Hepatocellular Carcinoma Microenvironment Research Articles

Overcoming resistance to immune checkpoint blockade in liver cancer - stronger together?

Primary liver cancer, represented mainly by hepatocellular carcinoma (HCC) and intrahepatic cholangiocellular carcinoma (CCA), is one of the most common and deadliest tumors worldwide. While surgical resection or liver transplantation are the best option in early disease stages, these tumors often present in advanced stages and systemic treatment is required to improve survival time. The emergence of immune checkpoint inhibitor therapy has had a positive impact especially on the treatment of advanced cancers, thereby establishing immunotherapy as part of first-line treatment in HCC and CCA. Nevertheless, low response rates reflect on the usually cold or immunosuppressed tumor microenvironment of primary liver cancer. In this review, we aim to summarize mechanisms of resistance leading to tumor immune escape with a special focus on the composition of tumor microenvironment in both HCC and CCA, also reflecting on recent important developments in ICI combination therapy. Furthermore, we discuss how combination of immune checkpoint inhibitors with established primary liver cancer treatments (e.g. multikinase inhibitors and chemotherapy) as well as more complex combinations with state-of-the-art therapeutic concepts may re-shape the tumor microenvironment, leading to higher response rates and long-lasting anti-tumor immunity for primary liver cancer patients.

Apparent diffusion coefficient and tissue stiffness are associated with different tumor microenvironment features of hepatocellular carcinoma.

To investigate associations between tissue diffusion, stiffness, and different tumor microenvironment features in resected hepatocellular carcinoma (HCC). Seventy-two patients were prospectively included for preoperative magnetic resonance (MR) diffusion-weighted imaging and MR elastography examination. The mean apparent diffusion coefficient (ADC) and stiffness value were measured on the central three slices of the tumor and peri-tumor area. Cell density, tumor-stroma ratio (TSR), lymphocyte-rich HCC (LR-HCC), and CD8 + T cell infiltration were estimated in resected tumors. The interobserver agreement of MRI measurements and subjective pathological evaluation was assessed. Variables influencing ADC and stiffness were screened with univariate analyses, and then identified with multivariable linear regression. The potential relationship between explored imaging biomarkers and histopathological features was assessed with linear regression after adjustment for other influencing factors. Seventy-two patients (male/female: 59/13, mean age: 56 ± 10.2 years) were included for analysis. Inter-reader agreement was good or excellent regarding MRI measurements and histopathological evaluation. No correlation between tumor ADC and tumor stiffness was found. Multivariable linear regression confirmed that cell density was the only factor associated with tumor ADC (Estimate = -0.03, p = 0.006), and tumor-stroma ratio was the only factor associated with tumor stiffness (Estimate = -0.18, p = 0.03). After adjustment for fibrosis stage (Estimate = 0.43, p < 0.001) and age (Estimate = 0.04, p < 0.001) in the multivariate linear regression, intra-tumoral CD8 + T cell infiltration remained a significant factor associated with peri-tumor stiffness (Estimate = 0.63, p = 0.02). Tumor ADC surpasses tumor stiffness as a biomarker of cellularity. Tumor stiffness is associated with tumor-stroma ratio and peri-tumor stiffness might be an imaging biomarker of intra-tumoral immune microenvironment. Tissue stiffness could potentially serve as an imaging biomarker of the intra-tumoral immune microenvironment of hepatocellular carcinoma and aid in patient selection for immunotherapy. Apparent diffusion coefficient reflects cellularity of hepatocellular carcinoma. Tumor stiffness reflects tumor-stroma ratio of hepatocellular carcinoma and is associated with tumor-infiltrating lymphocytes. Tumor and peri-tumor stiffness might serve as imaging biomarkers of intra-tumoral immune microenvironment.

Regulatory effect of Yinchenhao decoction on bile acid metabolism to improve the inflammatory microenvironment of hepatocellular carcinoma in mice.

Hepatocellular carcinoma (HCC) is a malignant tumor with extremely high mortality. The tumor microenvironment is the "soil" of its occurrence and development, and the inflammatory microenvironment is an important part of the "soil". Bile acid is closely related to the occurrence of HCC. Bile acid metabolism disorder is not only directly involved in the occurrence and development of HCC but also affects the inflammatory microenvironment of HCC. Yinchenhao decoction, a traditional Chinese medicine formula, can regulate bile acid metabolism and may affect the inflammatory microenvironment of HCC. To determine the effect of Yinchenhao decoction on bile acid metabolism in mice with HCC and to explore the possible mechanism by which Yinchenhao decoction improves the inflammatory microenvironment of HCC by regulating bile acid metabolism, we established mice model of orthotopic transplantation of hepatocellular carcinoma. These mice were treated with three doses of Yinchenhao decoction, then liver samples were collected and tested. Yinchenhao decoction can regulate the disorder of bile acid metabolism in liver cancer mice. Besides, it can improve inflammatory reactions, reduce hepatocyte degeneration and necrosis, and even reduce liver weight and the liver index. Taurochenodeoxycholic acid, hyodeoxycholic acid, and taurohyodeoxycholic acid are important molecules in the regulation of the liver inflammatory microenvironment, laying a foundation for the regulation of the liver tumor inflammatory microenvironment based on bile acids. Yinchenhao decoction may improve the inflammatory microenvironment of mice with HCC by ameliorating hepatic bile acid metabolism.

Tumor Microenvironment-Responsive Nanocapsule Delivery CRISPR/Cas9 to Reprogram the Immunosuppressive Microenvironment in Hepatoma Carcinoma.

Cancer immunotherapy has demonstrated significant efficacy in various tumors, but its effectiveness in treating Hepatocellular Carcinoma (HCC) remains limited. Therefore, there is an urgent need to identify a new immunotherapy target and develop corresponding intervention strategies. Bioinformatics analysis has revealed that growth differentiation factor 15 (GDF15) is highly expressed in HCC and is closely related to poor prognosis of HCC patients. The previous study revealed that GDF15 can promote immunosuppression in the tumor microenvironment. Therefore, knocking out GDF15 through gene editing could potentially reverse the suppressive tumor immune microenvironment permanently. To deliver the CRISPR/Cas9 system specifically to HCC, nanocapsules (SNC) coated with HCC targeting peptides (SP94) on their surface is utilized. These nanocapsules incorporate disulfide bonds (SNCSS) that release their contents in the tumor microenvironment characterized by high levels of glutathione (GSH). In vivo, the SNCSS target HCC cells, exert a marked inhibitory effect on HCC progression, and promote HCC immunotherapy. Mechanistically, CyTOF analysis showed favorable changes in the immune microenvironment of HCC, immunocytes with killer function increased and immunocytes with inhibitive function decreased. These findings highlight the potential of the CRISPR-Cas9 gene editing system in modulating the immune microenvironment and improving the effectiveness of existing immunotherapy approaches for HCC.

Impact of anesthesia-related genes on prognosis and tumor microenvironment in hepatocellular carcinoma: A comprehensive analysis.

Hepatocellular carcinoma (HCC), renowned for its bleak prognosis and high recurrence rates, necessitates innovative strategies for prognosis assessment and therapeutic intervention. In this pursuit, we systematically investigated the influence of anesthesia-related genes (ANARGs) on HCC outcomes. Leveraging data from The Cancer Genome Atlas (TCGA), our study scrutinized RNA sequencing data and clinical profiles from 374 HCC patients alongside 50 non-tumor liver samples to unravel ANARG expression patterns and their clinical relevance. Employing consensus clustering, we segregated HCC samples into two distinct subtypes based on ANARG profiles, unveiling significant survival disparities between them. Further differential expression analysis pinpointed pivotal genes and pathways distinguishing these subtypes, notably implicating lipid metabolism and the MTOR signaling pathway in HCC pathogenesis. A prognostic model, comprising five key ANARGs (DAGLA, CYP26B, HAVCR, G6PD and AKR1B), exhibited robust predictive capability for patient outcomes, validated across independent patient cohorts. Furthermore, immune infiltration analysis uncovered a nuanced interplay between ANARG expression and the tumor immune microenvironment, spotlighting variations in immune cell infiltration and function across the identified HCC subtypes. This comprehensive analysis underscores not only the prognostic significance of ANARGs in HCC but also their potential to modulate the tumor microenvironment, providing novel insights for tailoring anesthetic management and therapeutic strategies in HCC care. Our findings advocate for a more integrative approach to HCC management, amalgamating molecular profiling with traditional clinical parameters to refine patient stratification and personalize treatment strategies.

Multifaceted roles of lymphatic and blood endothelial cells in the tumor microenvironment of hepatocellular carcinoma: A comprehensive review.

The tumor microenvironment is a complex network of cells, extracellular matrix, and signaling molecules that plays a critical role in tumor progression and metastasis. Lymphatic and blood vessels are major routes for solid tumor metastasis and essential parts of tumor drainage conduits. However, recent studies have shown that lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) also play multifaceted roles in the tumor microenvironment beyond their structural functions, particularly in hepatocellular carcinoma (HCC). This comprehensive review summarizes the diverse roles played by LECs and BECs in HCC, including their involvement in angiogenesis, immune modulation, lymphangiogenesis, and metastasis. By providing a detailed account of the complex interplay between LECs, BECs, and tumor cells, this review aims to shed light on future research directions regarding the immune regulatory function of LECs and potential therapeutic targets for HCC.

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

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. 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). 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. 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.

Homologous-magnetic dual-targeted metal-organic framework to improve the Anti-hepatocellular carcinoma efficacy of PD-1 inhibitor

The insufficient abundance and weak activity of tumour-infiltrating lymphocytes (TILs) are two important reasons for the poor efficacy of PD-1 inhibitors in hepatocellular carcinoma (HCC) treatment. The combined administration of tanshinone IIA (TSA) and astragaloside IV (As) can up-regulate the abundance and activity of TILs by normalising tumour blood vessels and reducing the levels of immunosuppressive factors respectively. For enhancing the efficacy of PD-1 antibody, a magnetic metal–organic framework (MOF) with a homologous tumour cell membrane (Hm) coating (Hm@TSA/As-MOF) is established to co-deliver TSA&As into the HCC microenvironment. Hm@TSA/As-MOF is a spherical nanoparticle and has a high total drug-loading capacity of 16.13 wt%. The Hm coating and magnetic responsiveness of Hm@TSA/As-MOF provide a homologous-magnetic dual-targeting, which enable Hm@TSA/As-MOF to counteract the interference posed by ascites tumour cells and enhance the precision of targeting solid tumours. Hm coating also enable Hm@TSA/As-MOF to evade immune clearance by macrophages. The release of TSA&As from Hm@TSA/As-MOF can be accelerated by HCC microenvironment, thereby up-regulating the abundance and activity of TILs to synergistic PD-1 antibody against HCC. This study presents a nanoplatform to improve the efficacy of PD-1 inhibitors in HCC, providing a novel approach for anti-tumour immunotherapy in clinical practice.

Molecular Genealogy of Metabolic-associated Hepatocellular Carcinoma.

This review examines the latest epidemiological and molecular pathogenic findings of metabolic-associated hepatocellular carcinoma (HCC). Its increasing prevalence is a significant concern and reflects the growing burden of obesity and metabolic diseases, including metabolic dysfunction-associated steatotic liver disease, formerly known as nonalcoholic fatty liver disease, and type 2 diabetes. Metabolic-associated HCC has unique molecular abnormality and distinctive gene expression patterns implicating aberrations in bile acid, fatty acid metabolism, oxidative stress, and proinflammatory pathways. Furthermore, a notable frequency of single nucleotide polymorphisms in genes such as patatin-like phospholipase domain-containing 3, transmembrane 6 superfamily member 2, glucokinase regulator, and membrane-bound O-acyltransferase domain-containing 7 has been observed. The tumor immune microenvironment of metabolic-associated HCC is characterized by unique phenotypes of macrophages, neutrophils, and T lymphocytes. Additionally, the pathogenesis of metabolic-associated HCC is influenced by abnormal lipid metabolism, insulin resistance, and dysbiosis. In conclusion, deciphering the intricate interactions among metabolic processes, genetic predispositions, inflammatory responses, immune regulation, and microbial ecology is imperative for the development of novel therapeutic and preventative measures against metabolic-associated HCC.

Comprehensive analysis of m6A modification in immune infiltration, metabolism and drug resistance in hepatocellular carcinoma

N6-methyladenosine (m6A) is important in regulating mRNA stability, splicing, and translation, and it also contributes to tumor development. However, there is still limited understanding of the comprehensive effects of m6A modification patterns on the tumor immune microenvironment, metabolism, and drug resistance in hepatocellular carcinoma (HCC). In this study, we utilized unsupervised clustering based on the expression of 23 m6A regulators to identify m6A clusters. We identified differential m6A modification patterns and characterized m6A-gene-cluster A, which exhibited poorer survival rates, a higher abundance of Treg cells, and increased expression of TGFβ in the tumor microenvironment (TME). Additionally, m6A-gene-cluster A demonstrated higher levels of glycolysis activity, cholesterol metabolism, and fatty acid biosynthesis. We also found that the m6A score was associated with prognosis and drug resistance. Patients with a low m6A score experienced worse prognoses, which were linked to an abundance of Treg cells, upregulation of TGFβ, and increased metabolic activity. HCC patients with a higher m6A score showed improved prognosis following sorafenib treatment and immunotherapy. In conclusion, we reveals the association between m6A modification patterns and the tumor immune microenvironment, metabolism, and drug resistance in HCC. Furthermore, the m6A score holds potential as a predictive factor for the efficacy of targeted therapy and immunotherapy in HCC.

The role of alpha-fetoprotein in the tumor microenvironment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent malignant cancer worldwide, characterized by high morbidity and mortality rates. Alpha-fetoprotein (AFP) is a glycoprotein synthesized by the liver and yolk sac during fetal development. However, the serum levels of AFP exhibit a significant correlation with the onset and progression of HCC in adults. Extensive research has demonstrated that the tumor microenvironment (TME) plays a crucial role in the malignant transformation of HCC, and AFP is a key factor in the TME, promoting HCC development. The objective of this review was to analyze the existing knowledge regarding the role of AFP in the TME. Specifically, this review focused on the effect of AFP on various cells in the TME, tumor immune evasion, and clinical application of AFP in the diagnosis and treatment of HCC. These findings offer valuable insights into the clinical treatment of HCC.

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

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. 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. 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. 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.

Metabolome Profiling of Malignant Ascites Identifies Candidate Metabolic Biomarkers of Hepatocellular Carcinoma.

Malignant ascites is one of the severe complications of hepatocellular carcinoma, which can be regarded as a unique tumor microenvironment of hepatocellular carcinoma. The identification of novel biomarkers in malignant ascites could be crucial to differentiate patients with hepatocellular carcinoma and cirrhotic ascites. The study aimed to distinguish the metabolomics of malignant ascites in patients with hepatocellular carcinoma from that of non-malignant ascites (cirrhotic ascites). Liquid chromatography-mass spectrometry was performed to analyze the differentially distributed biomarkers in patients with malignant ascites and hepatocellular carcinoma (n = 39), as well as in patients with cirrhotic ascites, which were taken as controls (n = 36). A total of 20 differential metabolites associated with malignant ascites were identified, of which 8 metabolites were upregulated and 12 metabolites were downregulated (ratio < 0.5 or > 1.5, respectively). Moreover, pathway and enrichment analyses revealed nitrogen metabolism, urea cycle, phenylalanine, and tyrosine metabolism to be implicated in the formation of malignant ascites in patients with hepatocellular carcinoma. Our results suggest that the key factors associated with pathways, such as arachidonic acid, phenylalanine, and glutamic acid pathways, are potential ascitic fluidbased biomarkers for differentiating hepatocellular carcinoma with cirrhosis ascites; the results also provide a clinical pathophysiological interpretation of biomarkers and metabolic pathways relevant to disease status.

Hepatocellular carcinoma immune microenvironment and check point inhibitors-current status.

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver and has a high mortality rate. The Barcelona Clinic Liver Cancer staging system in addition to tumor staging also links the modality of treatment available to a particular stage. The recent description of the tumor microenvironment (TME) in HCC has provided a new concept of immunogenicity within the HCC. Virus-related HCC has been shown to be more immunogenic with higher expression of cytotoxic T lymphocytes and decreased elements for immunosuppression such as regulatory T cells. This immunogenic milieu provides a better response to immunotherapy especially immune checkpoint inhibitors (ICIs). In addition, the recent data on combining locoregional therapies and other strategies may convert the less immunogenic state of the TME towards higher immunogenicity. Therefore, data are emerging on the use of combinations of locoregional therapy and ICIs in unresectable or advanced HCC and has shown better survival outcomes in this difficult population.

Single-cell and spatial analyses revealed the co-location of cancer stem cells and SPP1+ macrophage in hypoxic region that determines the poor prognosis in hepatocellular carcinoma

In hepatocellular carcinoma (HCC), classical cancer stem cells (CSC) markers were shared by normal stem cells, targeting which may hinder hepatic regeneration and cause liver failure. Additionally, the spatial structure of CSC still remained elusive. To address these limitations, we undertook a comprehensive study combining single-cell data (56,022 cells from 20 samples) and spatial data (38,191 spots from eight samples) to obtain CSC signature and uncover its spatial structure. Utilizing the CytoTRACE algorithm, we discretely identified CSC, which displayed upregulated proliferation pathways regulated by HIF1A. A CSC signature of 107 genes was then developed using Weighted Gene Co-expression Network Analysis (WGCNA). Notably, HCC patients with high CSC levels exhibited an accumulation of SPP1+ macrophages (Macro_SPP1) expressing metalloproteinases (MMP9, MMP12, and MMP7) regulated by HIF1A, suggesting a hypoxic tumor region connecting Macro_SPP1 and CSC. Both CSC and Macro_SPP1 correlated with worse prognosis and undesirable immunotherapy response. Spatial analysis revealed the co-location of CSC and Macro_SPP1, with CD8 T cells excluded from the tumor region. The co-location area and non-tumor area of boundary exhibited a high level of hypoxia, with the HAVRC2 checkpoint highly expressed. Within the co-location area, the SPP1 signaling pathway was most active in cell-cell communication, with SPP1-CD44 and SPP1-ITGA/ITGB identified as the main ligand-receptor pairs. This study successfully constructed a CSC signature and demonstrated the co-location of CSC and Macro_SPP1 in a hypoxic region that exacerbates the tumor microenvironment in HCC.

Abstract 1526: The distinct tumor microenvironment of advanced hepatocellular carcinoma with high PVR or high PD-L1 expression

Abstract Background: The combination of anti-PD-L1 and anti-VEGF has been the standard-of-care for advanced hepatocellular carcinoma (HCC), while additionally targeting TIGIT is under active investigation. We aimed to explore the expression and clinical significance of PVR (CD155), the ligand of TIGIT, within the tumor microenvironment (TME) of advanced HCC. Methods: All HCC tumor specimens were obtained at the advanced stage of HCC. Immunohistochemistry (IHC) was employed to evaluate membranous staining of PVR and PD-L1 of tumor specimens, and semiquantitative scores with IHC 0, 1, 2, or 3 were given if &amp;lt; 1%, ≥1% but &amp;lt; 5%, ≥5% but &amp;lt; 10%, or ≥10% of tumor cells (TCs) and tumor-infiltrating immune cells (ICs) were PVR or PD-L1 positive. Selected tumor specimens underwent immune gene expression profiling using the nCounter Analysis System (NanoString Technologies, Inc.) with the PanCancer Immune Profiling Panel. Results: The study enrolled 67 advanced HCC patients, with median age of 59.2 years (male:female = 57:10). Forty-eight (71.6%) of the patients were hepatitis B virus (HBV) surface antigen positive, and 23 (34.3%) had received sorafenib before the study. Thirteen (19.4%) and 18 (26.9%) tumor tissues showed positive PVR expressions (IHC scores ≥1) in TCs and ICs, respectively; Seventeen (25.4%) and 36 (53.7%) tumor tissues showed positive PD-L1 expressions in TCs and ICs, respectively. The expressions of PVR in TCs or ICs showed no association with clinical factors, such as sex, age, HBV infection, sorafenib treatment, or overall survival. Interestingly, in both TCs and ICs, the intensities of PVR and PD-L1 expression were inversely correlated for those with at least IHC scores ≥1 in either PVR or PD-L1 (TCs, n = 26, r = -0.6966, p &amp;lt; 0.0001; ICs, n = 46, r = -0.4548, p = 0.0015). Immune profiling of the selected tumor specimens showed that those with high PVR but low PD-L1 expressions had significantly lower CD8 T cells scores, lower cytotoxic cells scores, and lower CD8/Treg ratio than those with high PD-L1 but low PVR expressions. Conclusions: There was an inverse correlation observed between expressions of PVR and PD-L1 in the advanced HCC tissues. The TME of those with high PVR expression was distinct from those with high PD-L1 expression. These findings may have important clinical implications for developing combination immunotherapy in advanced HCC. (This work is supported by grants from the Ministry of Science and Technology, Taiwan, MOST 110-2314-B-002-204-MY3). Citation Format: Li-Chun Lu, Yi-Hsuan Lee, Tsung-hao Liu, Yu-Yun Shao, Ann-Lii Cheng, Chih-Hung Hsu. The distinct tumor microenvironment of advanced hepatocellular carcinoma with high PVR or high PD-L1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1526.

Abstract 574: Adipocyte-derived FABP4 promotes non-alcoholic fatty liver disease (NAFLD)-induced hepatocellular carcinoma by driving ITGB1-mediated β-catenin activation

Abstract Nonalcoholic fatty liver disease (NAFLD)-induced hepatocellular carcinoma (HCC) is an emerging malignancy in the developed countries. However, the mechanisms contributing to its formation remain largely unknown. Given the role of cancer stem cells (CSCs) in tumor initiation and therapeutic resistance, we hypothesize that adipocytes, one of the key cellular factors within the tumor microenvironment of NAFLD-induced HCC, may play a critical role in HCC development and drug resistance by regulating liver CSCs. Using a co-culture system in which differentiated adipocytes were grown with HCC cells, we found that adipocytes enhanced the self-renewal ability of HCC cells through indirect paracrine secretion. HCC cells pre-incubated with conditioned medium from adipocytes showed enhanced liver CSC properties including self-renewal, tumorgenicity, invasiveness and chemo-resistance to doxorubicin and sorafenib. Secretome profiles showed that FABP4 was preferentially secreted by adipocytes and its level was further augmented when co-cultured with HCC cells. Concurrently, recombinant FABP4 enhanced CSC properties of HCC cells. Drastic delay in the onset of tumor development in FABP4−/- mice upon DEN-injected and high fat diet-induced mouse models of NAFLD-HCC. Mass spectrometry analysis revealed ITGB1 as a direct binding partner of FABP4 for the first time. This data, together with the observation of significant downregulation of the Wnt/β-catenin pathway in tumors of FABP4−/− mice, revealed the role of adipocyte-derived FABP4 promotes liver CSC function by activating the PI3K/Akt/β-catenin signaling pathway via ITGB1. Overexpression of ITGB1 led to poorer survival of HCC patients with NAFLD as a risk factor. The development of a monoclonal neutralizing antibody against FABP4 successfully blocked FABP4-driven CSC functions, implicating the targetability of FABP4 for the treatment of NAFLD-induced HCC. Citation Format: Carmen Oi Ning Leung, Shilpa Gurung, Katherine Po Sin Chung, Sze Man Chan, Terence Kin Wah Lee. Adipocyte-derived FABP4 promotes non-alcoholic fatty liver disease (NAFLD)-induced hepatocellular carcinoma by driving ITGB1-mediated β-catenin activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 574.

Abstract 7039: Leveraging single cell RNA-seq for uncovering immune landscape of hepatocellular carcinoma

Abstract The immune cell types as well as tumor microenvironment (TME) status may influence prognosis, metastasis, tumor progression, and clinical response to immunotherapies for cancer. Hepatocellular carcinoma (HCC) is a typical highly heterogeneous solid tumor with high morbidity and mortality worldwide, especially in China, however, the immune microenvironment of HCC, especially which caused by cancerogenic substance has not been clarified so far. In this study, we employed single-cell RNA sequencing (scRNA-seq) on diethylnitrosamine (DEN)-induced mouse HCC model to dissect the immune cell dynamics during tumorigenesis. Our findings reveal distinct immune profiles in both precancerous and cancerous lesions, indicating early tumor-associated immunological alterations. Notably, specific T and B cell subpopulations are preferentially enriched in the HCC TME. Furthermore, we identified a subpopulation of naïve B cells with high CD83 expression, correlating with improved prognosis in human HCC. These signature genes were validated in The Cancer Genome Atlas (TCGA) HCC RNA-seq dataset. Moreover, cell interaction analysis revealed that subpopulations of B cells in both mouse and human samples are activated and may potentially contribute to oncogenic processes. In summary, our study provides insights into the dynamic immune microenvironment and cellular networks in HCC pathogenesis, with a specific emphasis on naïve B cells. These findings emphasize the significance of targeting TME in HCC patients to prevent HCC pathological progression, which may give a new perspective on the therapeutics for the HCC. Citation Format: Jian He. Leveraging single cell RNA-seq for uncovering immune landscape of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7039.

Abstract 1408: Intrahepatic IgA complex induces polarization of cancer-associated fibroblasts to matrix phenotypes in the tumor microenvironment of HCC

Abstract Background and Aims: Cancer-associated fibroblasts (CAFs) are activated fibroblasts that play key roles in the tumor microenvironment (TME). Immunoglobulin A (IgA) contributes to inflammation and anti-tumor immunity dismantling in the human liver. We investigated the effects of the IgA complex on CAFs in the TME of hepatocellular carcinoma (HCC). Approach and Results: CAF dynamics in HCC TME were analyzed via single-cell RNA sequencing of HCC samples. CAFs were isolated from 40 HCC samples. The isolated CAFs were treated with mock or serum-derived IgA dimers. Following treatment with the mock or serum IgA dimers in vitro, co-culture experiments were performed using CAF and CD8+ T cells. Single-cell analysis showed that sub-cluster proportions in the CAF-fibroblast activation protein-α (FAP) matrix were significantly increased in patients with high serum IgA levels. We were performed flow cytometry on fresh surgical tissues and observed a significant increase in the mean fluorescence intensity (MFI) of FAP in the CD68+ cells from patients with high serum IgA levels compared to those with low serum IgA levels (p&amp;lt;0.001). We validated that the transferrin receptor (CD71) is expressed in CAFs (p&amp;lt;0.01). IgA-treated CAFs exhibited higher programmed death-ligand 1 (PD-L1) expression levels than mock-treated CAFs (p&amp;lt;0.05). Co-culture with CAFs induced the attenuation of the cytotoxic function of activated CD8+ T cell, and these cells co-cultured with IgA-treated CAFs exhibited increased expression levels of programmed death-1 (PD-1) compared to those co-cultured with mock-treated CAFs (p&amp;lt;0.05). Conclusions: Intrahepatic IgA induces polarization of HCC CAFs into more malignant matrix phenotype and attenuates cytotoxic T cell function. Our study uncovers five dynamic CAF subpopulations within HCC tissues and highlights their potential roles in tumor progression and immune suppression. Citation Format: Pil Soo Sung. Intrahepatic IgA complex induces polarization of cancer-associated fibroblasts to matrix phenotypes in the tumor microenvironment of HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1408.

Abstract 7398: Unveiling the tumor microenvironment of hepatocellular carcinoma using AI trained by spatial transcriptomics: A preliminary study to predict response to immunotherapy

Abstract Background. Immune checkpoint inhibitors (ICIs), particularly targeting the PD-1 pathway, are promising in treating hepatocellular carcinoma (HCC). However, their variable effectiveness among individuals calls for a better understanding of the tumor microenvironment (TME) and reliable predictive biomarkers. Here, we employ spatial transcriptomics (ST) to develop a deep-learning model that aims to investigate the TME in HCC using Hematoxylin and eosin (H&amp;E) staining images. Our focus is on identifying cell types within the TME that are linked to the response to ICI in a cohort of patients treated with nivolumab. Methods. Our study leveraged 21 Visium ST datasets from HCC to develop a model aimed at inferring the cellular composition of TME from H&amp;E images that matched with ST data. The comprehensive TME cell types enrichment score, encompassing Tumor Endothelial Cells (TECs), Tumor-Associated Macrophages (TAMs), B cells, T cells, and Cancer-Associated Fibroblasts (CAFs) in each spot, was calculated by the CellDART algorithm, a method that maps cell type by integrating with a reference scRNA-seq data. The model was trained in a patch-wise manner, utilizing a pre-trained ResNetRS50 as the backbone model. For the internal validation, Spearman correlation coefficients were evaluated for predicted cell types using H&amp;E, and the trained model was correlated with cell type enrichment scores estimated by ST datasets of 4 samples independent of the training set. The model was applied to H&amp;E images of 16 patients with HCC who underwent nivolumab treatment. The model predicted the 5 cell types enrichment score in the tumor and tumor-adjacent normal liver tissues. Results. The internal validation set results demonstrate robust Spearman correlations between our model predictions and actual cellular compositions in the TME. Specifically, we observe strong correlations for T cells (rho = 0.66), TAMs (rho=0.46), CAFs (rho=0.32), B cells (rho=0.30), and TECs (rho=0.24). When we applied the model to the H&amp;E images of the cohort of patients who underwent nivolumab, immunotherapy non-responders showed a significantly higher predicted enrichment score of CAFs in tumor-adjacent regions (Mann-Whitney test, p&amp;lt;.01). Upon stratifying patients based on stromal CAF prediction, those with low stromal CAF levels exhibited better overall survival (Log-rank test, p&amp;lt;.05). Conclusions. We present a deep learning model to analyze TME in HCC solely on H&amp;E images trained by ST data. Our findings showed a potential relationship between CAFs in tumor-adjacent regions and non-responders to nivolumab treatment in HCC. These insights underscore the potential to predict nivolumab treatment responders using H&amp;E images combined with the deep learning model. This approach could provide a significant advancement in personalized treatment strategies for HCC patients. Citation Format: Dongjoo Lee, Haenara Shin, Seungho Cook, Daeseung Lee, Hongyoon Choi, Won-Mook Choi, Changhoon Yoo, Kwon Joong Na. Unveiling the tumor microenvironment of hepatocellular carcinoma using AI trained by spatial transcriptomics: A preliminary study to predict response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7398.