanoparticle enhances antitumor immunity of neoantigen vaccine for hepatocellular carcinoma.

Rationale: Although neoantigen-based cancer vaccines have shown promise in various solid tumors, limited immune responses and clinical outcomes have been reported in patients with advanced disease. Cytosolic transport of neoantigen and adjuvant is required for the activation of intracellular Toll-like receptors (TLRs) and cross-presentation to prime neoantigen-specific CD8+T cells but remains a significant challenge. Methods: In this study, we aimed to develop a virus-like silicon vaccine (V-scVLPs) with a unique spike topological structure, capable of efficiently co-delivering a hepatocellular carcinoma (HCC)-specific neoantigen and a TLR9 agonist to dendritic cells (DCs) to induce a robust CD8+T cell response to prevent orthotopic tumor growth. We evaluated the antitumor efficacy of V-scVLPs by examining tumor growth and survival time in animal models, as well as analyzing tumor-infiltrating CD8+T cells and cytokine responses in the tumor microenvironment (TME). To evaluate the synergistic efficacy of V-scVLPs in combination with α-TIM-3 in HCC, we used an orthotopic HCC mouse model, a lung metastasis model, and a tumor rechallenge model after hepatectomy. Results: We found that V-scVLPs can efficiently co-deliver the hepatocellular carcinoma (HCC)-specific neoantigen and the TLR9 agonist to DCs via caveolin-mediated endocytosis. This advanced delivery strategy results in efficient lymph node draining of V-scVLPs to activate lymphoid DC maturation for promoting robust CD8+T cells and central memory T cells responses, which effectively prevents orthotopic HCC tumor growth. However, in the established orthotopic liver tumor models, the inhibitory receptor of TIM-3 was significantly upregulated in tumor-infiltrating CD8+T cells after immunization with V-scVLPs. Blocking the TIM-3 signaling further restored the antitumor activity of V-scVLPs-induced CD8+T cells, reduced the proportion of regulatory T cells, and increased the levels of cytokines to alter the tumor microenvironment to efficiently suppress established orthotopic HCC tumor growth, and inhibit lung metastasis as well as recurrence after hepatectomy. Conclusion: Overall, the developed novel spike nanoparticles with efficient neoantigen and adjuvant intracellular delivery capability holds great promise for future clinical translation to improve HCC immunotherapy.

Delivery of human natural killer cell-derived exosomes for liver cancer therapy: an in vivo study in subcutaneous and orthotopic animal models

Exosomes are nanosized extracellular vesicles secreted by various cell types, including those of the immune system, such as natural killer (NK) cells. They play a role in intercellular communication by transporting signal molecules between the cells. Recent studies have reported that NK cell-derived exosomes (NK-exo) contain cytotoxic proteins-induced cell death. However, the characteristics and potential functions of NK-exo, especially for the liver cancer are poorly understood. In this study, we investigated the anti-tumor effects of NK-exo in the primary liver cancer, hepatocellular carcinoma (HCC), using the orthotopic and subcutaneous tumor model. We found that NK-exo expressed both typical exosomal markers (e.g. CD63, CD81, and Alix) and cytotoxic proteins (e.g. perforin, granzyme B, FasL, and TRAIL). NK-exo were selectively taken up by HCC cells (e.g. Hep3B, HepG2, and Huh 7). Interestingly, Hep3B cells induced the highest cytotoxicity compared with HepG2 and Huh7 cells, and substantially enhanced the apoptosis by NK-exo. Furthermore, we demonstrated that NK-exo inhibited the phosphorylation of serine/threonine protein kinases (e.g. AKT and ERK1/2), and enhanced the activation of specific apoptosis markers (e.g. caspase-3, -7, -8, -9, and PARP) in Hep3B cells. NK-exo also exhibit the active targeting ability and potent therapeutic effects in both orthotopic and subcutaneous HCC mouse models. Overall, these results suggest that NK-exo indicate strong anti-tumor effects in HCC, which are mediated by novel regulatory mechanisms involved in serine/threonine kinase pathway-associated cell proliferation and caspase activation pathway-associated apoptosis.

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...