JHEP Rep. 2025 Jul 10;7(11):101518. doi: 10.1016/j.jhepr.2025.101518. eCollection 2025 Nov.

ABSTRACT

BACKGROUND & AIMS: To enhance our understanding of the tumor immune microenvironment (TIME) in hepatocellular carcinoma (HCC), we investigated the heterogeneity of T-cell exhaustion and its association with HBV integrations and direct oncogenic potential in HCC.

METHODS: We conducted a multi-omics analysis, including single-cell RNA sequencing, whole-exome sequencing, whole-transcriptome sequencing, and next-generation sequencing (NGS)-based HBV integration analysis, in eight patients with virus-related HCC. For validation, bulk RNA sequencing and NGS-based HBV integration analysis were performed in an independent cohort (n = 106).

RESULTS: Based on the expression scores of exhaustion markers in effector CD8⁺ T cells, patients were classified into high (n = 2) and low (n = 6) exhaustion groups (p <0.001). The high-exhaustion group exhibited higher clonal expansion (Gini index: 0.83 vs. 0.48, p = 0.006) and sharing of CD8⁺ T effector memory and cycling T cells with elevated exhaustion markers. This group also showed increased clonal expansion of CD4⁺ regulatory T cells and follicular helper T cells (p <0.001) with higher PDCD1 expression. In addition, the high-exhaustion group had higher TP53 mutation rates and signature scores for proliferation subtypes compared with the low-exhaustion group, who predominantly harbored TERT mutations. Moreover, the high-exhaustion group demonstrated more pronounced HBV integrations with elevated intrahepatic covalently closed circular DNA (cccDNA) and pregenomic (pg)RNA levels. Similarly, in the validation cohort, the high-exhaustion group (n = 28) demonstrated stronger proliferation subtype signatures (p <0.001), along with higher HBV integrations, S-fusion transcripts, and an increased intrahepatic viral reservoir (cccDNA/pgRNA) (p <0.05) compared with the low-exhaustion group (n = 78).

CONCLUSIONS: Our study revealed the heterogeneity in T-cell exhaustion in the TIME of HCC, along with differences in HBV integrations and molecular subtypes. These findings provide insight into the intricate relationship between high exhaustion, proliferation subtype, increased HBV integrations, and enhanced HBV-induced oncogenic potential in virus-related HCC.

IMPACT AND IMPLICATIONS: This study provides a comprehensive immune landscape of T-cell exhaustion using multi-omics analysis, offering critical insights into T cell heterogeneity in virus-related HCC. It establishes a strong association between higher HBV integration, enhanced oncogenic potential, T-cell exhaustion, and proliferation subtypes in HCC. Our results also establish a basis for personalized therapies tailored to the immune-exhaustion status within the TIME of each patient with HCC.

PMID:41113120 | PMC:PMC12529496 | DOI:10.1016/j.jhepr.2025.101518

Cell Death Dis. 2025 Oct 21;16(1):746. doi: 10.1038/s41419-025-08060-7.

ABSTRACT

The regulatory mechanisms governing transcriptional programs in the cancer genome remain elusive, particularly those concerning cell-type specificity. We carefully curated single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) data from eight distinct carcinoma tissues, including breast, skin, colon, endometrium, lung, ovary, liver, and kidney. Using single-cell multi-omics analysis, we identified extensive open chromatin regions and constructed peak-gene link networks, which can reveal distinct cancer gene regulation and genetic risks. We further explored conserved epigenetic regulation across cell types within cancer and elucidated their functional implications. Moreover, we identified cell-type-associated transcription factors (TFs) that regulate key cellular functions, such as the TEAD family of TFs, which widely control cancer-related signaling pathways in tumor cells. In colon cancer, we further identified tumor-specific TFs that are more highly activated in tumor cells than in normal epithelial cells, including CEBPG, LEF1, SOX4, TCF7, and TEAD4, which are pivotal in driving malignant transcriptional programs and represent potential therapeutic targets, as corroborated by single-cell sequencing data from multiple sources and in vitro experiments. Our findings provide a comprehensive understanding of the regulatory dynamics underlying carcinomas and offer valuable insights into potential therapeutic interventions.

PMID:41120274 | PMC:PMC12541060 | DOI:10.1038/s41419-025-08060-7

Front Immunol. 2025 Oct 2;16:1673098. doi: 10.3389/fimmu.2025.1673098. eCollection 2025.

ABSTRACT

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) continues to exhibit resistance to immunotherapy. In this study, we evaluated the efficacy of combining immunotherapy with chemotherapy for the treatment of advanced pancreatic cancer. Additionally, we employed a multimodal analytical approach to elucidate the immune landscape and conduct transcriptomic profiling in PDAC.

METHODS: A retrospective analysis was conducted on the clinical data of 52 patients diagnosed with advanced PDAC who underwent a combined treatment regimen of immunotherapy and chemotherapy. The study evaluated the objective response rate (ORR), disease control rate (DCR), and progression-free survival (PFS). To characterize the immune landscape in treatment-naive pancreatic ductal adenocarcinoma (PDAC) tumors and in the systemic circulation, flow cytometry, multiplex immunohistochemistry (mIHC), and whole transcriptome sequencing were employed.

RESULTS: The study reported an ORR of 32.7%, a DCR of 67.3%, and a 6-month PFS rate of 38.5%, with a median PFS of 5.5 months. Patients treated with a combination of immunotherapy and gemcitabine achieved the longest PFS. The first-line treatment cohort exhibited a significantly higher DCR (79.3% vs. 52.2%, P = 0.038) and a longer median PFS (6.6 vs. 3.5 months, P = 0.032) compared to the second-line treatment cohort. The efficacy of treatment varied depending on the drug combinations used. Flow cytometry analysis revealed a greater frequency of CD45- CD64+ cells in the peripheral blood of patients with progressive disease (PD) compared to those with a partial response (PR). Multiplex immunofluorescence (MIF) analysis indicated an increased intratumoral infiltration of CD8+ T cells and CD137+ CD8+ T cells in patients with PR. Whole transcriptome sequencing (WTSS) identified key genes involved in immune regulation, signal transduction, and digestive function. Hemopexin (HPX) and regulatory factor X-associated protein (RFXAP) were upregulated in PR patients and showed a positive correlation with survival, whereas Interleukin-6 (IL-6) expression was linked to poor prognosis.

CONCLUSIONS: These findings indicate that immunochemotherapy shows potential for the treatment of advanced PDAC. Our study elucidates the immune landscape associated with PDAC and provides critical insights for the identification of prospective therapeutic targets, which could guide the development of innovative combination immunotherapy strategies.

PMID:41112307 | PMC:PMC12528169 | DOI:10.3389/fimmu.2025.1673098

Mol Med Rep. 2026 Jan;33(1):6. doi: 10.3892/mmr.2025.13716. Epub 2025 Oct 17.

ABSTRACT

R‑loops, three‑stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced single‑stranded DNA, have emerged as important regulators of gene expression and genome maintenance. Although physiological R‑loops participate in normal cellular processes, their dysregulation can threaten genomic integrity by inducing DNA damage and replication stress. The present review explores the role of R‑loops in hepatocellular carcinoma (HCC), a malignancy characterized by marked genomic instability. In the present review, the formation mechanisms of R‑loops, their dual functions in transcriptional regulation and DNA damage, and their specific implications for HCC pathophysiology were discussed. HCC cells exhibit altered R‑loop homeostasis with aberrant accumulation linked to hepatitis B virus infection, inflammatory signaling and oncogene activation. The present review highlighted how HCC cells exploit or manage R‑loops to promote tumor progression, particularly through the epigenetic silencing of differentiation genes and modulation of replication stress responses. Furthermore, emerging therapeutic strategies targeting R‑loop biology were examined, including small molecules that induce synthetic lethality, gene‑based interventions and combination approaches that exploit R‑loop vulnerabilities. Challenges in targeting R‑loops and future directions, including multi‑omics profiling and biomarker development, were also addressed. Understanding the complex interplay between R‑loops and HCC offers promising avenues for novel diagnostic and therapeutic approaches for this malignancy.

PMID:41104860 | DOI:10.3892/mmr.2025.13716