Immune Responses In Hepatocellular Carcinoma Research Articles

Targeting mitochondrial quality control of T cells: Regulating the immune response in HCC.

Most of the primary hepatocellular carcinoma (HCC) develops from Viral Hepatitis including Hepatitis B virus, Hepatitis C Virus, and Nonalcoholic Steatohepatitis. Herein, T cells play crucial roles combined with chronic inflammation and chronic viral infection. However, T cells are gradually exhausted under chronic antigenic stimulation, which leads to T cell exhaustion in the tumor microenvironment, and the exhaustion is associated with mitochondrial dysfunction in T cells. Meanwhile, mitochondria play a crucial role in altering T cells’ metabolism modes to achieve desirable immunological responses, wherein mitochondria maintain quality control (MQC) and promote metabolism regulation in the microenvironment. Although immune checkpoint inhibitors have been widely used in clinical practice, there are some limitations in the therapeutic effect, thus combining immune checkpoint inhibitors with targeting mitochondrial biogenesis may enhance cellular metabolic adaptation and reverse the exhausted state. At present, several studies on mitochondrial quality control in HCC have been reported, however, there are gaps in the regulation of immune cell function by mitochondrial metabolism, particularly the modulating of T cell immune function. Hence, this review summarizes and discusses existing studies on the effects of MQC on T cell populations in liver diseases induced by HCC, it would be clued by mitochondrial quality control events.

Predicting prognosis and immune responses in hepatocellular carcinoma based on N7-methylguanosine-related long noncoding RNAs.

Background: Hepatocellular carcinoma (HCC), which has high rates of recurrence and metastasis and is the main reason and the most common tumor for cancer mortality worldwide, has an unfavorable prognosis. N7-methylguanosine (m7G) modification can affect the formation and development of tumors by affecting gene expression and other biological processes. In addition, many previous studies have confirmed the unique function of long noncoding RNAs (lncRNAs) in tumor progression; however, studies exploring the functions of m7G-related lncRNAs in HCC patients has been limited. Methods: Relevant RNA expression information was acquired from The Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov), and m7G-related lncRNAs were identified via gene coexpression analysis. Afterward, univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and multivariate regression analyses were implemented to construct an ideal risk model whose validity was verified using Kaplan–Meier survival, principal component, receiver operating characteristic (ROC) curve, and nomogram analyses. In addition, the potential functions of lncRNAs in the novel signature were explored through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and gene set enrichment analysis (GSEA). At last, in both risk groups and subtypes classified based on the expression of the risk-related lncRNAs, we analyzed the immune characteristics and drug sensitivity of patients. Results: After rigorous screening processes, we built a model based on 11 m7G-related lncRNAs for predicting patient overall survival (OS). The results suggested that the survival status of patients with high-risk scores was lower than that of patients with low-risk scores, and a high-risk score was related to malignant clinical features. Cox regression analysis showed that the m7G risk score was an independent prognostic parameter. Moreover, immune cell infiltration and immunotherapy sensitivity differed between the risk groups. Conclusion: The m7G risk score model constructed based on 11 m7G-related lncRNAs can effectively assess the OS of HCC patients and may offer support for making individualized treatment and immunotherapy decisions for HCC patients.

Construction of a Necroptosis-Associated Long Non-Coding RNA Signature to Predict Prognosis and Immune Response in Hepatocellular Carcinoma.

Background: Necroptosis is a form of programmed cell death, and studies have shown that long non-coding RNA molecules (lncRNAs) can regulate the process of necroptosis in various cancers. We sought to screen lncRNAs associated with necroptosis to predict prognosis and tumor immune infiltration status in patients with hepatocellular carcinoma (HCC). Methods: Transcriptomic data from HCC tumor samples and normal tissues were extracted from The Cancer Genome Atlas database. Necroptosis-associated lncRNAs were obtained by co-expression analysis. Necroptosis-associated lncRNAs were then screened by Cox regression and least absolute shrinkage and selection operator methods to construct a risk model for HCC. The models were also validated and evaluated by Kaplan-Meier analysis, univariate and multivariate Cox regression, and time-dependent receiver operating characteristic (ROC) curves. In addition, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment, gene set enrichment, principal component, immune correlation, and drug sensitivity analyses were applied to assess model risk groups. To further differentiate the immune microenvironment of different HCC subtypes, the entire dataset was divided into three clusters, based on necroptosis-associated lncRNAs, and a series of analyses performed. Results: We constructed a model comprising four necroptosis-associated lncRNAs: POLH-AS1, DUXAP8, AC131009.1, and TMCC1-AS1. Overall survival (OS) duration was significantly longer in patients classified as low-risk than those who were high-risk, according to our model. Univariate and multivariate Cox regression analyses further confirmed risk score stability. The analyzed models had area under the ROC curve values of 0.786, 0.713, and 0.639 for prediction of 1-, 3-, and 5-year OS, respectively, and risk score was significantly associated with immune cell infiltration and ESTIMATE score. In addition, differences between high and low-risk groups in predicted half-maximal inhibitory concentration values for some targeted and chemical drugs, providing a potential basis for selection of treatment approach. Finally, cluster analysis facilitated more refined differentiation of the immune microenvironment in patients with HCC and may allow prediction of the effectiveness of immune checkpoint inhibitors. Conclusions: This study contributes to understanding of the function of necroptosis-related lncRNAs in predicting the prognosis and immune infiltration status of HCC. The risk model constructed and cluster analysis provide a basis for predicting the prognosis of patients with HCC and to inform the selection of immunotherapeutic strategies.

Construction of a Novel Clinical Stage-Related Gene Signature for Predicting Outcome and Immune Response in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) with high heterogeneity is one of the most frequent malignant tumors. However, there were no studies to create a clinical stage-related gene signature for HCC patients. Differentially expressed genes (DEGs) associated with clinical stage of HCC were analyzed based on TCGA datasets. Functional enrichment analysis was carried out by the use of stage-related DEGs. Then, the least absolute shrinkage and selection operator (LASSO) regression and univariate Cox regression were performed to reduce the overfit and the number of genes for further analysis. Next, survival and ROC assays were carried out to demonstrate the model using TCGA. Functional analysis and immune microenvironment analysis related to stage-related DEGs were performed. Reverse transcriptase polymerase chain reaction (RT-PCR) and Cell Counting Kit-8 (CCK-8) assays were applied to examine the expression and function of PNCK in HCC. In this research, there were 21 DEGs between HCC specimens with stage (I-II) and HCC specimens with stage (III-IV), including 20 increased genes and 1 decreased genes. A novel seven-gene signature (including PITX2, PNCK, GLIS1, SCNN1G, MMP1, ZNF488, and SHISA9) was created for the prediction of outcomes of HCC patients. The ROC curves confirmed the prognostic value of the new model. Cox assays demonstrated that the seven-gene signature can independently forecast overall survival. The immune analysis revealed that patients with low risk score exhibited more immune activities. Moreover, we confirmed that PNCK expressions were distinctly increased in HCC, and its silence suppressed the proliferation of HCC cells. Overall, our research offered a robust and reliable gene signature which displayed an important value in the prediction of overall survival of HCC patients and might deliver more effective personalized therapies.

Abstract 3130: IL-27 signaling regulates anti-cancer immune response in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer with poor survival and limited therapeutic options. HCC development is accompanied by underlying chronic inflammation, which represents a major unifying mechanism for tumor promotion. While some tumor-promoting inflammatory mechanisms had been proposed, the identity of immune mechanisms controlling anti-cancer immunity in HCC remain poorly understood. Interleukin (IL)-27 receptor signaling plays an anti-inflammatory role in a variety of infectious and chronic inflammatory diseases. Here, using genetic and pharmacological approaches we found that IL-27 receptor (IL-27R) signaling promotes HCC development in vivo. Genetic loss of IL-27R suppressed HCC in both carcinogen-induced and non-alcoholic steatohepatitis (NASH)-driven models. Mechanistically, the pro-tumorigenic effect was mediated by an immunoregulatory role of IL-27R within the tumor microenvironment, particularly the suppression of cytotoxic Natural killer (NK) cells. Single-cell RNA analysis further established the role of IL-27R signaling in restraining cytotoxic populations of NK cells. IL-27R ablation enhanced the accumulation and activation of cytotoxic NK cells during acute liver injury and in HCC tumors, while depletion or functional impairment of NK cells abrogated the effect of genetic IL-27R disruption. Taken together, our data suggest an unexpected role of IL-27R signaling as a novel immunological checkpoint regulating innate cytotoxic cell activity and promoting development of HCC of different etiologies. Citation Format: Aleksandra M. Mazitova, Turan Aghayev, Jennifer Fang, Amiran Dzutsev, Giorgio Trinchieri, Kerry S. Campbell, Sergei I. Grivennikov, Ekaterina K. Koltsova. IL-27 signaling regulates anti-cancer immune response in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3130.

Establishment of a lncRNA-Based Prognostic Gene Signature Associated With Altered Immune Responses in HCC.

Hepatocellular carcinoma (HCC) is a common malignancy with higher mortality, and means are urgently needed to improve the prognosis. T cell exclusion (TCE) plays a pivotal role in immune evasion, and lncRNAs represent a large group of tumor development and progression modulators. Using the TCGA HCC dataset (n=374), we identified 2752 differentially expressed and 702 TCE-associated lncRNAs, of which 336 were in both groups. As identified using the univariate Cox regression analysis, those associated with overall survival (OS) were subjected to the LASSO-COX regression analysis to develop a prognosis signature. The model, which consisted of 11 lncRNAs and was named 11LNCPS for 11-lncRNA prognosis signature, was validated and performed better than two previous models. In addition to OS and TCE, higher 11LNCPS scores had a significant correlation with reduced infiltrations of CD8+ T cells and dendritic cells (DCs) and decreased infiltrations of Th1, Th2, and pro B cells. As expected, these infiltration alterations were significantly associated with worse OS in HCC. Analysis of published data indicates that HCCs with higher 11LNCPS scores were transcriptomically similar to those that responded better to PDL1 inhibitor. Of the 11LNCPS lncRNAs, LINC01134 and AC116025.2 seem more crucial, as their upregulations affected more immune cell types’ infiltrations and were significantly associated with TCE, worse OS, and compromised immune responses in HCC. LncRNAs in the 11LNCPS impacted many cancer-associated biological processes and signaling pathways, particularly those involved in immune function and metabolism. The 11LNCPS should be useful for predicting prognosis and immune responses in HCC.

Modulation of the Gut Microbiome to Improve Clinical Outcomes in Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma (HCC), the most common form of liver cancer, is the third leading cause of cancer-related mortality worldwide. Recent research indicates that altering the gut environment affects liver immune response and cancer progression, through what is known as the ‘gut–liver axis’. HCC patients have dampened anticancer responses, but through modulating the gut environment, there is potential to reinvigorate these ‘exhausted’ immune cells to target tumors. In promising research on melanoma, transplanting stool from healthy donors through faecal microbiota transplantation (FMT) resulted in increased response to immunotherapy treatment in patients who were previously nonresponding. However, manipulating the gut environment as a therapeutic option in HCC remains to be explored. In this review, we explore the mechanisms through which this occurs, including how the gut environment affects gut barrier function, bacterial sensing, and liver immune responses, and how FMT may be a potential therapy for HCC patients nonresponsive to immunotherapy.Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Recently, the gut microbiota has been shown to be closely linked to modulation of the immune and inflammatory responses, hence its potential as a therapeutic target. Although still under intense investigation, there exists a ‘gut–liver axis’ that links changes in the gut to the liver. In this regard, composition of gut microbiota and related metabolites, such as bile acids and short-chain fatty acids, have been shown to orchestrate key immune–metabolic events in liver disease and liver cancer. As hepatic immune cells are important determinants of antitumor responses, it is now increasingly recognized that the gut–liver axis plays a key role in influencing the intrahepatic immune response in HCC to favor a pro- or antitumor immune milieu. Hence, modulation of gut microbiota is potentially an attractive option to reinvigorate the antitumor responses. In this regard, promising evidence from melanoma preclinical and clinical studies has demonstrated the efficacy of gut-based intervention in reinvigorating the antitumor responses and improving responses to immunotherapy. However, the role of gut-based interventions as a therapeutic option in HCC remains to be elucidated. This review details how the gut microbiota and bacterial metabolites affect gut barrier function and ultimately immune response in HCC and raises the question of the potential of gut-based interventions as an adjunct therapy for patients with HCC receiving immunotherapy.

T-cell exhaustion and residency dynamics inform clinical outcomes in hepatocellular carcinoma

Despite recent translation of immunotherapies into clinical practice, the immunobiology of hepatocellular carcinoma (HCC), in particular the role and clinical relevance of exhausted and liver-resident T cells remain unclear. We therefore dissected the landscape of exhausted and resident T cell responses in the peripheral blood and tumor microenvironment of patients with HCC. Lymphocytes were isolated from the blood, tumor and tumor-surrounding liver tissue of patients with HCC (n= 40, n= 10 treated with anti-PD-1 therapy). Phenotype, function and response to anti-PD-1 were analyzed by mass and flow cytometry exvivo and invitro, tissue residence was further assessed by immunohistochemistry and imaging mass cytometry. Gene signatures were analyzed in silico. We identified significant enrichment of heterogeneous populations of exhausted CD8+ T cells (TEX) in the tumor microenvironment. Strong enrichment of severely exhausted CD8 T cells expressing multiple immune checkpoints in addition to PD-1 was linked to poor progression-free and overallsurvival. In contrast, PD-1 was also expressed on a subset of more functional and metabolically active CD103+ tissue-resident memory T cells (TRM) that expressed few additional immune checkpoints and were associated with better survival. TEX enrichment was independent of BCLC stage, alpha-fetoprotein levels or age as a variable for progression-free survival in our cohort. These findings were in line with in silico gene signature analysis of HCC tumor transcriptomes from The Cancer Genome Atlas. A higher baseline TRM/TEX ratio was associated with disease control in anti-PD-1-treated patients. Our data provide information on the role of peripheral and intratumoral TEX-TRM dynamics in determining outcomes in patients with HCC. The dynamics between exhausted and liver-resident T cells have implications for immune-based diagnostics, rational patient selection and monitoring during HCC immunotherapies. The role of the immune response in hepatocellular carcinoma (HCC) remains unclear. T cells can mediate protection against tumor cells but are frequently dysfunctional andexhausted in cancer. We found that patients with a predominance of exhausted CD8+ T cells (TEX) had poor survivalcompared to patients with a predominance of tissue-resident memory T cells (TRM). This correlated with the molecular profile, metabolic and functional status of these cell populations. The enrichment of TEX was independently associated with prognosis in addition to disease stage, age and tumor markers. A high TRM proportion was also associated with better outcomes following checkpoint therapy. Thus, these T-cell populations are novel biomarkers with relevance in HCC.

Inflamed and non-inflamed classes of HCC: a revised immunogenomic classification

ObjectiveWe previously reported a characterisation of the hepatocellular carcinoma (HCC) immune contexture and described an immune-specific class. We now aim to further delineate the immunogenomic classification of HCC to incorporate...

Prediction of Hepatocellular Carcinoma Prognosis and Immune Cell Infiltration Using Gene Signature Associated with Inflammatory Response.

It has been demonstrated that the inflammatory response influences cancer development and can be used as a prognostic biomarker in various tumors. However, the relevance of genes associated with inflammatory responses in hepatocellular carcinoma (HCC) remains unknown. The Cancer Genome Atlas (TCGA) database was analyzed using weighted gene coexpression network analysis (WGCNA) and differential analysis to discover essential inflammatory response-related genes (IFRGs). Cox regression studies, both univariate and multivariate, were employed to develop a prognostic IFRGs signature. Additionally, Gene Set Enrichment Analysis (GSEA) was used to deduce the biological function of the IFRGs signature. Finally, we estimated immune cell infiltration using a single sample GSEA (ssGSEA) and x-cell. Our results revealed that, among the major HCC IFRGs, two (DNASE1L3 and KLKB1) were employed to create a predictive IFRG signature. The IFRG signature could correctly predict overall survival (O.S) as per Kaplan-Meier time-dependent roc curves analysis. It was also linked to pathological tumor stage and T stage and might be used as a prognostic predictor in HCC. GSEA analysis concluded that the IFRG signature might influence the immune response in HCC. Immunological cell infiltration and immune checkpoint molecule expression differed in the high-risk and low-risk groups. As a result of our findings, DNASILE may play a role in the tumor microenvironment. However, more research is necessary to confirm the role of DNASE1L3 and KLKB1.

Personalized neoantigen vaccine prevents postoperative recurrence in hepatocellular carcinoma patients with vascular invasion

BackgroundClinically, prophylactic anti-recurrence treatments for hepatocellular carcinoma (HCC) patients after radical surgery are extremely limited. Neoantigen based vaccine can generate robust anti-tumor immune response in several solid tumors but whether it could induce anti-tumor immune response in HCC and serve as a safe and effective prophylactic strategy for preventing postoperative HCC recurrence still remain largely unclear.MethodsPersonalized neoantigen vaccine was designed and immunized for 10 HCC patients with high risk of postoperative recurrence in a prime-boost schedule. The safety and immune response were assessed through adverse events, tissue sequencing, ELISpot, TCR sequencing. The clinical response was evaluated by recurrence-free survival (RFS) and personalized circulating tumor DNA (ctDNA) sequencing.ResultsIn the 10 enrolled patients, no obvious adverse events were observed during neoantigen vaccinations. Until the deadline of clinical trial, 8 of 10 patients were confirmed with clinical relapse by imaging, the other 2 patients remained relapse-free. From receiving first neoantigen vaccination, the median RFS of 10 patients were 7.4 months. Among 7 patients received all planned neoantigen vaccinations, 5 of them demonstrated neoantigen-induced T cell responses and have significantly longer RFS after radical surgery than other 5 patients without responsive neoantigens or only with prime vaccination and propensity scores matching control patients (p = 0.035). Moreover, tracking personalized neoantigen mutations in ctDNA could provide real-time evaluation of clinical response in HCC patients during neoantigen vaccination and follow up.ConclusionPersonalized neoantigen vaccine is proved as a safe, feasible and effective strategy for HCC anti-recurrence, and its progression could be sensitively monitored by corresponding neoantigen mutations in ctDNA, and thus provided solid information for individualized medicine in HCC.Trial registrationThis study was registered at Chinese Clinical Trial Registry; Registration number: ChiCTR1900020990.

Construction of a Ferroptosis-Related Nine-lncRNA Signature for Predicting Prognosis and Immune Response in Hepatocellular Carcinoma.

Ferroptosis is an iron-dependent cell death process that plays important regulatory roles in the occurrence and development of cancers, including hepatocellular carcinoma (HCC). Moreover, the molecular events surrounding aberrantly expressed long non-coding RNAs (lncRNAs) that drive HCC initiation and progression have attracted increasing attention. However, research on ferroptosis-related lncRNA prognostic signature in patients with HCC is still lacking. In this study, the association between differentially expressed lncRNAs and ferroptosis-related genes, in 374 HCC and 50 normal hepatic samples obtained from The Cancer Genome Atlas (TCGA), was evaluated using Pearson’s test, thereby identifying 24 ferroptosis-related differentially expressed lncRNAs. The least absolute shrinkage and selection operator (LASSO) algorithm and Cox regression model were used to construct and validate a prognostic risk score model from both TCGA training dataset and GEO testing dataset (GSE40144). A nine-lncRNA-based signature (CTD-2033A16.3, CTD-2116N20.1, CTD-2510F5.4, DDX11-AS1, LINC00942, LINC01224, LINC01231, LINC01508, and ZFPM2-AS1) was identified as the ferroptosis-related prognostic model for HCC, independent of multiple clinicopathological parameters. In addition, the HCC patients were divided into high-risk and low-risk groups according to the nine-lncRNA prognostic signature. The gene set enrichment analysis enrichment analysis revealed that the lncRNA-based signature might regulate the HCC immune microenvironment by interfering with tumor necrosis factor α/nuclear factor kappa-B, interleukin 2/signal transducers and activators of transcription 5, and cytokine/cytokine receptor signaling pathways. The infiltrating immune cell subtypes, such as resting memory CD4(+) T cells, follicular helper T cells, regulatory T cells, and M0 macrophages, were all significantly different between the high-risk group and the low-risk group as indicated in Spearman’s correlation analysis. Moreover, a substantial increase in the expression of B7H3 immune checkpoint molecule was found in the high-risk group. Our findings provided a promising insight into ferroptosis-related lncRNAs in HCC and a personalized prediction tool for prognosis and immune responses in patients.

Abstract 1909: Pan-TGF-β inhibition with PD-1 blockade as a combination treatment strategy to augment anti-tumor immune response in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide, accounting for more than 700,000 deaths per year. Although transforming growth factor beta (TGF-β) can function as a tumor-suppressor in premalignant cells, patient data suggest that it plays a role in the progression of HCC. Recently, immunotherapy targeting PD-1 has been approved for HCC, however, success is limited to only ~17% objective response rate in the treated HCC patients. Analysis of genomic data from two HCC patient cohorts revealed that TGF-β pathway is highly activated in patients with advanced HCC. Collectively, these findings indicate that targeting TGF-β in combination with PD-1 blockade may be a compelling therapeutic strategy. Therefore, using two syngeneic mouse models of HCC (Hepa1-6 and H22), we examined the effect of a pan-TGF-β neutralizing antibody (1D11) in combination with PD-1 blockade. Results from our study show that co-inhibiting TGF-β with PD-1 resulted in significant tumor growth inhibition in both HCC models. Flow cytometry analysis of the tumor revealed the combination treatment group exhibited a reduction in potentially suppressive tumor-associated-macrophages relative to either single agent groups. In addition, we show that combination therapy was associated with increase in the intra-tumoral levels of proinflammatory cytokines - IFNγ, IL-1β, TNF-α as well as enhanced expression of anti-tumor immune related genes. Based on these data, clinical testing of SAR439459 (TGF-β antibody) and Cemiplimab (anti-PD1) combination has recently been initiated in HCC patients (NCT03192345). Citation Format: Natalia Malkova, Joachim Theilhaber, Mikhail Levit, Tatiana Tolstykh, Andrew Hebert, Kevin Atchison, Adalis Maisonet, Sara Sara Sinicropi-Yao, Alexei Protopopov, Jack Pollard, Sukhvinder Sidhu, Tun Tun Lin, Lily Pao, Dmitri Wiederschain, Sharad K. Sharma. Pan-TGF-β inhibition with PD-1 blockade as a combination treatment strategy to augment anti-tumor immune response in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1909.

Prospects and Challenges for T Cell-Based Therapies of HCC.

The scope of therapeutic options for the treatment of hepatocellular carcinoma (HCC) has recently been expanded by immunotherapeutic regimens. T cell-based therapies, especially in combination with other treatments have achieved far better outcomes compared to conventional treatments alone. However, there is an emerging body of evidence that eliciting T cell responses in immunotherapeutic approaches is insufficient for favorable outcomes. Immune responses in HCC are frequently attenuated in the tumor microenvironment (TME) or may even support tumor progress. Hence, therapies with immune checkpoint inhibitors or adoptive cell therapies appear to necessitate additional modification of the TME to unlock their full potential. In this review, we focus on immunotherapeutic strategies, underlying molecular mechanisms of CD8 T cell immunity, and causes of treatment failure in HCC of viral and non-viral origin. Furthermore, we provide an overview of TME features in underlying etiologies of HCC patients that mediate therapy resistance to checkpoint inhibition and discuss strategies from the literature concerning current approaches to these challenges.

Identification METTL18 as a Potential Prognosis Biomarker and Associated With Immune Infiltrates in Hepatocellular Carcinoma.

Methyltransferase-like 18 (METTL18), a METTL family member, is abundant in hepatocellular carcinoma (HCC). Studies have indicated the METTL family could regulate the progress of diverse malignancies while the role of METTL18 in HCC remains unclear. Data of HCC patients were acquired from the cancer genome atlas (TCGA) and gene expression omnibus (GEO). The expression level of METTL18 in HCC patients was compared with normal liver tissues by Wilcoxon test. Then, the logistic analysis was used to estimate the correlation between METTL18 and clinicopathological factors. Besides, Gene Ontology (GO), Gene Set Enrichment Analysis (GSEA), and single-sample Gene Set Enrichment Analysis (ssGSEA) were used to explore relevant functions and quantify the degree of immune infiltration for METTL18. Univariate and Multivariate Cox analyses and Kaplan–Meier analysis were used to estimate the association between METTL18 and prognosis. Besides, by cox multivariate analysis, a nomogram was conducted to forecast the influence of METTL18 on survival rates. METTL18-high was associated with Histologic grade, T stage, Pathologic stage, BMI, Adjacent hepatic tissue inflammation, AFP, Vascular invasion, and TP53 status (P < 0.05). HCC patients with METTL18-high had a poor Overall-Survival [OS; hazard ratio (HR): 1.87, P < 0.001), Disease-Specific Survival (DSS, HR: 1.76, P = 0.015), and Progression-Free Interval (PFI, HR: 1.51, P = 0.006). Multivariate analysis demonstrated that METTL18 was an independent factor for OS (HR: 2.093, P < 0.001), DSS (HR: 2.404, P = 0.015), and PFI (HR: 1.133, P = 0.006). Based on multivariate analysis, the calibration plots and C-indexes of nomograms showed an efficacious predictive effect for HCC patients. GSEA demonstrated that METTL18-high could activate G2M checkpoint, E2F targets, KRAS signaling pathway, and Mitotic Spindle. There was a positive association between the METTL18 and abundance of innate immunocytes (T helper 2 cells) and a negative relation to the abundance of adaptive immunocytes (Dendritic cells, Cytotoxic cells etc.). Finally, we uncovered knockdown of METTL18 significantly suppressed the proliferation, invasion, and migration of HCC cells in vitro. This research indicates that METTL18 could be a novel biomarker to evaluate HCC patients’ prognosis and an important regulator of immune responses in HCC.

Peritumoral Tertiary Lymphoid Structures Correlate With Protective Immunity and Improved Prognosis in Patients With Hepatocellular Carcinoma.

The existence of intratumoral tertiary lymphoid structure (iTLS) has been reported to correlative with favorable clinical outcomes for patients with hepatocellular carcinoma (HCC). However, little is known about the role of peritumoral TLS (pTLS). This study aimed to investigate the prognostic role of pTLS either alone or jointly with iTLS and the potential association with local immune response in HCC. The formation and cellular composition of TLS was evaluated by hematoxylin & eosin and immunohistochemistry. Evaluation of tumor-infiltrating immune cells and formation of germinal center (GC) inside TLS was performed by immunohistochemistry. The gene expression profiles were analyzed by real-time PCR. In a total of 360 patients from two independent cohorts, the pTLS was identified in most, whereas iTLS could be observed in only approximately 30% of HCC specimens. Patients with high pTLS densities were associated with improved outcomes, those present with characteristic morphology of GC, particularly, showing an even better prognosis. The combination of pTLS and iTLS allowed the identification of patients with best prognosis. Tumors with high pTLS density showed significantly increased expression of Th1-, Th17- and immune suppression-related genes, as well as significantly higher infiltration of CD3+, CD8+ and CD20+ cells and lower infiltration of FOXP3+, CD68+ and PD1+ cells. Conclusively, we provide evidence that pTLS is associated with intratumoral immune infiltration, highlighting the dynamic interplay between pTLS and immune cells recruitment. High pTLS density links to a tumor microenvironment with an active immune reaction and improved patient survival and represents a promising prognostic biomarker for HCC.

MicroRNAs as Modulators of Tumor Metabolism, Microenvironment, and Immune Response in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality. Molecular heterogeneity and absence of biomarkers helping patient allocation to the best therapeutic option contribute to poor prognosis in advanced stages. MicroRNAs’ (miRNAs) deregulated expression contributes to tumor development and progression and influences drug resistance in HCC. Accordingly, miRNAs have been extensively investigated as both biomarkers and therapeutic targets. The diagnostic and prognostic roles of circulating miRNAs have been ascertained, though with some inconsistencies across studies. From a therapeutic perspective, miRNA-based approaches demonstrated safety profiles and antitumor efficacy in HCC animal models. Nevertheless, caution should be used when transferring preclinical findings to the clinic, due to possible molecular inconsistency between animal models and the heterogeneous patterns of human diseases. A wealth of information is offered by preclinical studies exploring the mechanisms driving miRNAs’ aberrant expression, the molecular cascades triggered by miRNAs and the corresponding phenotypic changes. Ex-vivo analyses confirmed these results, further shedding light on the intricacy of the human disease often overcoming pre-clinical models. This complexity seems to be ascribed to the intrinsic heterogeneity of HCC, to different risk factors driving its development, as well as to changes across stages and previous treatments. Preliminary findings suggest that miRNAs associated with specific risk factors might be more informative in defined patients’ subgroups. The first issue to be considered when trying to envisage a possible translational perspective is the molecular context that often drives different miRNA functions, as clearly evidenced by “dual” miRNAs. Concerning the possible roles of miRNAs as biomarkers and therapeutic targets, we will focus on miRNAs’ involvement in metabolic pathways and in the modulation of tumor microenvironment, to support their exploitation in defined contexts.

Icaritin Induces Anti-tumor Immune Responses in Hepatocellular Carcinoma by Inhibiting Splenic Myeloid-Derived Suppressor Cell Generation

Recent studies have demonstrated that splenic extramedullary hematopoiesis (EMH) is an important mechanism for the accumulation of myeloid-derived suppressor cells (MDSCs) in tumor tissues, and thus contributes to disease progression. Icaritin, a prenylflavonoid derivative from plants of the Epimedium genus, has been implicated as a novel immune-modulator that could prolong the survival of hepatocellular carcinoma (HCC) patients. However, it is unclear whether icaritin achieves its anti-tumor effects via the regulation of MDSCs generated by EMH in HCC. Here, we investigated the anti-tumor potential of icaritin and its mechanism of action in murine HCC. Icaritin suppressed tumor progression and significantly prolonged the survival of mice-bearing orthotopic and subcutaneous HCC tumors. Rather than exerting direct cytotoxic activity against tumor cells, icaritin significantly reduced the accumulation and activation of tumoral and splenic MDSCs, and increased the number and activity of cytotoxic T cells. Mechanistically, icaritin downregulates the tumor-associated splenic EMH, thereby reducing the generation and activation of MDSCs. The inhibitory effects of icaritin on human MDSCs in vitro were verified in short-term culture with cord-blood derived hematopoietic precursors. Furthermore, icaritin synergistically enhanced the therapeutic efficacy of immune checkpoint blockade therapy in HCC mice. These findings revealed that icaritin dampens tumoral immunosuppression to elicit anti-tumor immune responses by preventing MDSC generation via the attenuation of EMH. Thus, icaritin may serve as a novel adjuvant or even a stand-alone therapeutic agent for the effective treatment of HCC.

Gut microbiota impact on the peripheral immune response in non-alcoholic fatty liver disease related hepatocellular carcinoma

The gut microbiota is reported to modulate the immune response in hepatocellular carcinoma (HCC). Here, we employ metagenomic and metabolomic studies to characterise gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD) related cirrhosis, with or without HCC, and evaluate its effect on the peripheral immune response in an ex vivo model. We find that dysbiosis characterises the microbiota of patients with NAFLD-cirrhosis, with compositional and functional shifts occurring with HCC development. Gene function of the microbiota in NAFLD-HCC supports short chain fatty acid production, and this is confirmed by metabolomic studies. Ex vivo studies show that bacterial extracts from the NAFLD-HCC microbiota, but not from the control groups, elicit a T cell immunosuppressive phenotype, characterised by expansion of regulatory T cells and attenuation of CD8 + T cells. Our study suggest that the gut microbiota in NAFLD-HCC is characterised by a distinctive microbiome/metabolomic profile, and can modulate the peripheral immune response.

Liraglutide activates nature killer cell-mediated antitumor responses by inhibiting IL-6/STAT3 signaling in hepatocellular carcinoma.

Inflammatory IL-6/STAT3 signaling is constitutively activated in diverse cancers and is associated with malignant cell proliferation, invasion and escape of antitumor immunosurveillance. Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, is commonly used to treat insulin-resistant diabetes. In this study, for the first time, we showed that liraglutide remarkably improved the antitumor immune responses in hepatocellular carcinoma (HCC). Furthermore, we showed that the antitumor activity was mediated by nature killer cells (NKs) but not CD8+ T cells. Finally, we showed that liraglutide enhanced NK-mediated cytotoxicity by suppressing the IL-6/STAT3 signaling pathway in HCC cells. Our findings unveil a novel therapeutic role of liraglutide by manipulating the innate immunity in cancer therapy.