Multiomics and multi-region spatial transcriptome analysis reveal cellular networks and pathways associated with HCC recurrence.

Background: Intratumour heterogeneity (ITH) is one of the key characteristics of cancer and is closely associated with patient prognosis, treatment resistance, and tumor metastasis. Nevertheless, the study of ITH in hepatocellular carcinoma (HCC) remains limited. Methods: The present study elucidated the influence of ITH on the tumor microenvironment (TME) in HCC. We applied Non-negative Matrix Factorization (NMF) analysis to a cohort of 78 single-cell RNA sequencing (scRNA-seq) HCC samples to systematically characterize ITH. Furthermore, by integrating spatial transcriptomics (ST) data from five HCC patients, we comprehensively analyzed the spatial organization and functional properties of distinct niches within HCC. We conducted a detailed analysis of the cell-type co-localization relationships within the TME and constructed a comprehensive atlas of HCC spatial organization. Results: We observed a co-localization relationship between hypoxia tumor cells, plasmalemma vesicle-associated protein (PLVAP+) endothelial cells (EC), and vascular endothelial growth factor A (VEGFA+) cancer-associated fibroblasts (CAF), suggesting a key role for hypoxia tumor cells in VEGFA+ CAF transformation and tumor angiogenesis. We identified a unique boundary region enriched with dendritic cells1 (DC1), interferon-expressing tumor cells, lymphatic EC, C–X–C Motif Chemokine Ligand 10 (CXCL10+) macrophages (Mac), and secreted phosphoprotein 1 (SPP1+) Mac located between the tumor-infiltrating immune cells and tumor regions. Furthermore, we found that CXCL10+ Mac and SPP1+ Mac, despite co-localizing in the boundary region, exhibit distinct functions, which may be attributed to their unique spatial locations, with the former being closer to the immune-infiltrated region and the latter more proximal to the tumor area. Conclusions: Our study highlights the critical role of spatial interactions between tumor cells and the microenvironment in HCC. The findings offer new insights into ITH and underscore the importance of spatial organization in understanding cancer biology and designing future precision therapies.

BackgroundHepatocellular carcinoma (HCC) poses a significant health burden globally, with high mortality rates despite various treatment options. Immunotherapy, particularly immune-checkpoint inhibitors (ICIs), has shown promise, but resistance and metastasis remain major challenges. Understanding the intricacies of the tumor microenvironment (TME) is imperative for optimizing HCC management strategies and enhancing patient prognosis.MethodsThis study employed a comprehensive approach integrating multi-omics approaches, including single-cell RNA sequencing (scRNA-seq), bulk RNA sequencing (Bulk RNA-seq), and validation in clinical samples using spatial transcriptomics (ST) and multiplex immunohistochemistry (mIHC). The analysis aimed to identify key factors influencing the immunosuppressive microenvironment associated with HCC metastasis and immunotherapy resistance.ResultsHMGB2 is significantly upregulated in HCCTrans, a transitional subgroup associated with aggressive metastasis. Furthermore, HMGB2 expression positively correlates with an immunosuppressive microenvironment, particularly evident in exhausted T cells. Notably, HMGB2 expression correlated positively with immunosuppressive markers and poor prognosis in HCC patients across multiple cohorts. ST combined with mIHC validated the spatial expression patterns of HMGB2 within the TME, providing additional evidence of its role in HCC progression and immune evasion.ConclusionHMGB2 emerges as a critical player of HCC progression, metastasis, and immunosuppression. Its elevated expression correlates with aggressive tumor behavior and poor patient outcomes, suggesting its potential as both a therapeutic target and a prognostic indicator in HCC management.

Ubiquitination, a critical post-translational modification, plays a pivotal role in regulating protein stability and activity, influencing various aspects of cancer development, including metabolic reprogramming, immune evasion, and tumor progression. However, the specific role of ubiquitination in hepatocellular carcinoma (HCC), particularly in relation to the tumor microenvironment (TME), remains poorly understood. This study aims to systematically explore the role of ubiquitination in shaping the TME of HCC, with a focus on its impact on cancer progression and immune modulation. We performed bioinformatics analysis by integrating multiple publicly available HCC datasets to assess the ubiquitination status across various cell types in the TME, including plasma cells, fibroblasts, endothelial cells, and epithelial-mesenchymal transition (EMT) cells. Ubiquitination scores were calculated to categorize these cell types, and survival data, along with spatial transcriptomics, were employed to evaluate how different levels of ubiquitination influence HCC progression. In vitro experiments, such as transwell, CCK8, and wound healing assays, were used to further investigate the role of the key ubiquitination gene UBE2C in HCC phenotypes. Our study revealed that ubiquitination-related genes are significantly upregulated in HCC tissues, with high expression levels correlating with poor prognosis in patients. Pathway analysis showed that these genes are enriched in key processes such as cell cycle regulation, DNA repair, metabolic reprogramming, and p53 signaling. These pathways contribute to the TME by promoting tumor cell proliferation, facilitating matrix remodeling, and enhancing angiogenesis. Notably, UBE2C, a critical ubiquitination enzyme, appears to play a key role in immune evasion, potentially by inhibiting anti-tumor immune responses and reducing the immune system's ability to recognize and eliminate tumor cells. Furthermore, experimental data confirmed that UBE2C overexpression promotes HCC cell proliferation, invasion, and metastasis, further supporting its role in tumor progression and TME remodeling. This study reveals the multifaceted regulatory roles of ubiquitination in HCC. Ubiquitination not only supports proliferation and anti-apoptotic functions within tumor cells but also promotes tumor progression by modulating the activity of immune and stromal cells. Among all ubiquitination-related genes, UBE2C emerges as a potential prognostic biomarker and therapeutic target in HCC, offering new directions for precision treatment of HCC in the future.

PurposeHighly complex tumor microenvironment makes hepatocellular carcinoma (HCC) as one of the most malignant tumors worldwide. The role of cellular senescence in HCC has been gradually recognized. The present study aimed to comprehensively elucidate the senescence-related features of HCC in single-cell and spatial dimension.MethodsSingle-cell RNA sequencing (scRNA-Seq) data was used to clarify the heterogeneity of senescence-related genes (SRGs) among multiple cell types within HCC. Spatial transcriptome RNA sequencing (stRNA-Seq) data was used for depicting SRGs features in spatial dimension. A prognostic model based on SRGs was constructed by using of bulk sequencing (bulk-Seq) data of HCC. The cell-cell interaction of senescent endothelial cells (ECs) in tumor microenvironment was analyzed. Then, the role of senescent ECs was verified through in vitro and in vivo experiments.ResultsThe level of senescence demonstrated substantial heterogeneity among different cell types within tumor microenvironment of HCC, where ECs exhibited the most prominent senescent phenotype. Senescent ECs activated specific regulatory pathways through communicating with other cell types, with a potential impact on tumor progression. Spatial analysis revealed senescent ECs mainly located in the core region of HCC. The interaction of senescent ECs and immune cells implicated their role in tumor progression and immunotherapeutic response. In addition, CDKN2A was identified as an independent risk factor for HCC prognosis by constructing a prognostic model. Patients with high risk displayed an even worse outcome. The experimental verification indicated senescence of ECs determined by CDKN2A exhibited a secretory phenotype. Furthermore, senescent ECs with CDKN2A overexpression promote the proliferation and migration of HCC.ConclusionThe present study recognizes the critical effect of senescent ECs defined by CDKN2A in the promotion of tumor progression, which sheds new light on the investigation of ECs senescence in HCC.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00432-024-06017-5.

Lung cancer remains one of the leading causes of cancer-related deaths worldwide, with lung adenocarcinoma being the most common pathological type.1, 2 Clinically, many lung adenocarcinomas are detected at an early stage as ground-glass nodules. Although some nodules may remain indolent for a long time, a considerable portion eventually progresses into invasive malignancies. Understanding the molecular and cellular events driving this malignant transformation is crucial for achieving early intervention and improving patient cure rates. Traditionally, tumour endothelial cells were thought to primarily drive angiogenesis in advanced tumours. However, increasing evidence shows that even in early, seemingly quiescent lesions, these cells may undergo subtle functional changes. In this article, the authors identified an endothelial cell subpopulation—tip cells—that promotes early lung adenocarcinoma progression, providing deeper insights into the functional heterogeneity of tumour endothelial cells.3 Traditional high-throughput transcriptomic approaches, such as bulk RNA sequencing (RNA-seq), have played an indispensable role in tumour research over the past few decades. However, conventional bulk RNA-seq technologies can only provide the average gene expression profile of all cells within a tissue sample, thereby failing to capture cellular heterogeneity. Single-cell RNA-seq (scRNA-seq) overcomes this limitation by isolating and profiling individual cells from tissues. This technology enables the identification of rare cellular subpopulations—often overlooked by bulk sequencing—and allows detailed dissection of intratumoural gene expression heterogeneity. Consequently, scRNA-seq has helped uncover key genes, such as oncogenic drivers and immune evasion-related genes, that underlie tumour initiation, progression and drug resistance.4, 5 Nevertheless, scRNA-seq loses the spatial context of cells within tissues. Spatial transcriptomics (ST) perfectly compensates for this shortcoming by revealing which genes are activated in specific tissue regions and by mapping single-cell data back into the spatial context, forming a more comprehensive tissue atlas.6-8 This study integrates both scRNA-seq and ST approaches, confirming the existence of ‘tip cells’ at the single-cell level and, through spatial localisation, revealing their positional relationships with adjacent tumour cells.3 These findings provide strong spatial evidence for the hypothesis that bidirectional communication exists between tip cells and tumour cells—that is, some tip cells express transforming growth factor beta 1 (TGFβ1) and act directly on tumour cells. This multi-omics integration strategy represents one of the most powerful tools currently available for unraveling the complexity of tumour biology. A tumour is not merely a simple accumulation of cancer cells but a complex ecosystem composed of various cell types and non-cellular components—collectively known as the tumour microenvironment (TME).9, 10 The TME not only passively supports tumour growth, but also actively participates in and drives tumour initiation, invasion, immune escape and therapy resistance. This study focuses on a component of the TME that has long been oversimplified—tumour endothelial cells—and successfully reveals a novel function of a key subpopulation, the tip cells, within lung adenocarcinoma.3 This discovery offers dual insights. First, it emphasises that even within well-studied TME cell types, there exists immense functional heterogeneity and previously unrecognised subtypes. Tip cells, as a functional subtype of endothelial cells, exhibit independent pro-tumour function in early invasion, redefining our understanding of endothelial cell roles in tumourigenesis. Second, it raises new questions: within the vast TME, do other cell subpopulations exist—like tip cells—that are functionally critical but have yet to be identified and understood? This study provides a successful research paradigm, demonstrating that applying new technologies to re-examine established cell types or discover novel cellular states within the TME opens new and fertile ground for discovery. In summary, this cutting-edge study, leveraging scRNA-seq and ST, precisely links the early invasive mechanism of lung adenocarcinoma to a specific endothelial cell subpopulation and its key molecule, plasma vesicle-associated protein (PLVAP).3 It further reveals a novel mechanism of bidirectional crosstalk between tip cells and tumour cells via TGFβ1. This work not only deepens our understanding of early lung adenocarcinoma development but, more importantly, exemplifies how emerging technologies empower scientists to decode the complex ecosystem of the TME—making an outstanding contribution to the elucidation of early tumour development mechanisms. Lewei Duan, Yidu Hu and Jiaqi Liang wrote the initial draft of this manuscript. Cheng Zhan reviewed the manuscript. All the authors approved the final version of this letter. This work was supported by grants from the National Natural Science Foundation of China (no. 82473184) and the Shanghai Sailing Program (no. 24YF2704100). The authors declare they have no conflicts of interest. Not applicable.

Emerging Role of Semaphorins as Major Regulatory Signals and Potential Therapeutic Targets in Cancer

Year in review: Liver cancer research in 2022: tumor microenvironment takes the central stage.

P61 Transcriptomic analysis of endothelium from human hepatocellular carcinoma highlights its potential to suppress anti-tumour immune responses

How should patients with hepatocellular carcinoma recurrence after liver transplantation be treated?

Cancer-associated fibroblasts (CAFs), as a major stromal component of the tumor microenvironment (TME) in hepatocellular carcinoma (HCC), play critical roles in regulating tumor progression, immune evasion, and therapeutic responses. However, research on CAFs in HCC remains fragmented, and systematic synthesis with multidimensional mechanistic integration is still lacking. This study aims to systematically elucidate the key roles and mechanisms of CAFs in therapeutic resistance, metastasis, and immune microenvironment remodeling in HCC by integrating bibliometric analysis with multi-omics and spatial transcriptomic data. Original research articles related to CAFs and HCC were systematically retrieved from the Web of Science Core Collection. Bibliometric analyses were conducted using the R package Bibliometrix to assess annual publication trends, country- and institution-level collaboration networks, author and journal distributions, and keyword evolution patterns. In addition, transcriptomic datasets from GEO, TCGA, and ICGC, together with spatial transcriptomic data, were integrated to identify CAF-related differentially expressed genes, characterize their spatial localization, and evaluate correlations between CAF-related gene expression and immune cell infiltration, thereby exploring their potential biological functions. A total of 266 original studies were included. Since 2018, publications in the CAF–HCC field have increased markedly, accompanied by expanding international collaboration. Keyword analyses indicated that “resistance,” “metastasis,” “tumor microenvironment,” and “immune regulation” have remained central research themes. Mechanistic integration suggested that CAFs promote HCC stemness maintenance, immunosuppression, and pre-metastatic niche formation via pathways including TGF-β, IL-6/STAT3, and VEGFA. Multi-omics analyses identified 34 key genes commonly upregulated in both CAFs and HCC tissues. Spatial transcriptomic profiling further revealed that MMP9, SPP1, MMP12, and TREM2 exhibited pronounced spatial enrichment in tumor core regions and the tumor–stroma interface, with significant co-localization with immune cells such as macrophages and regulatory T cells. Among these, TREM2 showed the strongest positive correlation with macrophage infiltration, suggesting a potential role in CAF–immune cell crosstalk and the establishment of an immunosuppressive microenvironment in HCC. CAFs exert multilayered and dynamic regulatory effects on therapeutic resistance, metastasis, and immune microenvironment remodeling in HCC.

Hepatocellular carcinoma (HCC), a highly heterogeneous malignant tumor associated with a poor prognosis, is a common cause of cancer-related deaths worldwide, with a limited survival benefit for patients despite ongoing therapeutic breakthroughs. Coronavirus disease 2019 (COVID-19), a severe infectious disease caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), is a global pandemic and a serious threat to human health. The increased susceptibility to SARS-CoV-2 infection and a poor prognosis in patients with cancer necessitate the exploration of the potential link between the two. No studies have investigated the relationship of COVID-19 genes with the prognosis and tumor development in patients with HCC. We screened prognosis-related COVID-19 genes in HCC, performed molecular typing, developed a stable and reliable COVID-19 genes signature for predicting survival, characterized the immune microenvironment in HCC patients, and explored new molecular therapeutic targets. Datasets of HCC patients, including RNA sequencing data and clinical information, were obtained from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO) databases. Prognosis-related COVID-19 genes were identified by univariate Cox analysis. Molecular typing of HCC was performed using the consensus non-negative matrix factorization method (cNMF), followed by the analysis of survival, tumor microenvironment, and pathway enrichment for each subtype. Prognostic signatures were constructed using LASSO-Cox regression models, and receiver operating characteristic (ROC) curves were used to validate the predictive performance of the signature. The same approach was used for the test and external validation sets. Seven software packages were applied to determine the abundance of immune infiltration in HCC patients and investigate its relationship with the risk scores. Gene set enrichment analysis (GSEA) was used to explore the potential mechanisms by which the COVID-19 genes affect hepatocarcinogenesis and prognosis. Three types of machine learning methods were combined to identify the most critical genes in the signature and localize their expression at the single cell level. We identified 53 prognosis-related COVID-19 genes and classified HCC into two molecular subtypes (C1, C2) by using the NMF method. The prognosis of C2 was significantly better than that of C1, and the two subtypes differed remarkably in terms of the tumor immune microenvironment and biological functions. The 17 COVID-19 genes were screened using the LASSO regression method to develop a 17 COVID-19 genes signature, which demonstrated a good predictive performance for 1-, 2- and 3-year OS of patients with HCC. The risk score as an independent prognostic factor for HCC has better predictive accuracy than traditional clinical variables. Patients in the TCGA cohort were categorized by risk score into the high- and low-risk groups, with the high-risk group mainly enriched in the immune modulation-related pathways and the low-risk group mainly enriched in the metabolism-related pathways, suggesting that the COVID-19 genes may affect disease progression and prognosis by regulating the tumor immune microenvironment and metabolism in HCC. NOL10 was identified as the most critical gene in the signature and hypothesized to be a potential therapeutic target for HCC. Objectively, the COVID-19 genes signature developed in this study, as an independent prognostic factor in HCC patients, is closely associated with the prognosis and tumor immune microenvironment of HCC patients and indicates that they may regulate the development of HCC in multiple ways, providing us with new perspectives for understanding the molecular mechanisms of HCC and finding effective therapeutic targets.

The tumor microenvironment in hepatocellular carcinoma: Current status and therapeutic targets

BackgroundThe highly heterogeneity of the tumor microenvironment (TME) in hepatocellular carcinoma (HCC) results in diverse clinical outcomes and therapeutic responses. This study aimed to investigate potential intercellular crosstalk and its impact on clinical outcomes and therapeutic responses. MethodsSingle-cell RNA sequencing (scRNA-seq), spatial transcriptomics (ST) and bulk RNA sequencing (RNA-seq) datasets were integrated to comprehensively analyze the intercellular interactions within the TME. Multiplex immunohistochemistry was conducted to validate the intercellular interactions. A machine learning-based integrative procedure was used in bulk RNA-seq datasets to generate a risk model to predict prognosis and therapeutic responses. ResultsSurvival analyses based on the bulk RNA-seq datasets revealed the negative impact of the naïve Cluster of Differentiation 4+ (CD4) T cells and Secreted Phosphoprotein 1+ (SPP1) macrophages on prognosis. Furthermore, their intricate intercellular crosstalk and spatial colocalization were also observed by scRNA-seq and ST analyses. Based on this crosstalk, a machine learning model, termed the naïve CD4+ T cell and SPP1+ macrophage prognostic score (TMPS), was established in the bulk-RNA seq datasets for prognostic prediction. The TMPS achieved C-index values of 0.785, 0.715, 0.692 and 0.857, respectively, across 4 independent cohorts. A low TMPS was associated with a significantly increased survival rates, improved response to immunotherapy and reduced infiltration of immunosuppressive cells, such as. regulatory T cells. Finally, 8 potential sensitive drugs and 6 potential targets were predicted for patients based on their TMPS. ConclusionThe crosstalk between naïve CD4+ T cells and SPP1+ macrophages play a crucial role in the TME. TMPS can reflect this crosstalk and serve as a valuable tool for prognostic stratification and guiding clinical decision-making.

Can We Move on From the Discussion of Direct Antiviral Agents and Risk of Hepatocellular Carcinoma Recurrence?

Background: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Curative treatments in early-stage HCC are hampered by high recurrence rates, and targeted therapies against HCC are rare. Little is known about the impact of tumor microenvironment (TME) in this tumor. Manipulation of the immune system by tumors leads to compromised antigen recognition and an immunosuppressive microenvironment. One immunomodulatory strategy employed by malignant tumors is the expression of the programmed cell death receptor 1 ligand (PD-L1), whose receptor is expressed on activated T cells (PD-1). The use of immune checkpoint inhibitors (ICI) has shown antitumor activity and unprecedented rates of durable clinical responses in various cancer types. In HCC, nivolumab and pembrolizumab are described as possible second line therapies. Expression of PD-1 and PD-L1 in the TME has prognostic importance and can predict response to ICI. The aim of our study was to investigate the impact of PD-1 and PD-L1 expression on HCC recurrence and the relation to their cancer-immune phenotypes. Materials and methods: Immunohistochemical staining was performed on archival tissue from 91 HCC, primary and recurrence, using the following antibodies: NAT105 (PD-1) and Ventana SP263 (PD-L1). On the basis of protein expression, tumors were classified as immunologically active, excluded or deserted. PD-1 and PD-L1 immunoreactivity was evaluated as the proportion of positive staining of the total number of immune cells (0%, <1% and >1%). Results: Basic tumor characteristics were not related to recurrence of HCC. However, the immune-excluded subclass showed significant association with recurrence in HCC. The majority of non-recurrent HCC showed PD-1 expression >1% (78%; 38/49), also statistically significant. Conversely, recurrent HCC showed low PD-1 expression (0% or <1%) in 42% (19/45) of the cases. Of note, HCC classified as immune active were enriched for PD-1 positive (>1%) (77%; 49/64). An association between HCC recurrence and PD-L1 expression on tumor cells was not found. Conclusion: In concordance with previous reports, we found an association between low frequency of PD-1 expression and increased HCC recurrence, as well as the lack of impact of PD-L1 expression on HCC recurrence. Furthermore, our results suggest that HCC recurrence may be more frequent in immune-exclusive tumors. Pembrolizumab as adjuvant instead of 2nd line therapy should be further investigated. Citation Format: Gabriel Fridolin Hess, Mairene Coto-Llerena, Jasmin Zeindler, Silvio Däster, Simone Muenst, Jürg Vosbeck, Caner Ercan, Otto Kollmar, Salavtore Piscuoglio, Savas Deniz Soysal. PD1 expression and immune-active lymphocytes can predict recurrence of 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 6146.