KHDRBS1 as a novel prognostic signaling biomarker influencing hepatocellular carcinoma cell proliferation, migration, immune microenvironment, and drug sensitivity.

Background: Human tumors pose significant challenges, with targeted therapy against specific molecular targets or signaling pathways being a mainstay alongside surgical resection. Previous studies have implicated KHDRBS1 in the oncogenesis of certain human tumors such as colorectal and prostate cancers, underscoring its potential as a therapeutic target. However, the comprehensive expression pattern of KHDRBS1 in hepatocellular carcinoma (HCC) warrants further exploration. Methods: Integrating and analyzing multi-omics, multi-cohort data from public databases, coupled with clinical samples and molecular biology validation, we elucidate the oncogenic role of KHDRBS1 in HCC progression. Additionally, leveraging HCC single-cell sequencing data, we segregate malignant cells into KHDRBS1-positive and negative subsets, uncovering significant differences in their expression profiles and functional roles. Results: Our study identifies KHDRBS1 as a tumor-promoting factor in HCC, with its positivity correlating with tumor progression. Furthermore, we highlight the clinical significance of KHDRBS1-positive malignant cells, aiming to further propel its clinical utility. Conclusion: KHDRBS1 plays a key role in HCC development. This study provides crucial insights for further investigation into KHDRBS1 as a therapeutic target in HCC.

Prognostic value of CD8+T cells related genes and exhaustion regulation of Notch signaling pathway in hepatocellular carcinoma.

Immunotherapy has emerged as the primary treatment modality for patients with advanced Hepatocellular carcinoma (HCC). However, its clinical efficacy remains limited, benefiting only a subset of patients, while most exhibit immune tolerance and face a grim prognosis. The infiltration of immune cells plays a pivotal role in tumor initiation and progression. In this study, we conducted an analysis of immune cell infiltration patterns in HCC patients and observed a substantial proportion of CD8+T cells. Leveraging the weighted gene co-expression network analysis (WGCNA), we identified 235 genes associated with CD8+T cell and constructed a risk prediction model. In this model, HCC patients were stratified into a high-risk and low-risk group. Patients in the high-risk group exhibited a lower survival rate, predominantly presented with intermediate to advanced stages of cancer, displayed compromised immune function, showed limited responsiveness to immunotherapy, and demonstrated elevated expression levels of the Notch signaling pathway. Further examination of clinical samples demonstrated an upregulation of the Notch1+CD8+T cell exhaustion phenotype accompanied by impaired cytotoxicity and cytokine secretion functions that worsened with increasing Notch activation levels. Our study not only presents a prognostic model but also highlights the crucial involvement of the Notch pathway in CD8+T cell exhaustion-a potential target for future immunotherapeutic interventions.

A prognostic signature established based on genes related to tumor microenvironment for patients with hepatocellular carcinoma.

BACKGROUND: Complex cellular signaling network in the tumor microenvironment (TME) could serve as an indicator for the prognostic classification of hepatocellular carcinoma (HCC) patients. METHODS: Univariate Cox regression analysis was performed to screen prognosis-related TME-related genes (TRGs), based on which HCC samples were clustered by running non-negative matrix factorization (NMF) algorithm. Furthermore, the correlation between different molecular HCC subtypes and immune cell infiltration level was analyzed. Finally, a risk score (RS) model was established by LASSO and Cox regression analyses (CRA) using these TRGs. Functional enrichment analysis was performed using gene set enrichment analysis (GSEA). RESULTS: HCC patients were divided into three molecular subtypes (C1, C2, and C3) based on 704 prognosis-related TRGs. HCC subtype C1 had significantly better OS than C2 and C3. We selected 13 TRGs to construct the RS model. Univariate and multivariate CRA showed that the RS could independently predict patients' prognosis. A nomogram integrating the RS and clinicopathologic features of the patients was further created. We also validated the reliability of the model according to the area under the receiver operating characteristic (ROC) curve value, concordance index (C-index), and decision curve analysis. The current findings demonstrated that the RS was significantly correlated with CD8+ T cells, monocytic lineage, and myeloid dendritic cells. CONCLUSION: This study provided TRGs to help classify patients with HCC and predict their prognoses, contributing to personalized treatments for patients with HCC.

Single-cell data revealed exhaustion of characteristic NK cell subpopulations and T cell subpopulations in hepatocellular carcinoma.

BACKGROUND: The treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) have been a major medical challenge. Unraveling the landscape of tumor immune infiltrating cells (TIICs) in the immune microenvironment of HCC is of great significance to probe the molecular mechanisms. METHODS: Based on single-cell data of HCC, the cell landscape was revealed from the perspective of TIICs. Special cell subpopulations were determined by the expression levels of marker genes. Differential expression analysis was conducted. The activity of each subpopulation was determined based on the highly expressed genes. CTLA4+ T-cell subpopulations affecting the prognosis of HCC were determined based on survival analysis. A single-cell regulatory network inference and clustering analysis was also performed to determine the transcription factor regulatory networks in the CTLA4+ T cell subpopulations. RESULTS: 10 cell types were identified and NK cells and T cells showed high abundance in tumor tissues. Two NK cells subpopulations were present, FGFBP2+ NK cells, B3GNT7+ NK cells. Four T cells subpopulations were present, LAG3+ T cells, CTLA4+ T cells, RCAN3+ T cells, and HPGDS+ Th2 cells. FGFBP2+ NK cells, and CTLA4+ T cells were the exhaustive subpopulation. High CTLA4+ T cells contributed to poor prognostic outcomes and promoted tumor progression. Finally, a network of transcription factors regulated by NR3C1, STAT1, and STAT3, which were activated, was present in CTLA4+ T cells. CONCLUSION: CTLA4+ T cell subsets in HCC exhibited functional exhaustion characteristics that probably inhibited T cell function through a transcription factor network dominated by NR3C1, STAT1, and STAT3.

The sorafenib resistance-related gene signature predicts prognosis and indicates immune activity in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death worldwide. Most patients with advanced HCC acquire sorafenib resistance. Drug resistance reflects the heterogeneity of tumors and is the main cause of tumor recurrence and death.We identified and validated sorafenib resistance related-genes (SRGs) as prognostic biomarkers for HCC. We obtained SRGs from the Gene Expression Omnibus and selected four key SRGs using the least absolute shrinkage and selection operator, random forest, and Support Vector Machine-Recursive feature elimination machine learning algorithms. Samples from the The Cancer Genome Atlas (TCGA)-HCC were segregated into two groups by consensus clustering. Following difference analysis, 19 SRGs were obtained through univariate Cox regression analysis, and a sorafenib resistance model was constructed for risk stratification and prognosis prediction. In multivariate Cox regression analysis, the risk score was an independent predictor of overall survival (OS). Patients classified as high-risk were more sensitive to other chemotherapy drugs and showed a higher expression of the common immune checkpoints. Additionally, the expression of drug-resistance genes was verified in the International Cancer Genome Consortium cohort. A nomogram model with a risk score was established, and its prediction performance was verified by calibration chart analysis of the TCGA-HCC cohort. We conclude that there is a significant correlation between sorafenib resistance and the tumor immune microenvironment in HCC. The risk score could be used to identify a reliable prognostic biomarker to optimize the therapeutic benefits of chemotherapy and immunotherapy, which can be helpful in the clinical decision-making for HCC patients.

Role of CCR1/5/7 in hepatocellular carcinoma: a study on prognostic evaluation, molecular subtyping, and association with immune infiltration.

This study aims to assess the prognostic value of the C-C motif chemokine receptor (CCR) gene family in hepatocellular carcinoma (HCC) and its relationship with immune infiltration and molecular subtypes of HCC. The evaluation of the GSE14520 dataset and TCGA database confirmed the prognostic significance of CCR. Building upon the correlation between CCR1, CCR5, and CCR7 and favorable prognosis, we further validated the prognostic importance of CCR1, CCR5, and CCR7 in ICGC database and an independent cohort from Guangxi autonomous region. Then, we constructed a risk prognosis model. Additionally, we observed significant positive correlations between CCR1, CCR5, and CCR7 and the infiltration of B cells, T cells, and macrophages in HCC. Subsequently, we conducted CCK assays, Transwell assays, and colony formation assays to evaluate the molecular biological functions of CCR1, CCR5, and CCR7. These experiments further confirmed that upregulation of CCR1, CCR5, and CCR7 can individually inhibit the proliferation, migration, and stemness of HCC cells. By analyzing the relationship between expression levels and tumor mutation frequency, we discovered that patients with high CCR1 expression were more likely to be classified as non-proliferative HCC. Similar conclusions were observed for CCR5 and CCR7. The association of CCR1, CCR5, and CCR7 with the molecular subtypes of HCC suggests that they may serve as intermediary molecules linking immune status and molecular subtypes in HCC. In summary, CCR1, CCR5, and CCR7 have the potential to serve as prognostic biomarkers for HCC and regulate HCC progression by influencing immune cell infiltration.

RECQL4 Inhibits Radiation-Induced Tumor Immune Awakening via Suppressing the cGAS-STING Pathway in Hepatocellular Carcinoma.

Many patients with hepatocellular carcinoma (HCC) respond poorly to radiotherapy despite remarkable advances in treatment. A deeper insight into the mechanism of sensitivity of HCC to this therapy is urgently required. It is demonstrated that RECQL4 is upregulated in the malignant cells of patients with HCC. Elevated RECQL4 levels reduce the sensitivity of HCC to radiotherapy by repairing radiation-induced double-stranded DNA (dsDNA) fragments. Mechanistically, the inhibitory effect of RECQL4 on radiotherapy is due to the reduced recruitment of dendritic cells and CD8+ T cells in the tumor microenvironment (TME). RECQL4 disrupts the radiation-induced transformation of the TME into a tumoricidal niche by inhibiting the cGAS-STING pathway in dendritic cells. Knocking out STING in dendritic cells can block the impact of RECQL4 on HCC radiosensitivity. Notably, high RECQL4 expressions in HCC is significantly associated with poor prognosis in multiple independent cohorts. In conclusion, this study highlights how HCC-derived RECQL4 disrupts cGAS-STING pathway activation in dendritic cells through DNA repair, thus reducing the radiosensitivity of HCC. These findings provide new perspectives on the clinical treatment of HCC.

Radix Bupleuri-Radix Paeoniae Alba Inhibits the Development of Hepatocellular Carcinoma through Activation of the PTEN/PD-L1 Axis within the Immune Microenvironment.

OBJECTIVE: This study investigated how Radix Bupleuri-Radix Paeoniae Alba (BP) was active against hepatocellular carcinoma (HCC). METHODS: Traditional Chinese medicine systems pharmacology (TCMSP) database was employed to determine the active ingredients of BP and potential targets against HCC. Molecular docking analysis verified the binding activity of PTEN with BP ingredients. H22 cells were used to establish an HCC model in male balb/c mice. Immunofluorescence staining, immunohistochemistry, flow cytometry, western blotting, enzyme-linked immunosorbent assay, and real-time quantitative PCR were used to study changes in proliferation, apoptosis, PTEN levels, inflammation, and T-cell differentiation in male balb/c mice. RESULTS: The major active ingredients in BP were found to be quercetin, kaempferol, isorhamnetin, stigmasterol, and beta-sitosterol. Molecular docking demonstrated that these five active BP ingredients formed a stable complex with PTEN. BP exhibited an anti-tumor effect in our HCC mouse model. BP was found to increase the CD8+ and IFN-γ+/CD4+ T cell levels while decreasing the PD-1+/CD8+ T and Treg cell levels in HCC mice. BP up-regulated the IL-6, IFN-γ, and TNF-α levels but down-regulated the IL-10 levels in HCC mice. After PTEN knockdown, BP-induced effects were abrogated. CONCLUSION: BP influenced the immune microenvironment through activation of the PTEN/PD-L1 axis, protecting against HCC.

Overexpression of SH2D1A promotes cancer progression and is associated with immune cell infiltration in hepatocellular carcinoma via bioinformatics and in vitro study.

BACKGROUND: SH2 domain containing 1A (SH2D1A) expression has been linked to cancer progression. However, the functions of SH2D1A in hepatocellular carcinoma (HCC) have not been reported. METHODS: The effects of SH2D1A on the proliferation, migration, and invasion of HCC cells and the related pathways were re-explored in cell models with SH2D1A overexpression using the CCK-8, migration and invasion assays and western blotting. The functions and mechanisms of genes co-expressed with SH2D1A were analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The relationship between SH2D1A expression and immune microenvironment features in HCC was explored. RESULTS: Elevated SH2D1A expression promoted cell proliferation, migration, and invasion, which was related to the overexpression of p-Nf-κB and BCL2A1 protein levels in HCC. SH2D1A expression was related to the immune, stromal, and ESTIMATE scores, and the abundance of immune cells, such as B cells, CD8+ T cells, and T cells. SH2D1A expression was significantly related to the expression of immune cell markers, such as PDCD1, CD8A, and CTLA4 in HCC. CONCLUSION: SH2D1A overexpression was found to promote cell growth and metastasis via the Nf-κB signaling pathway and may be related to the immune microenvironment in HCC. The findings indicate that SH2D1A can function as a biomarker in HCC.

The aryl hydrocarbon receptor maintains antitumor activity of liver resident natural killer cells after partial hepatectomy in C57BL/6J mice.

BACKGROUND: Liver-resident natural killer (lr-NK) cells are distinct from conventional NK cells and exhibit higher cytotoxicity against hepatoma via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the mechanism by which partial hepatectomy (PH) significantly suppresses TRAIL expression in lr-NK cells remains unclear. METHODS: This study aimed to investigate the PH influence on the function and characteristics of liver-resident NK (lr-NK) cells using a PH mouse model. RESULTS: Here, we report that PH alters the differentiation pattern of NK cells in the liver, and an aryl hydrocarbon receptor (AhR) molecule is involved in these changes. Treatment with the AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) inhibited the maturation of NK cells. FICZ increased the immature subtype proportion of NK cells with high TRAIL activity and decreased the mature subtype of NK cells with low TRAIL activity. Consequently, FICZ increased the expression of TRAIL and cytotoxic activity of NK cells in the liver, and this effect was confirmed even after hepatectomy. The participation of AhR promoted FoxO1 expression in the mTOR signaling pathway involved in the maturation of NK cells, resulting in TRAIL expression. CONCLUSION: Our findings provide direct in-vivo evidence that partial hepatectomy affects lrNK cell activity through NK cell differentiation in the liver. Perioperative therapies using an AhR agonist to improve NK cell function may reduce the recurrence of hepatocellular carcinoma after hepatectomy.

Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin.

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Characterization of the Clinical Significance and Immunological Landscapes of a Novel TMEMs Signature in Hepatocellular Carcinoma and the Contribution of TMEM201 to Hepatocarcinogenesis.

Aberrant transmembrane protein (TMEM) expression is implicated in tumor progression, but its functional role in hepatocellular carcinoma (HCC) is unclear. Thus, we aim to characterize the functional contributions of TMEM in HCC. In this study, four novel TMEM-family genes (TMEMs), TMEM106C, TMEM201, TMEM164, and TMEM45A, were screened to create a TMEMs signature. These candidate genes are distinguished between patients with varying survival statuses. High-risk HCC patients had a significantly worse prognosis and more advanced clinicopathological characteristics in both the training and validation groups. The GO and KEGG analyses unveiled that the TMEMs signature might play a crucial role in cell-cycle-relevant and immune-related pathways. We found that the high-risk patients had lower stromal scores and a more immunosuppressive tumor microenvironment with massive infiltration of macrophages and Treg cells, whereas the low-risk group had higher stromal scores and gamma delta T-cell infiltration. Moreover, the expression level of suppressive immune checkpoints increased as the TMEM-signature scores increased. Furthermore, the in vitro experiments validated TMEM201, one feature of the TMEMs signature, and facilitated HCC proliferation, survival, and migration. The TMEMs signature provided a more precise prognostic evaluation of HCC and reflected the immunological status of HCC. Of the TMEMs signature studied, TMEM201 was found to significantly promote HCC progression.

Pan-cancer analysis of SYNGR2 with a focus on clinical implications and immune landscape in liver hepatocellular carcinoma.

BACKGROUND: Synaptogyrin-2 (SYNGR2), as a member of synaptogyrin gene family, is overexpressed in several types of cancer. However, the role of SYNGR2 in pan-cancer is largely unexplored. METHODS: From the TCGA and GEO databases, we obtained bulk transcriptomes, and clinical information. We examined the expression patterns, prognostic values, and diagnostic value of SYNGR2 in pan-cancer, and investigated the relationship of SYNGR2 expression with tumor mutation burden (TMB), microsatellite instability (MSI), immune infiltration, and immune checkpoint (ICP) genes. The gene set enrichment analysis (GSEA) software was used to perform pathway analysis. Besides, we built a nomogram of liver hepatocellular carcinoma patients (LIHC) and validated its prediction accuracy. RESULTS: SYNGR2 was highly expressed in most cancers. The high expression of SYNGR2 significantly reduced the overall survival (OS), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI) in multiple types of cancer. Also, receiver operating characteristic (ROC) curve analysis demonstrated that SYNGR2 showed high accuracy in distinguishing cancerous tissues from normal ones. Moreover, SYNGR2 expression was correlated with TMB, MSI, immune scores, and immune cell infiltrations. We also analyzed the association of SYNGR2 with immunotherapy response in LIHC. Finally, a nomogram including SYNGR2 and pathologic T, N, M stage was built and exhibited good predictive power for the OS, DSS, and PFI of LIHC patients. CONCLUSION: Overall, SYNGR2 is a critical oncogene in various tumors. SYNGR2 participates in the carcinogenic progression, and may contribute to the immune infiltration in tumor microenvironment. Our study suggests that SYNGR2 can serve as a predictor related to prognosis in pan-cancer, especially LIHC.

Prognostic and Immunological Significance of the Molecular Subtypes and Risk Signatures Based on Cuproptosis in Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) remains a challenging medical problem. Cuproptosis is a novel form of cell death that plays a crucial role in tumorigenesis, angiogenesis, and metastasis. However, it remains unclear whether cuproptosis-related genes (CRGs) influence the outcomes and immune microenvironment of HCC patients. Method: From The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases, we obtained the mRNA expression file and related clinical information of HCC patients. We selected 19 CRGs as candidate genes for this study according to previous literature. We performed a differential expression analysis of the 19 CRGs between malignant and precancerous tissue. Based on the 19 CRGs, we enrolled cluster analysis to identify cuproptosis-related subtypes of HCC patients. A prognostic risk signature was created utilizing univariate Cox regression and least absolute shrinkage and selection operator (LASSO) regression analyses. We employed independent and stratification survival analyses to investigate the predictive value of this model. The functional enrichment features, mutation signatures, immune profile, and response to immunotherapy of HCC patients were also investigated according to the two molecular subtypes and the prognostic signature. Results: We found that 17 CRGs significantly differed in HCC versus normal samples. Cluster analysis showed two distinct molecular subtypes of cuproptosis. Cluster 1 is preferentially related to poor prognosis, high activity of immune response signaling, high mutant frequency of TP53, and distinct immune cell infiltration versus cluster 2. Through univariate and LASSO Cox regression analyses, we created a cuproptosis-related prognostic risk signature containing LIPT1, DLAT, MTF1, GLS, and CDKN2A. High-risk HCC patients were shown to have a worse prognosis. The risk signature was proved to be an independent predictor of prognosis in both the TCGA and ICGC datasets, according to multivariate analysis. The signature also performed well in different stratification of clinical features. The immune cells, which included regulatory T cells (Treg), B cells, macrophages, mast cells, NK cells, and aDCs, as well as immune functions containing cytolytic activity, MHC class I, and type II IFN response, were remarkably distinct between the high-risk and low-risk groups. The tumor immune dysfunction and exclusion (TIDE) score suggested that high-risk patients had a higher response rate to immune checkpoint inhibitors than low-risk patients. Conclusion: This research discovered the potential prognostic and immunological significance of cuproptosis in HCC, improved the understanding of cuproptosis, and may deliver new directions for developing more efficacious therapeutic techniques for HCC patients.

A novel liver zonation phenotype-associated molecular classification of hepatocellular carcinoma.

Background: Liver zonation is a unique phenomenon in which the liver exhibits distinct functions among hepatocytes along the radial axis of the lobule. This phenomenon can cause the sectionalized initiation of several liver diseases, including hepatocellular carcinoma (HCC). However, few studies have explored the zonation features of HCC. Methods: Four single-cell RNA sequencing datasets were used to identify hepatocyte-specific zonation markers. Integrative analysis was then performed with a training RNA-seq cohort (616 HCC samples) and an external validating microarray cohort (285 HCC samples) from the International Cancer Genome Consortium, The Cancer Genome Atlas, Gene Expression Omnibus, and EMBL's European Bioinformatics Institute for clustering using non-negative matrix factorization consensus clustering based on zonation genes. Afterward, we evaluated the prognostic value, clinical characteristics, transcriptome and mutation features, immune infiltration, and immunotherapy response of the HCC subclasses. Results: A total of 94 human hepatocyte-specific zonation markers (39 central markers and 55 portal markers) were identified for the first time. Subsequently, three subgroups of HCC, namely Cluster1, Cluster2, and Cluster3 were identified. Cluster1 exhibited a non-zonational-like signature with the worst prognosis. Cluster2 was intensively associated with a central-like signature and exhibited low immune infiltration and sensitivity toward immune blockade therapy. Cluster3 was intensively correlated with a portal-like signature with the best prognosis. Finally, we identified candidate therapeutic targets and agents for Cluster1 HCC samples. Conclusion: The current study established a novel HCC classification based on liver zonation signature. By classifying HCC into three clusters with non-zonational-like (Cluster1), central-like (Cluster2), and portal-like (Cluster3) features, this study provided new perspectives on the heterogeneity of HCC and shed new light on delivering precision medicine for HCC patients.

Sunitinib suppresses M2 polarization of macrophages in tumor microenvironment to regulate hepatocellular carcinoma progression.

This work aimed to investigate the role and mechanism of Sunitinib (Sun) in suppressing M2 polarization of macrophages in tumor microenvironment (TME). IL-4 was applied to induce the M2 polarization of RAW264.7 cells, followed by treatment with Sun at 50 and 100 nM. Flow cytometry (FCM) was conducted to detect the proportion of F4/80 + CD206 + cells. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of IL-10, Arg-1 and VEGF. Immunofluorescence (IF) staining was carried out to detect the expression of CD206 and Arg-1. Besides, western-Blot (WB) assay was performed to measure the levels of p-JAK1 and p-STAT6 proteins. After polarization, the macrophage culture medium was employed to culture hepatocellular carcinoma (HCC) Hca-F cells. Thereafter, Transwell assays were conducted to examine cell invasion and migration, whereas plate clone formation assay was carried out to detect the clone forming capacity. In further experiments, cells were treated with the STAT6 inhibitor, or STAT6 inhibitor + Sun. Then, the polarization levels of RAW264.7 cells were detected. Moreover, this study established the xenograft tumor mouse model. Later, CD206 and Ki67 expression, IL-10, Arg-1 and VEGF expression levels in tissues, and p-JAK1 and p-STAT6 protein levels were detected by histochemical staining. Sun suppressed the M2 polarization of RAW264.7 cells. Compared with IL-4 treatment, the proportion of F4/80 + CD206 + cells decreased. Meanwhile, the levels of IL-10, Arg-1 and VEGF were downregulated, and the phosphorylation level of JAK1-STAT6 signaling was suppressed. After being cocultured with Hca-F, the malignant behaviors of HCC cells were suppressed after Sun treatment. Similarly, STAT6 inhibitor treatment suppressed the M2 polarization, while the combined application of Sun did not further restrain the polarization level. In the mouse model, Sun suppressed the expression of CD206 and Ki67, simultaneously inhibiting the polarization of JAK1-STAT6 signaling. Sunitinib can suppress the M2 polarization of macrophages to exert the anti-HCC effect, which is its another anticancer mechanism.

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

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

Risk modeling of single-cell transcriptomes reveals the heterogeneity of immune infiltration in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common primary hepatic malignancies. E2F transcription factors play an important role in the tumorigenesis and progression of HCC, mainly through the RB/E2F pathway. Prognostic models for HCC based on gene signatures have been developed rapidly in recent years; however, their discriminating ability at the single-cell level remains elusive, which could reflect the underlying mechanisms driving the sample bifurcation. In this study, we constructed and validated a predictive model based on E2F expression, successfully stratifying patients with HCC into two groups with different survival risks. Then we used a single-cell dataset to test the discriminating ability of the predictive model on infiltrating T cells, demonstrating remarkable cellular heterogeneity as well as altered cell fates. We identified distinct cell subpopulations with diverse molecular characteristics. We also found that the distribution of cell subpopulations varied considerably across onset stages among patients, providing a fundamental basis for patient-oriented precision evaluation. Moreover, single-sample gene set enrichment analysis revealed that subsets of CD8+ T cells with significantly different cell adhesion levels could be associated with different patterns of tumor cell dissemination. Therefore, our findings linked the conventional prognostic gene signature to the immune microenvironment and cellular heterogeneity at the single-cell level, thus providing deeper insights into the understanding of HCC tumorigenesis.

Disrupted BRCA1-PALB2 interaction induces tumor immunosuppression and T-lymphocyte infiltration in HCC through cGAS-STING pathway.

BACKGROUND AND AIMS: BRCA1 (BRCA1 DNA repair associated) and PALB2 (partner and localizer of BRCA2) interact with each other to promote homologous recombination and DNA double-strand breaks repair. The disruption of this interaction has been reported to play a role in tumorigenesis. However, its precise function in HCC remains poorly understood. APPROACH AND RESULTS: We demonstrated that mice with disrupted BRCA1-PALB2 interaction were more susceptible to HCC than wild-type mice. HCC tumors arising from these mice showed plenty of T-lymphocyte infiltration and a better response to programmed cell death 1 (PD-1) antibody treatment. Mechanistically, disruption of the BRCA1-PALB2 interaction causes persistent high level of DNA damage in HCC cells, leading to activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway in both malignant hepatocytes and M1 macrophages in the tumor microenvironment. The activated cGAS-STING pathway induces programmed cell death 1 ligand 1 expression via the STING-interferon regulatory factor 3 (IRF3)-signal transducer and activator of transcription 1 pathway, causing immunosuppression to facilitate tumorigenesis and tumor progression. Meanwhile, M1 macrophages with an activated cGAS-STING pathway could recruit T lymphocytes through the STING-IRF3 pathway, leading to T-lymphocyte infiltration in tumors. After normalizing immune responses by PD-1 antibody treatment, the infiltrating T lymphocytes attack tumor cells rapidly and effectively. CONCLUSIONS: This study reveals that persistent DNA damage caused by a defective BRCA pathway induces tumor immunosuppression and T-lymphocyte infiltration in HCC through the cGAS-STING pathway, providing insight into tumor immune microenvironment remodeling that may help improve HCC response to PD-1 antibody treatment.

IFNγ-IL-17+ CD8 T cells contribute to immunosuppression and tumor progression in human hepatocellular carcinoma.

IL-17-producing CD8 (Tc17) T cells have been shown to play an important role in infection and chronic inflammation, however their implications in hepatocellular carcinoma (HCC) remain elusive. In this study, we performed cytometry by time-of-flight (CyTOF) and revealed the distinctive immunological phenotypes of two IFNγ+ and IFNγ- Tc17 subsets that were preferentially enriched in human HCC. Single-cell RNA-sequencing analysis further revealed regulatory circuits governing the different phenotypes of these Tc17 subsets. In particular, we discovered that IFNγ- Tc17 subset demonstrated pro-tumoral characteristics and expressed higher levels of CCL20. This corresponded to increased tumor infiltration of T regulatory cells (Treg) validated by immunohistochemistry in another independent HCC cohort, demonstrating the immunosuppressive functions of IFNγ- Tc17 subset. Most importantly, higher intra-tumoral proportions of IFNγ- Tc17 were associated with poorer prognosis in patients with HCC and this was further validated in The Cancer Genome Atlas (TCGA) HCC cohort. Taken together, this compendium of transcriptomic and proteomic data of Tc17 subsets sheds light on the immunosuppressive phenotypes of IFNγ- Tc17 and its implications in HCC progression.