DNA methylation regulator-based molecular subtyping and tumor microenvironment characterization in hepatocellular carcinoma.

Backgroud: Although recent studies have reported the regulation of the immune response in hepatocellular carcinoma (HCC) through DNA methylation, the comprehensive impact methylation modifications on tumor microenvironment characteristics and immunotherapy efficacy has not been fully elucidated. Methods: In this research, we conducted a comprehensive assessment of the patterns of DNA methylation regulators and the profiles of the tumor microenvironment (TME) in HCC, focusing on 21 specific DNA methylation regulators. We subsequently developed a unique scoring system, a DNA methylation score (DMscore), to assess the individual DNA methylation modifications among the three distinct methylation patterns for differentially expressed genes (DEGs). Results: Three distinct methylation modification patterns were identified with distinct TME infiltration characteristics. We demonstrated that the DMscore could predict patient subtype, TME infiltration, and patient prognosis. A low DMscore, characterized by an elevated tumor mutation burden (TMB), hepatitis B virus (HBV)/hepatitis C virus (HCV) infection, and immune activation, indicates an inflamed tumor microenvironment phenotype with a 5-year survival rate of 7.8%. Moreover, a low DMscore appeared to increase the efficacy of immunotherapy in the anti-CTLA-4/PD-1/PD-L1 cohort. Conclusions: In brief, this research has enhanced our understanding of the correlation between modifications in DNA methylation patterns and the profile of the tumor microenvironment in individuals diagnosed with HCC. The DMscore may serve as an alternative biomarker for survival and efficacy of immunotherapy in patients with HCC.

Multi-Omics profiling identifies aldehyde dehydrogenase 2 as a critical mediator in the crosstalk between Treg-mediated immunosuppression microenvironment and hepatocellular carcinoma.

Dysregulation of the aldehyde dehydrogenase (ALDH) family has been implicated in various pathological conditions, including cancer. However, a systematic evaluation of ALDH alterations and their therapeutic relevance in hepatocellular carcinoma (HCC) remains lacking. Herein, we found that 15 of 19 ALDHs were transcriptionally dysregulated in HCC tissues compared to normal liver tissues. A four gene signature, including ALDH2, ALDH5A1, ALDH6A1, and ALDH8A1, robustly predicted prognosis and defined a high-risk subgroup exhibiting immunosuppressive features like regulatory T cell (Tregs) infiltration. Single-cell profiling revealed selective overexpression of tumor necrosis factor receptor superfamily member 18 (TNFRSF18) on Tregs, upregulated in high-risk HCC patients. We identified ALDH2 as a tumor suppressor in HCC, with three novel phosphorylation sites mediated by protein kinase C zeta that enhanced enzymatic activity. Mechanistically, ALDH2 suppressed Tregs differentiation by inhibiting ß-catenin/TGF-ß1 signaling in HCC. Collectively, our integrated multi-omics analysis defines an ALDH-Tregs-TNFRSF18 axis that contributes to HCC pathogenesis and represents potential therapeutic targets for this aggressive malignancy.

Identification of vitamin D-related signature for predicting the clinical outcome and immunotherapy response in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers globally, seriously endangering people health. Vitamin D was significantly associated with tumor progression and patients' prognosis. Integrative 10 machine learning algorithms were used to develop a Vitamin D-related signature (VRS) with one training cohort and 3 testing cohorts. The performance of VRS in predicting the immunology response was verified using several predicting approaches. The optimal VRS was constructed by stepCox + superPC algorithm. VRS acted as a risk factor for HCC patients. HCC patients with high-risk score had a poor clinical outcome and the AUCs of 1-, 3-, and 5-year ROC were 0.786, 0.755, and 0.786, respectively. A higher level of CD8 + cytotoxic T cells and B cells was obtained in HCC patients with low-risk score. There is higher PD1&CTLA4 immunophenoscore and TMB score in low-risk score in HCC patients. Lower TIDE score and tumor escape score was found in HCC cases with low-risk score. The IC50 value of camptothecin, docetaxel, crizotinib, dasatinib, and erlotinib was lower in HCC cases with high-risk score. HCC patients with high-risk score had a higher score of cancer-related hallmarks, including angiogenesis, glycolysis, and NOTCH signaling. Our study proposed a novel VRS for HCC, which served as an indicator for predicting clinical outcome and immunotherapy responses in HCC.

Identification of PANoptosis-related subtypes, construction of a prognosis signature, and tumor microenvironment landscape of hepatocellular carcinoma using bioinformatic analysis and experimental verification.

Background: Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide. PANoptosis is a recently unveiled programmed cell death pathway, Nonetheless, the precise implications of PANoptosis within the context of HCC remain incompletely elucidated. Methods: We conducted a comprehensive bioinformatics analysis to evaluate both the expression and mutation patterns of PANoptosis-related genes (PRGs). We categorized HCC into two clusters and identified differentially expressed PANoptosis-related genes (DEPRGs). Next, a PANoptosis risk model was constructed using LASSO and multivariate Cox regression analyses. The relationship between PRGs, risk genes, the risk model, and the immune microenvironment was studies. In addition, drug sensitivity between high- and low-risk groups was examined. The expression profiles of these four risk genes were elucidate by qRT-PCR or immunohistochemical (IHC). Furthermore, the effect of CTSC knock down on HCC cell behavior was verified using in vitro experiments. Results: We constructed a prognostic signature of four DEPRGs (CTSC, CDCA8, G6PD, and CXCL9). Receiver operating characteristic curve analyses underscored the superior prognostic capacity of this signature in assessing the outcomes of HCC patients. Subsequently, patients were stratified based on their risk scores, which revealed that the low-risk group had better prognosis than those in the high-risk group. High-risk group displayed a lower Stromal Score, Immune Score, ESTIMATE score, and higher cancer stem cell content, tumor mutation burden (TMB) values. Furthermore, a correlation was noted between the risk model and the sensitivity to 56 chemotherapeutic agents, as well as immunotherapy efficacy, in patient with. These findings provide valuable guidance for personalized clinical treatment strategies. The qRT-PCR analysis revealed that upregulated expression of CTSC, CDCA8, and G6PD, whereas downregulated expression of CXCL9 in HCC compared with adjacent tumor tissue and normal liver cell lines. The knockdown of CTSC significantly reduced both HCC cell proliferation and migration. Conclusion: Our study underscores the promise of PANoptosis-based molecular clustering and prognostic signatures in predicting patient survival and discerning the intricacies of the tumor microenvironment within the context of HCC. These insights hold the potential to advance our comprehension of the therapeutic contribution of PANoptosis plays in HCC and pave the way for generating more efficacious treatment strategies.

Involvement of S100A6/S100A11 in T-Cell Immune Regulatory in HCC Revealed by Single Cell RNA-seq.

Background: Immunotherapy plays a significant role in the treatment of hepatocellular carcinoma (HCC). Members of the S100 protein family (S100s) have been widely implicated in the pathogenesis and progression of tumors. However, the exact mechanism by which S100s contribute to tumor immunity remains unclear. Methods: To explore the role of S100s in HCC immune cells, we collected and comparatively analyzed single-cell RNA sequencing (scRNA-seq) data of HCC and hepatitis B virus-associated HCC. By mapping cell classification and searching for S100s binding targets and downstream targets. Results: S100A6/S100A11 was differentially expressed in tumor T cells and involved in the nuclear factor (NF) κB pathway. Further investigation of the TCGA dataset revealed that patients with low S100A6/S100A11 expression had a better prognosis. Temporal cell trajectory analysis showed that the activation of the NF-κB pathway is at a critical stage and has an important impact on the tumor microenvironment. Conclusion: Our study revealed that S100A6/S100A11 could be involved in regulating the differentiation and cellular activity of T-cell subpopulations in HCC, and its low expression was positively correlated with prognosis. It may provide a new direction for immunotherapy of HCC and a theoretical basis for future clinical applications.

Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells.

BACKGROUND: Dysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC. METHODS: The clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells. RESULTS: HCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells. CONCLUSIONS: IL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

Fluorescence imaging sheds light on the immune evasion mechanisms of hepatic stellate cells mediated by superoxide anion.

Whether and how the reactive oxygen species generated by hepatic stellate cells (HSCs) promote immune evasion of hepatocellular carcinoma (HCC) remains mysterious. Therefore, investigating the function of superoxide anion (O2•-), the firstly generated reactive oxygen species, during the immune evasion become necessary. In this work, we establish a novel in situ imaging method for visualization of O2•- changes in HSCs based on a new two-photon fluorescence probe TPH. TPH comprises recognition group for O2•- and HSCs targeting peptides. We observe that O2•- in HSCs gradually rose, impairing the infiltration of CD8+ T cells in HCC mice. Further studies reveal that the cyclin-dependent kinase 4 is deactivated by O2•-, and then cause the up-regulation of PD-L1. Our work provides molecular insights into HSC-mediated immune evasion of HCC, which may represent potential targets for HCC immunotherapy.

RRP8, associated with immune infiltration, is a prospective therapeutic target in hepatocellular carcinoma.

BACKGROUND: Ribosomal RNA Processing 8 (RRP8) is a nucleolar Rossman fold-like methyltransferase that exhibits increased expression in many malignant tumours. However, the role of RRP8 in hepatocellular carcinoma (HCC) is still uncertain. We explored the relationships between RRP8 and prognosis and immune infiltration, as well as the putative pathological function and mechanism of RRP8 in HCC. METHODS: Analysis of RRP8 expression across cancers was performed by using multiple databases. Associations between RRP8 expression and clinicopathological factors were further examined. Gene enrichment analysis was used to identify various putative biological activities and regulatory networks of RRP8 in HCC. The relationship between RRP8 expression and immune infiltration was confirmed by single-sample gene set enrichment analysis (ssGSEA). Univariate and multivariate Cox regression analyses were conducted to assess the impact of clinical variables on patient outcomes. Furthermore, a nomogram was constructed to estimate survival probability based on multivariate Cox regression analysis. Functional validation of RRP8 in HCC was performed with two different systems: doxycycline-inducible shRNA knockdown and CRISPR-Cas9 knockout. RESULTS: RRP8 was markedly overexpressed in HCC clinical specimens compared to adjacent normal tissues. Further analysis demonstrated that RRP8 was directly connected to multiple clinical characteristics and strongly associated with various immune markers in HCC. Moreover, elevated RRP8 expression indicated an unfavourable prognosis. Our functional studies revealed that both knockdown and knockout of RRP8 dramatically attenuated liver cancer cells to proliferate and migrate. Knockout of RRP8 decreased the phosphorylation of MEK1/2 and ß-catenin-(Y654) signalling pathway components; downregulated downstream signalling effectors, including Cyclin D1 and N-cadherin; and upregulated E-cadherin. CONCLUSIONS: RRP8 is strongly implicated in immune infiltration and could be a potential therapeutic target in HCC.

A comprehensive analysis of the oncogenic and prognostic role of TBC1Ds in human hepatocellular carcinoma.

Backgrounds: TBC1D family members (TBC1Ds) are a group of proteins that contain the Tre2-Bub2-Cdc16 (TBC) domain. Recent studies have shown that TBC1Ds are involved in tumor growth, but no analysis has been done of expression patterns and prognostic values of TBC1Ds in hepatocellular carcinoma (HCC). Methods: The expression levels of TBC1Ds were evaluated in HCC using the TIMER, UALCN and Protein Atlas databases. The correlation between the mRNA levels of TBC1Ds and the prognosis of patients with HCC in the GEPIA database was then analyzed. An enrichment analysis then revealed genes that potentially interact with TBC1Ds. The correlation between levels of TBC1Ds and tumor-infiltrating immune cells (TIICs) in HCC were studied using the TIMER 2.0 database. Finally, a series of in vitro assays verified the role of TBC1Ds in HCC progression. Results: This study revealed the upregulated expression of TBC1Ds in HCC and the strong positive correlation between the mRNA levels of TBC1Ds and poor prognosis of patients with HCC. The functions of TBC1Ds were mainly related to autophagy and the AMPK pathway. There was also a significant correlation between level of TBC1Ds and tumor-infiltrating immune cells (TIICs) in HCC. The promoting role of TBC1Ds in HCC progression was verified in vitro assays. Conclusion: The results of this analysis indicate that TBC1Ds may serve as new biomarkers for early diagnosis and treatment of HCC.

Constructing immune and prognostic features associated with ADCP in hepatocellular carcinoma and pan-cancer based on scRNA-seq and bulk RNA-seq.

Aim: Despite the significant therapeutic outcomes achieved in systemic treatments for liver hepatocellular carcinoma (LIHC), it is an objective reality that only a low proportion of patients exhibit an improved objective response rate (ORR) to current immunotherapies. Antibody-dependent cellular phagocytosis (ADCP) immunotherapy is considered the new engine for precision immunotherapy. Based on this, we aim to develop an ADCP-based LIHC risk stratification system and screen for relevant targets. Method: Utilizing a combination of single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data, we screened for ADCP modulating factors in LIHC and identified differentially expressed genes along with their involved functional pathways. A risk scoring model was established by identifying ADCP-related genes with prognostic value through LASSO Cox regression analysis. The risk scoring model was then subjected to evaluations of immune infiltration and immunotherapy relevance, with pan-cancer analysis and in vitro experimental studies conducted on key targets. Results: Building on the research by Kamber RA et al., we identified GYPA, CLDN18, and IRX5 as potential key target genes regulating ADCP in LIHC. These genes demonstrated significant correlations with immune infiltration cells, such as M1-type macrophages, and the effectiveness of immunotherapy in LIHC, as well as a close association with clinical pathological staging and patient prognosis. Pan-cancer analysis revealed that CLDN18 was prognostically and immunologically relevant across multiple types of cancer. Validation through tissue and cell samples confirmed that GYPA and CLDN18 were upregulated in liver cancer tissues and cells. Furthermore, in vitro knockdown of CLDN18 inhibited the malignancy capabilities of liver cancer cells. Conclusion: We have identified an ADCP signature in LIHC comprising three genes. Analysis based on a risk scoring model derived from these three genes, coupled with subsequent experimental validation, confirmed the pivotal role of M1-type macrophages in ADCP within LIHC, establishing CLDN18 as a critical ADCP regulatory target in LIHC.

Pan-cancer analysis of LRRC59 with a focus on prognostic and immunological roles in hepatocellular carcinoma.

BACKGROUND: LRRC59 is a leucine-rich repeats-containing protein located in the endoplasmic reticulum (ER), it serves as a prognostic marker in several cancers. However, there has been no systematic analysis of its role in the tumor immune microenvironment, nor its predictive value of prognosis and immunotherapy response in different cancers. METHODS: A comprehensive pan-cancer analysis of LRRC59 was conducted from various databases to elucidate the associations between its expression and the prognosis of cancer, genetic alterations, tumor metabolism, and tumor immunity. Additionally, further functional assays were performed in hepatocellular carcinoma (HCC) to study its biological role in regulating cell proliferation, migration, apoptosis, cell cycle arrest, and sensitivity to immunotherapy. RESULTS: The pan-cancer analysis reveals a significant upregulation of LRRC59 in pan-cancer, and its overexpression is correlated with unfavorable prognosis in cancer patients. LRRC59 is negatively correlated with immune cell infiltration, tumor purity estimation, and immune checkpoint genes. Finally, the validation in HCC demonstrates LRRC59 is significantly overexpressed in cancer tissue and cell lines, and its knockdown inhibits cell proliferation and migration, promotes cell apoptosis, induces cell cycle arrest, and enhances the sensitivity to immunotherapy in HCC cells. CONCLUSIONS: LRRC59 emerges as a novel potential prognostic biomarker across malignancies, offering promise for anti-cancer drugs and immunotherapy.

Ferroptosis, pyroptosis and necroptosis in hepatocellular carcinoma immunotherapy: Mechanisms and immunologic landscape (Review).

Hepatocellular carcinoma (HCC), one of the leading causes of cancer­related mortality worldwide, is challenging to identify in its early stages and prone to metastasis, and the prognosis of patients with this disease is poor. Treatment options for HCC are limited, with even radical treatments being associated with a risk of recurrence or transformation in the short term. Furthermore, the multi­tyrosine kinase inhibitors approved for first­line therapy have marked drawbacks, including drug resistance and side effects. The rise and breakthrough of immune checkpoint inhibitors (ICIs) have provided a novel direction for HCC immunotherapy but these have the drawback of low response rates. Since avoiding apoptosis is a universal feature of cancer, the induction of non­apoptotic regulatory cell death (NARCD) is a novel strategy for HCC immunotherapy. At present, NARCD pathways, including ferroptosis, pyroptosis and necroptosis, are novel potential forms of immunogenic cell death, which have synergistic effects with antitumor immunity, transforming immune 'cold' tumors into immune 'hot' tumors and exerting antitumor effects. Therefore, these pathways may be targeted as a novel treatment strategy for HCC. In the present review, the roles of ferroptosis, pyroptosis and necroptosis in antitumor immunity in HCC are discussed, and the relevant targets and signaling pathways, and the current status of combined therapy with ICIs are summarized. The prospects of targeting ferroptosis, pyroptosis and necroptosis in HCC immunotherapy are also considered.

The pattern of tumor progression on first-line immune checkpoint inhibitor-based systemic therapy for Chinese advanced hepatocellular carcinoma -CLEAP 004 study.

Background: For decades, stratification criteria for first-line clinical studies have been highly uniform. However, there is no principle or consensus for restratification after systemic treatment progression based on immune checkpoint inhibitors (ICIs). The aim of this study was to assess the patterns of disease progression in patients with advanced hepatocellular carcinoma (HCC) who are not eligible for surgical intervention, following the use of immune checkpoint inhibitors. Methods: This is a retrospective study that involved patients with inoperable China liver stage (CNLC) IIIa and/or IIIb. The patients were treated at eight centers across China between January 2017 and October 2022. All patients received at least two cycles of first-line treatment containing immune checkpoint inhibitors. The patterns of disease progression were assessed using RECIST criteria 1.1. Different progression modes have been identified based on the characteristics of imaging progress. The study's main outcome measures were post-progression survival (PPS) and overall survival (OS). Survival curves were plotted using the Kaplan-Meier method to compare the difference among the four groups. Subgroup analysis was conducted to compare the efficacy of different immunotherapy combinations. Variations in the efficacy of immunotherapy have also been noted across patient groups exhibiting alpha-fetoprotein (AFP) levels equal to or exceeding 400ng/mL, in contrast to those with AFP levels below 400ng/mL. Results: The study has identified four distinct patterns of progress, namely p-IIb, p-IIIa, p-IIIb, and p-IIIc. Diverse patterns of progress demonstrate notable variations in both PPS and OS. The group p-IIb had the longest PPS of 12.7m (95% 9.3-16.1) and OS 19.6m (95% 15.6-23.5), the remaining groups exhibited p-IIIb at PPS 10.5 months (95%CI: 7.9-13.1) and OS 19.2 months (95%CI 15.1-23.3). Similarly, p-IIIc at PPS 5.7 months (95%CI: 4.2-7.2) and OS 11.0 months (95%CI 9.0-12.9), while p-IIIa at PPS 3.4 months (95%CI: 2.7-4.1) and OS 8.2 months (95%CI 6.8-9.5) were also seen. Additional stratified analysis was conducted and showed there were no differences of immunotherapy alone or in combination in OS (HR= 0.92, 95%CI: 0.59-1.43, P=0.68) and PPS (HR= 0.88, 95%CI: 0.57-1.36, P=0.54); there was no significant difference in PPS (HR=0.79, 95% CI: 0.55-1.12, P=0.15) and OS (HR=0.86, 95% CI: 0.61-1.24, P=0.39) for patients with AFP levels at or over 400ng/mL. However, it was observed that patients with AFP levels above 400ng/mL experienced a shorter median progression of PPS (8.0 months vs. 5.0 months) after undergoing immunotherapy. Conclusion: In this investigation of advanced hepatocellular carcinoma among Chinese patients treated with immune checkpoint inhibitors, we identified four distinct progression patterns (p-IIb, p-IIIa, p-IIIb and p-IIIc) that showed significant differences in PPS and OS. These findings demonstrate the heterogeneity of disease progression and prognosis after immunotherapy failure. Further validation in large cohorts is necessary to develop prognostic models that integrate distinct progression patterns to guide subsequent treatment decisions. Additionally, post-immunotherapy progression in patients with AFP levels ≥400ng/mL indicates a shortened median PPS. These findings provide valuable insights for future personalized treatment decisions.

Comprehensive Analysis of the Immunosuppressive Function of Regulatory T Cells in Human Hepatocellular Carcinoma Tissues.

BACKGROUND: Immune-based therapies are commonly employed to combat hepatocellular carcinoma (HCC). However, the presence of immune-regulating elements, especially regulatory T cells (Tregs), can dramatically impact the treatment efficacy. A deeper examination of the immune-regulation mechanisms linked to these inhibitory factors and their impact on HCC patient outcomes is warranted. METHODS: We employed multicolor fluorescence immunohistochemistry (mIHC) to stain Foxp3, cytokeratin, and nuclei on an HCC tissue microarray (TMA). Leveraging liver cancer transcriptome data from TCGA, we built a prognostic model focused on Treg-associated gene sets and represented it with a nomogram. We then sourced liver cancer single-cell RNA sequencing data (GSE140228) from the GEO database, selectively focusing on Treg subsets, and conducted further analyses, including cell-to-cell communication and pseudo-time trajectory examination. RESULTS: Our mIHC results revealed a more substantial presence of Foxp3+Tregs in HCC samples than in adjacent normal tissue samples (P < .001). An increased presence of Foxp3+Tregs in HCC samples correlated with unfavorable patient outcomes (HR = 1.722, 95% CI:1.023-2.899, P = .041). The multi-factorial prognosis model we built from TCGA liver cancer data highlighted Tregs as a standalone risk determinant for predicting outcomes (HR = 3.84, 95% CI:2.52-5.83, P < .001). Re-analyzing the scRNA-seq dataset (GSE140228) showcased distinctive gene expression patterns in Tregs from varying tissues. Interactions between Tregs and other CD4+T cell types were predominantly governed by the CXCL13/CXCR3 signaling pathway. Communication pathways between Tregs and macrophages primarily involved MIF-CD74/CXCR4, LGALS9/CD45, and PTPRC/MRC1. Additionally, macrophages could influence Tregs via HLA-class II and CD4 interactions. CONCLUSION: An elevated presence of Tregs in HCC samples correlated with negative patient outcomes. Elucidating the interplay between Tregs and other immune cells in HCC could provide insights into the modulatory role of Tregs within HCC tissues.

Predicting effect of anti-PD-1/PD-L1 inhibitors therapy for hepatocellular carcinoma by detecting plasma metabolite based on UHPLC-MS.

Introduction: Anti-PD-1/PD-L1 inhibitors therapy has become a promising treatment for hepatocellular carcinoma (HCC), while the therapeutic efficacy varies significantly among effects for individual patients are significant difference. Unfortunately, specific predictive biomarkers indicating the degree of benefit for patients and thus guiding the selection of suitable candidates for immune therapy remain elusive.no specific predictive biomarkers are available indicating the degree of benefit for patients and thus screening the preferred population suitable for the immune therapy. Methods: Ultra-high-pressure liquid chromatography-mass spectrometry (UHPLC-MS) considered is an important method for analyzing biological samples, since it has the advantages of high rapid, high sensitivity, and high specificity. Ultra-high-pressure liquid chromatography-mass spectrometry (UHPLC-MS) has emerged as a pivotal method for analyzing biological samples due to its inherent advantages of rapidity, sensitivity, and specificity. In this study, potential metabolite biomarkers that can predict the therapeutic effect of HCC patients receiving immune therapy were identified by UHPLC-MS. Results: A partial least-squares discriminant analysis (PLS-DA) model was established using 14 glycerophospholipid metabolites mentioned above, and good prediction parameters (R2 = 0.823, Q2 = 0.615, prediction accuracy = 0.880 and p < 0.001) were obtained. The relative abundance of glycerophospholipid metabolite ions is closely related to the survival benefit of HCC patients who received immune therapy. Discussion: This study reveals that glycerophospholipid metabolites play a crucial role in predicting the efficacy of immune therapy for HCC.

Predictive value of a stemness-based classifier for prognosis and immunotherapy response of hepatocellular carcinoma based on bioinformatics and machine-learning strategies.

Objective: Significant advancements have been made in hepatocellular carcinoma (HCC) therapeutics, such as immunotherapy for treating patients with HCC. However, there is a lack of reliable biomarkers for predicting the response of patients to therapy, which continues to be challenging. Cancer stem cells (CSCs) are involved in the oncogenesis, drug resistance, and invasion, as well as metastasis of HCC cells. Therefore, in this study, we aimed to create an mRNA expression-based stemness index (mRNAsi) model to predict the response of patients with HCC to immunotherapy. Methods: We retrieved gene expression and clinical data of patients with HCC from the GSE14520 dataset and the Cancer Genome Atlas (TCGA) database. Next, we used the "one-class logistic regression (OCLR)" algorithm to obtain the mRNAsi of patients with HCC. We performed "unsupervised consensus clustering" to classify patients with HCC based on the mRNAsi scores and stemness subtypes. The relationships between the mRNAsi model, clinicopathological features, and genetic profiles of patients were compared using various bioinformatic methods. We screened for differentially expressed genes to establish a stemness-based classifier for predicting the patient's prognosis. Next, we determined the effect of risk scores on the tumor immune microenvironment (TIME) and the response of patients to immune checkpoint blockade (ICB). Finally, we used qRT-PCR to investigate gene expression in patients with HCC. Results: We screened CSC-related genes using various bioinformatics tools in patients from the TCGA-LIHC cohort. We constructed a stemness classifier based on a nine-gene (PPARGC1A, FTCD, CFHR3, MAGEA6, CXCL8, CABYR, EPO, HMMR, and UCK2) signature for predicting the patient's prognosis and response to ICBs. Further, the model was validated in an independent GSE14520 dataset and performed well. Our model could predict the status of TIME, immunogenomic expressions, congenic pathway, and response to chemotherapy drugs. Furthermore, a significant increase in the proportion of infiltrating macrophages, Treg cells, and immune checkpoints was observed in patients in the high-risk group. In addition, tumor cells in patients with high mRNAsi scores could escape immune surveillance. Finally, we observed that the constructed model had a good expression in the clinical samples. The HCC tumor size and UCK2 genes expression were significantly alleviated and decreased, respectively, by treatments of anti-PD1 antibody. We also found knockdown UCK2 changed expressions of immune genes in HCC cell lines. Conclusion: The novel stemness-related model could predict the prognosis of patients and aid in creating personalized immuno- and targeted therapy for patients in HCC.

Epigenetic silencing of LDHB promotes hepatocellular carcinoma by remodeling the tumor microenvironment.

Lactate dehydrogenase B (LDHB) reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate and expressed in various malignancies. However, the role of LDHB in modulating immune responses against hepatocellular carcinoma (HCC) remains largely unknown. Here, we found that down-regulation of lactate dehydrogenase B (LDHB) was coupled with the promoter hypermethylation and knocking down the DNA methyltransferase 3A (DNMT 3A) restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Moreover, immune checkpoint inhibitors (ICIs) have shown considerable promise for HCC treatment and patients with higher LDHB expression responded better to ICIs. Finally, we found that overexpression of LDHB suppressed HCC growth in immunocompetent but not in immunodeficient mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and therapeutic target for HCC immunotherapy.

Immunosuppressive tumor microenvironment and immunotherapy of hepatocellular carcinoma: current status and prospectives.

Hepatocellular carcinoma (HCC) is a major health concern worldwide, with limited therapeutic options and poor prognosis. In recent years, immunotherapies such as immune checkpoint inhibitors (ICIs) have made great progress in the systemic treatment of HCC. The combination treatments based on ICIs have been the major trend in this area. Recently, dual immune checkpoint blockade with durvalumab plus tremelimumab has also emerged as an effective treatment for advanced HCC. However, the majority of HCC patients obtain limited benefits. Understanding the immunological rationale and exploring novel ways to improve the efficacy of immunotherapy has drawn much attention. In this review, we summarize the latest progress in this area, the ongoing clinical trials of immune-based combination therapies, as well as novel immunotherapy strategies such as chimeric antigen receptor T cells, personalized neoantigen vaccines, oncolytic viruses, and bispecific antibodies.

NETosis in tumour microenvironment of liver: From primary to metastatic hepatic carcinoma.

BACKGROUND: Hepatocellular carcinoma is a common and highly lethal tumour. The tumour microenvironment (TME) plays an important role in the progression and metastasis of hepatocellular carcinoma (HCC). A cell death mechanism, termed NETosis, has been found to play an important role in the TME of HCC. SUMMARY: This review article focuses on the role of NETosis in the TME of HCC, a novel form of cell death in which neutrophils capture and kill microorganisms by releasing a type of DNA meshwork fibres called "NETs". This process is associated with neutrophil activation, local inflammation and cytokines. The study suggests that NETs play a multifaceted role in the development and metastasis of HCC. The article also discusses the role of NETs in tumour proliferation and metastasis, epithelial-mesenchymal transition (EMT), and surgical stress. In addition, the article discusses the interaction of NETosis with other immune cells in the TME and related therapeutic strategies. A deeper understanding of NETosis can help us better understand the complexity of the immune system and provide a new therapeutic basis for the treatment and prevention of HCC. KEY INFORMATION: In conclusion, NETosis is important in the TME of liver. NETs have been shown to contribute to the progression and metastasis of liver cancer. The interaction between NETosis and immune cells in the TME, as well as related therapies, are important areas of research.

A fully biodegradable spherical nucleic acid nanoplatform for self-codelivery of doxorubicin and miR122 for innate and adaptive immunity activation.

Facile construction of a fully biodegradable spherical nucleic acid (SNA) nanoplatform is highly desirable for clinical translations but remains rarely explored. We developed herein the first polycarbonate-based biodegradable SNA nanoplatform for self-codelivery of a chemotherapeutic drug, doxorubicin (DOX), and a human liver-specific miR122 for synergistic chemo-gene therapy of hepatocellular carcinoma (HCC). Ring-opening polymerization (ROP) of a carbonate monomer leads to a well-defined polycarbonate backbone for subsequent DOX conjugation to the pendant side chains via acidic pH-cleavage Schiff base links and miR122 incorporation to the chain termini via click coupling, affording an amphiphilic polycarbonate-DOX-miR122 conjugate, PBis-Mpa30-DOX-miR122 that can self-assemble into stabilized SNA. Besides the desired biodegradability, another notable merit of this nanoplatform is the use of miR122 not only for gene therapy but also for enhanced innate immune response. Together with the ICD-triggering effect of DOX, PBis-Mpa30-DOX-miR122 SNA-mediated DOX and miR122 codelivery leads to synergistic immunogenicity enhancement, resulting in tumor growth inhibition value (TGI) of 98.1 % significantly higher than those of the groups treated with only drug or gene in a Hepa1-6-tumor-bearing mice model. Overall, this study develops a useful strategy toward biodegradable SNA construction, and presents a drug and gene-based self-codelivery SNA with synergistic immunogenicity enhancement for efficient HCC therapy. STATEMENT OF SIGNIFICANCE: Facile construction of a fully biodegradable SNA nanoplatform is useful for in vivo applications but remains relatively unexplored likely due to the synthetic challenge. We report herein construction of a polycarbonate-based SNA nanoplatform for co-delivering a chemotherapeutic drug, DOX, and a human liver-specific miR-122 for synergistic HCC treatment. In addition to the desired biodegradability properties, this SNA nanoplatform integrates DOX-triggered ICD and miR-122-enhanced innate immunity for simultaneously activating adaptive and innate immunities, which leads to potent antitumor efficiency with a TGI value of 98.1 % in a Hepa1-6-tumor-bearing mice model.