Distinct Epigenetic Modifications of Regulatory T Cells in High-Frequency versus Rare Metastatic Sites of Hepatocellular Carcinoma

Distinct Epigenetic Modifications of Regulatory T Cells in High-Frequency versus Rare Metastatic Sites of Hepatocellular Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Distinct Epigenetic Modifications of Regulatory T Cells in High-Frequency versus Rare Metastatic Sites of Hepatocellular Carcinoma Jing Guo, Hua Li, Minghao Li, Mingjian Yang, Hao Wei, Zhendong Guo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9566783/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 4 You are reading this latest preprint version Abstract Metastatic hepatocellular carcinoma (HCC) exhibits pronounced heterogeneity across anatomical sites, posing significant challenges for effective treatment. In this study, we systematically characterized the immune and epigenetic landscapes of primary HCC tumors and their metastatic lesions, including common metastatic sites—bone, lung, and adrenal gland—and rarer intestinal metastases. Using single-cell RNA sequencing and multiplex immunofluorescence, we identified a marked enrichment of regulatory T cells (Tregs) in frequent metastatic sites, accompanied by downregulation of critical epigenetic activation pathways. In contrast, intestinal metastases exhibited significantly lower Treg infiltration and pronounced upregulation of the epigenetic regulators BRD4 and KMT2A, indicative of a distinct transcriptional activation state. These findings reveal substantial heterogeneity in both immune composition and site-specific epigenetic regulation, suggesting that Treg-mediated immunosuppression and differential epigenetic activity jointly shape the metastatic microenvironment. Our results provide novel insights into the molecular mechanisms underlying metastatic tropism and highlight the potential for developing tailored therapeutic strategies targeting immune and epigenetic modulators in advanced HCC. Biological sciences/Cancer Biological sciences/Computational biology and bioinformatics Health sciences/Oncology Hepatocellular carcinoma Metastasis Immune microenvironment Regulatory T cells (Tregs) Epigenetic activation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Metastatic progression remains a major clinical challenge in hepatocellular carcinoma (HCC), one of the most prevalent and lethal malignancies worldwide [1,2]. Despite advances in early detection and therapeutic interventions, distant metastases continue to represent the primary cause of mortality among HCC patients [3,4]. Notably, metastatic lesions exhibit substantial heterogeneity in their anatomical distribution, biological behavior, and response to treatment [5]. Frequent metastatic sites—including bone, lung, and adrenal gland—are commonly observed and associated with poor prognosis [6,7], whereas rare sites such as the intestine display distinct clinical features and outcomes [8]. A deeper understanding of the molecular and cellular mechanisms underlying this heterogeneity is essential for improving patient management and developing effective therapeutic strategies. The tumor immune microenvironment (TME) plays a pivotal role in regulating cancer progression and metastatic dissemination [9]. Among diverse immune cell populations, regulatory T cells (Tregs) are established mediators of immune suppression, promoting tumor immune evasion and facilitating metastatic outgrowth [10]. Accumulating evidence indicates that the abundance and functional states of Tregs vary across metastatic sites, potentially shaping site-specific immune landscapes and influencing metastatic tropism [11,12]. However, the precise mechanisms driving immune microenvironment remodeling during HCC metastasis—particularly in uncommon locations such as intestinal lesions—remain poorly characterized. Epigenetic regulation has emerged as a critical determinant of cancer cell plasticity, immune modulation, and metastatic adaptation [13]. Transcriptional regulators such as BRD4 and KMT2A have been implicated in controlling gene expression programs that govern tumor progression and immune interactions [14,15]. The interplay between epigenetic modifications and immune cell function within the TME may contribute to the distinct molecular profiles observed across different metastatic lesions [16]. Nevertheless, comprehensive characterization of epigenetic states in conjunction with immune profiling across various metastatic sites in HCC remains limited. Given these knowledge gaps, this study aims to systematically investigate immune and epigenetic heterogeneity in primary HCC tumors and their metastatic counterparts. By integrating single-cell RNA sequencing with multiplex immunofluorescence imaging, we compare cellular composition, Treg dynamics, and epigenetic activation status among primary tumors, frequent metastatic sites (bone, lung, adrenal gland), and rare intestinal metastases. Our findings provide novel insights into the molecular basis of metastatic heterogeneity and may guide the development of site-specific therapeutic approaches for advanced liver cancer. 2. Materials and Methods 2.1 Sample Collection and Clinical Information Tumor tissue samples and corresponding metastatic lesions were obtained from patients diagnosed with liver cancer at designated medical centers. Clinical data, including patient demographics, tumor histology, and metastatic site information, were systematically recorded. All procedures were conducted in accordance with institutional ethical guidelines, and informed consent was obtained from all participants. 2.2 Acquisition and Secondary Analysis of Single-cell RNA Sequencing Data Previously published single-cell RNA sequencing datasets for primary liver cancer and metastatic lesions—including those in the lung, bone, adrenal gland, and intestine—were retrieved from public repositories. Raw data were processed and subjected to quality control using standardized bioinformatics pipelines. Only high-quality cells and genes were retained for downstream analyses. 2.3 Cell Subtype Identification and Clustering Analysis Cell populations were identified based on canonical marker gene expression profiles. Clustering analysis was performed using Seurat or comparable software packages to distinguish major cell types, such as T cells, B cells, macrophages, fibroblasts, and other stromal components. 2.4 Uniform Manifold Approximation and Projection (UMAP) Dimensionality Reduction and Visualization UMAP was applied for dimensionality reduction and visualization of single-cell transcriptomic data. UMAP plots were generated to illustrate cellular distribution and transcriptional heterogeneity across different lesion types. 2.5 Immune Cell Subpopulation Proportion Analysis The proportion of immune cell subpopulations, particularly regulatory T cells, was quantified in both primary and metastatic lesions. Statistical comparisons were conducted to evaluate differences in cell abundance across distinct tumor microenvironments. 2.6 Multiplex Immunofluorescence Staining and Imaging Multiplex immunofluorescence staining was performed on formalin-fixed, paraffin-embedded tissue sections to detect FOXP3 and additional immune markers. Fluorescence images were acquired via confocal microscopy, and cellular localization was analyzed using dedicated image processing software. 2.6 RNA SCOPE In Situ Hybridization Assay RNAscope in situ hybridization was employed to detect expression of epigenetic regulators, including KMT2A and BRD4, in tissue sections. Hybridization signals were visualized and quantitatively assessed within FOXP3-positive cells to determine spatial gene expression patterns. 2.7 Quantification of Gene Expression and Signal Intensity Analysis Gene expression levels derived from single-cell and spatial assays were quantified using normalized counts or fluorescence intensity measurements. Comparative analyses were conducted across lesion sites and cell populations to identify differential expression. 2.8 Gene Set Enrichment Analysis (GSEA) GSEA was performed to investigate the functional state of regulatory T cells. Enrichment scores were calculated for key biological axes, including immune suppression, tissue residency, metabolic reprogramming, and epigenetic regulation. 2.9 Pathway and Functional Axis Analysis Functional axis analysis was conducted to assess activation of critical signaling pathways in regulatory T cells across metastatic sites. Results were visualized using heatmaps and box plots to highlight inter-site variations. 2.10 Statistical Analysis All statistical analyses were carried out using R or GraphPad Prism. Group differences were evaluated using Student’s t-test, ANOVA, or non-parametric tests as appropriate. P-values below the conventional significance threshold were considered statistically significant. 2.11 Ethics Statement and Data Management All experimental procedures involving human tissues were approved by the institutional review board. Data management practices adhered to relevant privacy and security regulations. All datasets used in this study are available upon reasonable request. 3. Results 3.1 Single-cell transcriptomic profiling reveals distinct immune landscapes between primary and high-frequency metastatic lesions To comprehensively characterize the cellular composition of the tumor microenvironment, single-cell RNA sequencing was performed on primary liver cancer lesions and high-frequency metastatic sites—including the adrenal gland, bone, and lung. UMAP visualization revealed clear clustering of major cell populations such as T cells, B cells, macrophages, and fibroblasts across different tissue origins (Figure 1A–C). Quantitative analysis demonstrated a significantly higher proportion of T cells in high-frequency metastatic lesions compared to primary tumors (Figure 1B, 1C). Further characterization of T cell subsets showed marked enrichment of regulatory T cells (Tregs) in metastatic sites relative to primary lesions (Figure 1D, 1E). These findings indicate profound remodeling of the immune microenvironment during metastatic progression, with Treg expansion emerging as a hallmark feature in high-frequency metastatic lesions. 3.2 Overlapping Differential Gene Analysis and Functional Enrichment between Bone, Lung, Adrenal Metastases and Primary Tumor To investigate molecular similarities among different metastatic sites and the primary tumor, overlapping differentially expressed genes (DEGs) were analyzed in comparisons of bone (MAG), lung (ML), adrenal gland (MB) metastases versus the primary tumor (HCC). A Venn diagram (Figure 2A) shows 795, 438, and 217 unique DEGs in MAG vs HCC, ML vs HCC, and MB vs HCC, respectively, with 1,220 DEGs shared across all three metastatic sites, indicating substantial molecular overlap. Additional shared DEGs between each pair of sites further support biological consistency among metastases. Gene Ontology (GO) enrichment analysis of these overlapping DEGs revealed significant enrichment in immune-related processes, particularly “T cell differentiation,” “lymphocyte differentiation,” and “positive regulation of T cell activation” (Figure 2B). Cellular component and molecular function categories highlighted associations with ATPase complexes, SWI/SNF superfamily complexes, and transcription coactivator activity. The prominence of T cell-related GO terms suggests enhanced T cell functionality in metastatic lesions, consistent with the observed increase in T cell and Treg proportions. Additionally, enrichment in ubiquitination and chromatin remodeling pathways indicates altered epigenetic regulation in metastatic contexts. KEGG pathway analysis confirmed enrichment in immune signaling pathways, including “PD-L1/PD-1 checkpoint,” “T cell receptor signaling,” and “Th17 cell differentiation” (Figure 2C), as well as chromatin remodeling and hepatocellular carcinoma-associated pathways. Collectively, these results highlight immune regulation—particularly T cell activity—and epigenetic modification as key features of metastatic lesions, offering mechanistic insights into tumor dissemination. 3.3 Integrated functional module enrichment analysis of Treg cells highlights epigenetic suppression in metastatic sites To comprehensively characterize the functional landscape of Treg cells in metastatic lesions, DEGs were first identified by comparing bone, lung, and adrenal gland metastases individually with primary HCC. Subsequently, common DEGs across all three metastatic sites were determined through intersection analysis. These shared DEGs were subjected to GSEA across four functional modules: tissue residency, immune suppression, metabolic reprogramming, and epigenetic modification. GSEA results revealed consistent and pronounced downregulation of epigenetic modification pathways in Treg cells from all high-frequency metastatic lesions, both individually (Figure A-C) and in combined analysis (Figure A-C). In contrast, no significant changes were observed in the other three modules—tissue residency, immune suppression, and metabolic reprogramming—between metastatic and primary lesions (data not shown). 3.4 Reduced Treg cell infiltration in rare intestinal metastases compared to high-frequency metastatic sites To further characterize the immune microenvironment of rare metastatic sites, multiplex immunofluorescence (mIF) staining was conducted on liver cancer intestinal metastases using markers for FOXP3, CD4, α-SMA, FAP, TGF-β, and Pan-CK (Figure 4A). Quantitative image analysis enabled precise identification and enumeration of total CD4⁺ T cells, FOXP3⁺ cells, and double-positive CD4⁺FOXP3⁺ regulatory T cells (Tregs) within the metastatic lesions (Figure 4B). The densities were 1.15% and 0.78% per mm² for CD4⁺ and FOXP3⁺ cells, respectively, with Tregs (CD4⁺FOXP3⁺) comprising only 0.30% per mm² of the analyzed area. To contextualize these findings, the Treg proportion in intestinal metastases (determined by mIF) was compared to that in high-frequency metastatic sites (bone, lung, adrenal gland) based on single-cell sequencing data (Figure 4C). The analysis revealed that Treg infiltration in rare intestinal metastases was significantly lower than in high-frequency metastatic lesions. This suggests a distinct immune landscape in rare metastatic sites characterized by reduced Treg presence, which may contribute to their unique biological behavior. 3.5 Epigenetic Activation Markers BRD4 and KMT2A Are Upregulated in Rare Intestinal Metastases To further investigate the molecular characteristics of rare metastatic sites, RNA-SCOPE multiplex immunofluorescence staining was performed for the epigenetic transcriptional activation markers BRD4 and KMT2A (Figure 5). Expression levels of both BRD4 and KMT2A were markedly elevated in intestinal metastases compared to bone metastases—a representative high-frequency metastatic site. Merged images show increased nuclear localization and higher abundance of BRD4 and KMT2A in intestinal lesions, whereas expression in bone metastases was substantially lower. Together with prior immune profiling (Figure 4), these findings indicate that intestinal metastases are characterized by reduced Treg infiltration and enhanced epigenetic activation. Upregulation of BRD4 and KMT2A may underlie the unique transcriptional and immunological features of rare metastatic lesions, potentially influencing their biological behavior and therapeutic response. 4. Discussion In this study, single-cell transcriptomic analysis and multiplex immunofluorescence imaging were integrated to systematically characterize the immune and epigenetic landscapes of primary HCC and its metastatic lesions. The results revealed substantial heterogeneity in both immune cell composition and epigenetic regulation across different metastatic sites. Notably, high-frequency metastatic sites such as bone, lung, and adrenal gland exhibited significant enrichment of regulatory T cells (Tregs) and downregulation of epigenetic activation pathways. In contrast, rare intestinal metastases were characterized by reduced Treg infiltration and marked upregulation of the epigenetic activation markers BRD4 and KMT2A. Role of Treg cells in metastatic progression The expansion of Treg cells in high-frequency metastatic lesions highlights their pivotal role in promoting metastatic progression through immune suppression. Tregs are well established for their ability to suppress anti-tumor immune responses, thereby facilitating tumor cell survival and dissemination [17,18]. These findings align with prior reports linking increased Treg abundance to immune evasion and poor prognosis in multiple cancers [18,19]. The pronounced enrichment of Tregs in bone, lung, and adrenal gland metastases suggests that these tissues provide a permissive microenvironment for tumor growth, potentially mediated by enhanced immunosuppressive signaling [20,21]. Conversely, the markedly lower proportion of Tregs in intestinal metastases indicates a divergent immune landscape that may restrict tumor cell colonization or outgrowth at this uncommon site. Epigenetic regulation and site-specific differences Integrated analysis demonstrated consistent downregulation of epigenetic modification pathways in Tregs from high-frequency metastatic lesions, supported by both transcriptomic data and gene set enrichment analyses. This suppression of epigenetic activity may contribute to the stabilization of Treg identity and function within immunosuppressive niches, further enabling metastatic progression [22]. In contrast, rare intestinal metastases displayed strong upregulation of epigenetic activation markers BRD4 and KMT2A, indicative of a more transcriptionally active state [22,23]. The elevated expression and nuclear localization of these markers suggest that heightened epigenetic activation plays a critical role in shaping the unique molecular and immunological profile of intestinal metastases. These observations underscore the importance of site-specific epigenetic regulation in modulating the tumor microenvironment and influencing metastatic behavior [20,23]. Unique features and implications of rare intestinal metastases The combination of reduced Treg infiltration and increased epigenetic activation in intestinal metastases reflects a distinct biological state that may account for the rarity and atypical clinical behavior of these lesions [23]. A less immunosuppressive microenvironment could constrain metastatic establishment, while enhanced epigenetic activation might drive alternative transcriptional programs affecting tumor cell survival or therapeutic response [22]. These site-specific disparities emphasize the complexity of metastatic evolution and suggest that rare metastatic lesions may require individualized therapeutic strategies differing from those effective at common metastatic sites [19,24]. Clinical implications and future directions These findings have important implications for developing precision medicine approaches targeting metastatic HCC. The differential distribution of Tregs and epigenetic activation markers across metastatic sites indicates that immune and epigenetic modulators could be applied in a site-directed manner to enhance treatment efficacy [19,24,25]. For instance, targeting Treg-mediated immunosuppression may be particularly advantageous in managing high-frequency metastatic lesions, whereas inhibitors of epigenetic activation could be explored for rare sites such as intestinal metastases [22]. Future studies should validate these observations in larger patient cohorts and investigate the mechanistic links between immune regulation, epigenetic dynamics, and metastatic tropism [20,21,26]. Limitations This study is limited by the relatively small sample size and the absence of functional validation for key molecular findings. Further investigation using in vivo models and mechanistic experiments will be required to fully elucidate the roles of Treg cells and epigenetic regulators in metastatic progression and site-specific adaptation [26]. Conclusion In summary, this study elucidates the immune and epigenetic heterogeneity of metastatic HCC, uncovering distinct regulatory mechanisms that govern metastasis at high-frequency versus rare sites. These findings deepen the mechanistic understanding of metastatic biology and may guide the development of novel, site-specific therapeutic strategies for advanced liver cancer[24,25]. Declarations Competing interests The authors declare no competing interests. Informed Consent Statement This study was conducted in accordance with the Declaration of Helsinki.Informed consent was obtained from all patients or their legal guardians prior to sample collection and participation in the study. All clinical data were de-identified to protect patient privacy, and all procedures adhered to relevant ethical and regulatory requirements for human research. Authorship Contribution Statement Jing Guo and Hua Li are co-first authors and contributed equally to this work. They were responsible for writing the original draft and editing the manuscript. Minghao Li,Mingjian Yang and Hao Wei undertook clinical data curation, including data collection, collation, organization, and verification. Zhendong Guo performed bioinformatics data analysis. Jianbin Zhuang and Changliang Wu contributed to the conceptualization and study design, and also provided supervision. All authors have read and approved the final manuscript, and agree to be accountable for all aspects of the work ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Data Availability Statement All single-cell RNA sequencing datasets analyzed in this study are deposited in official national public repositories. Access to these sequencing datasets requires independent official application and approval from the repository authority, and the corresponding authors are not authorized to redistribute the raw sequencing data directly. All other relevant original experimental data, including multiplex immunofluorescence imaging data, RNAscope in situ hybridization results, and associated clinical metadata generated in this work, are available from the corresponding authors (Jianbin Zhuang and Changliang Wu) upon reasonable request. Funding This work was supported by 1.Development and promotion of appropriate technology of traditional Chinese medicine in Guangxi Zhuang Autonomous Region，Grant No.：GZSY2025049 2.Guangxi Zhuang Autonomous Region Health Commission self-funded research projects，Grant No.:Z-A20230445 3.Guangxi International Zhuang Medical Hospital, Hospital-level Project, Grant No.：2023GZYJKT001 4.Guangxi Key Laboratory of Medical Genetics and Genomics Research, Open project, Grant No.：GXKMGG202202 References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians 71:209-249. https://doi.org/10.3322/caac.21660 Villanueva A (2019) Hepatocellular carcinoma. New England Journal of Medicine 380:1450-1462. https://doi.org/10.1056/NEJMra1713263 Forner A, Reig M, Bruix J (2018) Hepatocellular carcinoma. Lancet 391:1301-1314. https://doi.org/10.1016/S0140-6736(18)30010-2 Llovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roessler S, Schwabe RF, Heikenwalder M, El-Serag HB, Peck-Radosavljevic M, Zhu AX, Gores GJ, Finn RS (2021) Hepatocellular carcinoma. Nature Reviews Disease Primers 7:6. https://doi.org/10.1038/s41572-020-00240-3 Budhu A, Forgues M, Ye QH, Jia HL, He P, Zanetti KA, Kammula US, Chen Y, Qin LX, Tang ZY, Wang XW (2006) Prediction of venous metastasis, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell 10:99-111. https://doi.org/10.1016/j.ccr.2006.06.016 Katyal S, Oliver JH III, Peterson MS, Ferris JV, Carr BS, Baron RL (2000) Extrahepatic metastases of hepatocellular carcinoma. Radiology 216:698-703. https://doi.org/10.1148/radiology.216.3.r00se24698 Natsuizaka M, Omura T, Akaike T, Kuwata Y, Yamazaki K, Sato T, Kanda T, Arai M, Ishikawa K, Fujita N, Takei Y, Saito H, Ohto M (2005) Clinical features of hepatocellular carcinoma with extrahepatic metastases. World Journal of Gastroenterology 11:4685-4689. https://doi.org/10.3748/wjg.v11.i31.4685 Imianowski CJ, Chen Q, Workman CJ, Vignali DAA (2025) Regulatory T cells in the tumour microenvironment. Nature Reviews Cancer 25:703–722. https://doi.org/10.1038/s41568-025-00000-0 Zheng X, Fang Z, Liu X, Deng S, Zhou P, Wang X, Zhang C, Li Z, Xu X, Liu M, Li J, Wang F, Wang Y, Chen J (2020) Increased regulatory T cells are associated with immune suppression and poor prognosis in hepatocellular carcinoma. Cell and Molecular Immunology 17:807-820. https://doi.org/10.1038/s41423-019-0324-z Togashi Y, Shitara K, Nishikawa H (2019) Regulatory T cells in cancer immunosuppression—implications for anticancer therapy. Nature Reviews Clinical Oncology 16:356-371. https://doi.org/10.1038/s41571-019-0175-7 Plitas G, Konopacki C, Wu K, Bos PD, Morrow M, Putintseva EV, Chudakov DM, Rudensky AY (2016) Regulatory T cells exhibit distinct features in human breast cancer. Cell 165:1123-1135. https://doi.org/10.1016/j.cell.2016.04.056 Saito T, Nishikawa H, Yamashita T, Nagano H, Sugiyama M, Nakagawa K, Maeda Y, Okudaira H, Honda M, Kaneko S (2021) Treg heterogeneity and function in cancer: a review. Immunity 54:1047-1063. https://doi.org/10.1016/j.immuni.2021.04.014 Flavahan WA, Gaskell E, Bernstein BE (2017) Epigenetic plasticity and the hallmarks of cancer. Science 357:eaal2380. https://doi.org/10.1126/science.aal2380 Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T, Hickman TT, Felletar I, Philpott M, Munro S, McKeown MR, Wang Y, Christie AL, West N, Cameron MJ, Schwartz B, Heightman TD, La Thangue NB, French CA, Wiest O, Bradner JE, Knapp S (2010) Selective inhibition of BET bromodomains. Nature 468:1067-1073. https://doi.org/10.1038/nature09504 Li B, Carey M, Workman JL (2007) The Role of Chromatin during Transcription. Cell 128:707-719. https://doi.org/10.1016/j.cell.2007.01.015 Dawson MA, Kouzarides T (2012) Cancer epigenetics: from mechanism to therapy. Cell 150:12-27. https://doi.org/10.1016/j.cell.2012.06.013 Wolf D, Sopper S, Pircher A, Gastl G, Wolf AM (2015) Treg(s) in Cancer: Friends or Foe? J Cell Physiol 230:2598-2605. https://doi.org/10.1002/jcp.25016 Koyama S, Akbay EA, Li YY, Herter-Sprie GS, Buczkowski KA, Richards WG, Gandhi L, Red-Horse K, Cortinovis D, Sorrentino L, Wong KK, Dranoff G, Freeman GJ, Jänne PA, Hodi FS, Freeman GJ (2016) Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nature Communications 7:10501. https://doi.org/10.1038/ncomms10501 Harding JJ, El Dika I, Abou-Alfa GK (2019) Immunotherapy in hepatocellular carcinoma: current status and future directions. Hepatology 70:1652-1657. https://doi.org/10.1002/hep.30337 Zhou J, Zhou Y, Yin Y, He Y, Chen L, Liu L, Zhang G, Zhang X, Wang Y, Wang X, Zhang H, Wang Z, Li J, Wang J, Wang X (2022) Crosstalk between tumor microenvironment and cancer cells in hepatocellular carcinoma: the role of exosomes. Cancer Letters 524:36-45. https://doi.org/10.1016/j.canlet.2021.10.035 Chen DS, Mellman I (2017) Elements of cancer immunity and the cancer–immune set point. Nature 541:321-330. https://doi.org/10.1038/nature21349 Ahuja N, Sharma AR, Baylin SB (2016) Epigenetic Therapeutics: A New Weapon in the War Against Cancer. Annu Rev Med 67:73-89. https://doi.org/10.1146/annurev-med-111314-035900 Sia D, Hoshida Y, Villanueva A, Roayaie S, Ferrer J, Tabak B, Peix J, Sole M, Tovar V, Alsinet C, Cornella H, Klotzle B, Thung S, Fiel MI, Llovet JM (2013) Integrative molecular analysis of intrahepatic cholangiocarcinoma reveals two distinct subgroups and therapeutic opportunities. Gastroenterology 144:829-840. https://doi.org/10.1053/j.gastro.2012.12.039 Finn RS, Qin S, Ikeda M, Galle PR, Ducreux M, Kim TY, Kudo M, Breder V, Merle P, Kaseb AO, Li D, Verret W, Xu DZ, Hernandez S, Liu J, Huang C, Mulla S, Wang Y, Lim HY, Zhu AX (2020) Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma. New England Journal of Medicine 382:1894-1905. https://doi.org/10.1056/NEJMoa1915745 Sharma P, Allison JP (2015) The future of immune checkpoint therapy. Science 348:56-61. https://doi.org/10.1126/science.aaa8172 Gupta S, Saha S, Guha S, Saha SK, Saha S, Saha S (2021) Liver transplantation for non-hepatocellular carcinoma malignancies: current status and future prospects. World Journal of Gastroenterology 27:4497-4510. https://doi.org/10.3748/wjg.v27.i27.4497 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 06 May, 2026 Editor assigned by journal 30 Apr, 2026 Submission checks completed at journal 30 Apr, 2026 First submitted to journal 29 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9566783","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":631824217,"identity":"1332f712-3ec7-4742-a2cd-b7887d6e1735","order_by":0,"name":"Jing Guo","email":"","orcid":"","institution":"Tianjin Third Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Guo","suffix":""},{"id":631824220,"identity":"c66e7c54-016b-4f36-8971-6d56a9fb1660","order_by":1,"name":"Hua Li","email":"","orcid":"","institution":"Guangxi International Zhuang Medicine Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Li","suffix":""},{"id":631824223,"identity":"dfc044de-db87-41be-b55e-cce992f6bd16","order_by":2,"name":"Minghao Li","email":"","orcid":"","institution":"Tianjin Third Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Minghao","middleName":"","lastName":"Li","suffix":""},{"id":631824232,"identity":"86f6761d-51f8-4ab9-af7c-a688f013a3bf","order_by":3,"name":"Mingjian Yang","email":"","orcid":"","institution":"Tianjin Third Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mingjian","middleName":"","lastName":"Yang","suffix":""},{"id":631824234,"identity":"4e2f0ca0-ae33-467b-bf66-e8ecfa860f87","order_by":4,"name":"Hao Wei","email":"","orcid":"","institution":"Tianjin Third Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Wei","suffix":""},{"id":631824235,"identity":"211a66f1-0671-4a34-83fe-6195c51de604","order_by":5,"name":"Zhendong Guo","email":"","orcid":"","institution":"Chengde Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhendong","middleName":"","lastName":"Guo","suffix":""},{"id":631824236,"identity":"6a7e9405-d64f-4bf4-a56c-3f21dfdee2b4","order_by":6,"name":"Jianbin Zhuang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYFCCA1CavbHxwQdiNPDAtfAcbjacQZwWGJBIb5PmIEaLPePxx5952+zs+mc+bJBmYLCT020gaMsZA8OZbcnJM24nNhgXMCQbmx0grIUh4eO2A8kG0okNyTMYDiRuI6zl+AOQsmQDyYMNh3mI03LAsAFoi52BBGNjM3FaDpwxZpz5LzlB4kxiM+MMAyL8wj4DGGI8Z+zs+duPP//xocJOjqAWBgmIisQGMGVASDkI8EPU2hOjdhSMglEwCkYoAACSqkbfWb4SKwAAAABJRU5ErkJggg==","orcid":"","institution":"Tianjin Third Central Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jianbin","middleName":"","lastName":"Zhuang","suffix":""},{"id":631824239,"identity":"e505df9e-e78d-4284-8cd6-fb21bf845fb6","order_by":7,"name":"Changliang Wu","email":"","orcid":"","institution":"Guangxi Hospital Division of The First Affiliated Hospital, Sun Yat-sen University","correspondingAuthor":false,"prefix":"","firstName":"Changliang","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-04-29 13:53:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9566783/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9566783/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108236731,"identity":"97c3d90a-811c-4028-965a-37a2fefdc62f","added_by":"auto","created_at":"2026-04-30 19:08:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":160665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell landscape of primary and metastatic liver cancer lesions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Circos UMAP plot showing integrated clustering of single cells from four tissue types. (B, C) UMAP clustering and proportional distribution of major cell types in primary liver cancer and metastatic lesions (adrenal gland, bone, lung). (D, E) UMAP clustering and proportion of T cell subpopulations, highlighting increased Treg frequency in metastatic lesions.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9566783/v1/05eeb603132769783475291d.jpg"},{"id":108491323,"identity":"0fc75ebf-a33e-4fcd-a4ec-3de1595dff3d","added_by":"auto","created_at":"2026-05-05 09:53:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":227946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverlapping Differentially Expressed Genes and Enrichment Analysis between Metastatic Sites and Primary Tumor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Venn diagram showing the number of unique and shared differentially expressed genes (DEGs) among bone (MAG), lung (ML), adrenal gland (MB) metastases, and the primary tumor (HCC). The diagram highlights substantial overlap in DEGs across metastatic sites. (B) Gene Ontology (GO) enrichment analysis of the overlapping DEGs. The bar plot displays the top significantly enriched biological processes, with emphasis on immune-related functions such as T cell differentiation and activation. (C) KEGG pathway enrichment analysis of the overlapping DEGs. The bubble plot illustrates key signaling pathways, including immune regulation (PD-L1/PD-1 checkpoint, T cell receptor signaling) and chromatin remodeling, that are significantly enriched in metastatic lesions.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9566783/v1/8f68b48cf8714d5cf81d9b6d.jpg"},{"id":108491861,"identity":"6c1f94f1-7e12-4576-bd52-e56fc72538b8","added_by":"auto","created_at":"2026-05-05 09:56:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":159686,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEpigenetic modification module is significantly altered in metastatic lesions compared to primary tumor.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene set enrichment analysis (GSEA) results for the epigenetic modification module are shown for four comparisons: (A) MB (adrenal gland metastasis) vs. HCC, (B) ML (lung metastasis) vs. HCC, (C) MAG (bone metastasis) vs. HCC, and (D) combined metastatic lesions vs. HCC. The running enrichment score curve and ranked gene list are displayed for each comparison.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9566783/v1/ee6ca83ce78c48523254787a.jpg"},{"id":108803716,"identity":"8048cfb7-b93f-426a-83a5-9fc54c7acd95","added_by":"auto","created_at":"2026-05-08 15:04:46","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":436067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatial distribution and quantification of CD4⁺ and FOXP3⁺ T cells in metastatic lesions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Multiplex immunofluorescence image showing the spatial localization of CD4⁺ and FOXP3⁺ cells in metastatic lesions, with regions of interest outlined in red. (B) Quantitative summary of CD4⁺, FOXP3⁺, and CD4⁺/FOXP3⁺ cell numbers and densities (No./area and Ratio/area) per mm². (C) Treg cell ratio (%) in different metastatic subregions (MB, ML, MAG, MI), highlighting regional differences in Treg infiltration.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9566783/v1/2953d84b71f03f8362d19d30.jpg"},{"id":108491515,"identity":"ccd74ce9-5554-41e4-9a85-16a5c8226a5b","added_by":"auto","created_at":"2026-05-05 09:54:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":75853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-localization of BRD4 and KMT2A in metastatic subregions detected by multiplex immunofluorescence.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative images of metastatic intestine (MI) region showing merged signals (MERGE), BRD4 (red), KMT2A (green), and nuclei stained with DAPI (blue). (B) Representative images of metastatic bulk (MB) region with corresponding MERGE, BRD4, KMT2A, and DAPI channels. Scale bars are indicated in each panel. Co-localization of BRD4 and KMT2A is observed in both MI and MB regions, suggesting potential interaction in metastatic lesions.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9566783/v1/757903775842664e020bc1f5.jpg"},{"id":108808940,"identity":"15c4bd2d-c3d0-4dc1-9ad3-cf74ff193a8d","added_by":"auto","created_at":"2026-05-08 15:47:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1241336,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9566783/v1/448a736a-9901-4abf-aa75-3479b3d8f2db.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Distinct Epigenetic Modifications of Regulatory T Cells in High-Frequency versus Rare Metastatic Sites of Hepatocellular Carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMetastatic progression remains a major clinical challenge in hepatocellular carcinoma (HCC), one of the most prevalent and lethal malignancies worldwide [1,2]. Despite advances in early detection and therapeutic interventions, distant metastases continue to represent the primary cause of mortality among HCC patients [3,4]. Notably, metastatic lesions exhibit substantial heterogeneity in their anatomical distribution, biological behavior, and response to treatment [5]. Frequent metastatic sites—including bone, lung, and adrenal gland—are commonly observed and associated with poor prognosis [6,7], whereas rare sites such as the intestine display distinct clinical features and outcomes [8]. A deeper understanding of the molecular and cellular mechanisms underlying this heterogeneity is essential for improving patient management and developing effective therapeutic strategies.\u003c/p\u003e\n\u003cp\u003eThe tumor immune microenvironment (TME) plays a pivotal role in regulating cancer progression and metastatic dissemination [9]. Among diverse immune cell populations, regulatory T cells (Tregs) are established mediators of immune suppression, promoting tumor immune evasion and facilitating metastatic outgrowth [10]. Accumulating evidence indicates that the abundance and functional states of Tregs vary across metastatic sites, potentially shaping site-specific immune landscapes and influencing metastatic tropism [11,12]. However, the precise mechanisms driving immune microenvironment remodeling during HCC metastasis—particularly in uncommon locations such as intestinal lesions—remain poorly characterized.\u003c/p\u003e\n\u003cp\u003eEpigenetic regulation has emerged as a critical determinant of cancer cell plasticity, immune modulation, and metastatic adaptation [13]. Transcriptional regulators such as BRD4 and KMT2A have been implicated in controlling gene expression programs that govern tumor progression and immune interactions [14,15]. The interplay between epigenetic modifications and immune cell function within the TME may contribute to the distinct molecular profiles observed across different metastatic lesions [16]. Nevertheless, comprehensive characterization of epigenetic states in conjunction with immune profiling across various metastatic sites in HCC remains limited.\u003c/p\u003e\n\u003cp\u003eGiven these knowledge gaps, this study aims to systematically investigate immune and epigenetic heterogeneity in primary HCC tumors and their metastatic counterparts. By integrating single-cell RNA sequencing with multiplex immunofluorescence imaging, we compare cellular composition, Treg dynamics, and epigenetic activation status among primary tumors, frequent metastatic sites (bone, lung, adrenal gland), and rare intestinal metastases. Our findings provide novel insights into the molecular basis of metastatic heterogeneity and may guide the development of site-specific therapeutic approaches for advanced liver cancer.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Sample Collection and Clinical Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumor tissue samples and corresponding metastatic lesions were obtained from patients diagnosed with liver cancer at designated medical centers. Clinical data, including patient demographics, tumor histology, and metastatic site information, were systematically recorded. All procedures were conducted in accordance with institutional ethical guidelines, and informed consent was obtained from all participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Acquisition and Secondary Analysis of Single-cell RNA Sequencing Data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreviously published single-cell RNA sequencing datasets for primary liver cancer and metastatic lesions—including those in the lung, bone, adrenal gland, and intestine—were retrieved from public repositories. Raw data were processed and subjected to quality control using standardized bioinformatics pipelines. Only high-quality cells and genes were retained for downstream analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Cell Subtype Identification and Clustering Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell populations were identified based on canonical marker gene expression profiles. Clustering analysis was performed using Seurat or comparable software packages to distinguish major cell types, such as T cells, B cells, macrophages, fibroblasts, and other stromal components.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Uniform Manifold Approximation and Projection (UMAP) Dimensionality Reduction and Visualization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUMAP was applied for dimensionality reduction and visualization of single-cell transcriptomic data. UMAP plots were generated to illustrate cellular distribution and transcriptional heterogeneity across different lesion types.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Immune Cell Subpopulation Proportion Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proportion of immune cell subpopulations, particularly regulatory T cells, was quantified in both primary and metastatic lesions. Statistical comparisons were conducted to evaluate differences in cell abundance across distinct tumor microenvironments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Multiplex Immunofluorescence Staining and Imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultiplex immunofluorescence staining was performed on formalin-fixed, paraffin-embedded tissue sections to detect FOXP3 and additional immune markers. Fluorescence images were acquired via confocal microscopy, and cellular localization was analyzed using dedicated image processing software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 RNA SCOPE In Situ Hybridization Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNAscope in situ hybridization was employed to detect expression of epigenetic regulators, including KMT2A and BRD4, in tissue sections. Hybridization signals were visualized and quantitatively assessed within FOXP3-positive cells to determine spatial gene expression patterns.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Quantification of Gene Expression and Signal Intensity Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression levels derived from single-cell and spatial assays were quantified using normalized counts or fluorescence intensity measurements. Comparative analyses were conducted across lesion sites and cell populations to identify differential expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Gene Set Enrichment Analysis (GSEA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGSEA was performed to investigate the functional state of regulatory T cells. Enrichment scores were calculated for key biological axes, including immune suppression, tissue residency, metabolic reprogramming, and epigenetic regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Pathway and Functional Axis Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional axis analysis was conducted to assess activation of critical signaling pathways in regulatory T cells across metastatic sites. Results were visualized using heatmaps and box plots to highlight inter-site variations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were carried out using R or GraphPad Prism. Group differences were evaluated using Student’s t-test, ANOVA, or non-parametric tests as appropriate. P-values below the conventional significance threshold were considered statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Ethics Statement and Data Management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving human tissues were approved by the institutional review board. Data management practices adhered to relevant privacy and security regulations. All datasets used in this study are available upon reasonable request.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Single-cell transcriptomic profiling reveals distinct immune landscapes between primary and high-frequency metastatic lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo comprehensively characterize the cellular composition of the tumor microenvironment, single-cell RNA sequencing was performed on primary liver cancer lesions and high-frequency metastatic sites—including the adrenal gland, bone, and lung. UMAP visualization revealed clear clustering of major cell populations such as T cells, B cells, macrophages, and fibroblasts across different tissue origins (Figure 1A–C). Quantitative analysis demonstrated a significantly higher proportion of T cells in high-frequency metastatic lesions compared to primary tumors (Figure 1B, 1C). Further characterization of T cell subsets showed marked enrichment of regulatory T cells (Tregs) in metastatic sites relative to primary lesions (Figure 1D, 1E). These findings indicate profound remodeling of the immune microenvironment during metastatic progression, with Treg expansion emerging as a hallmark feature in high-frequency metastatic lesions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Overlapping Differential Gene Analysis and Functional Enrichment between Bone, Lung, Adrenal Metastases and Primary Tumor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate molecular similarities among different metastatic sites and the primary tumor, overlapping differentially expressed genes (DEGs) were analyzed in comparisons of bone (MAG), lung (ML), adrenal gland (MB) metastases versus the primary tumor (HCC). A Venn diagram (Figure 2A) shows 795, 438, and 217 unique DEGs in MAG vs HCC, ML vs HCC, and MB vs HCC, respectively, with 1,220 DEGs shared across all three metastatic sites, indicating substantial molecular overlap. Additional shared DEGs between each pair of sites further support biological consistency among metastases.\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO) enrichment analysis of these overlapping DEGs revealed significant enrichment in immune-related processes, particularly “T cell differentiation,” “lymphocyte differentiation,” and “positive regulation of T cell activation” (Figure 2B). Cellular component and molecular function categories highlighted associations with ATPase complexes, SWI/SNF superfamily complexes, and transcription coactivator activity. The prominence of T cell-related GO terms suggests enhanced T cell functionality in metastatic lesions, consistent with the observed increase in T cell and Treg proportions. Additionally, enrichment in ubiquitination and chromatin remodeling pathways indicates altered epigenetic regulation in metastatic contexts.\u003c/p\u003e\n\u003cp\u003eKEGG pathway analysis confirmed enrichment in immune signaling pathways, including “PD-L1/PD-1 checkpoint,” “T cell receptor signaling,” and “Th17 cell differentiation” (Figure 2C), as well as chromatin remodeling and hepatocellular carcinoma-associated pathways. Collectively, these results highlight immune regulation—particularly T cell activity—and epigenetic modification as key features of metastatic lesions, offering mechanistic insights into tumor dissemination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Integrated functional module enrichment analysis of Treg cells highlights epigenetic suppression in metastatic sites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo comprehensively characterize the functional landscape of Treg cells in metastatic lesions, DEGs were first identified by comparing bone, lung, and adrenal gland metastases individually with primary HCC. Subsequently, common DEGs across all three metastatic sites were determined through intersection analysis. These shared DEGs were subjected to GSEA across four functional modules: tissue residency, immune suppression, metabolic reprogramming, and epigenetic modification. GSEA results revealed consistent and pronounced downregulation of epigenetic modification pathways in Treg cells from all high-frequency metastatic lesions, both individually (Figure A-C) and in combined analysis (Figure A-C). In contrast, no significant changes were observed in the other three modules—tissue residency, immune suppression, and metabolic reprogramming—between metastatic and primary lesions (data not shown).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Reduced Treg cell infiltration in rare intestinal metastases compared to high-frequency metastatic sites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further characterize the immune microenvironment of rare metastatic sites, multiplex immunofluorescence (mIF) staining was conducted on liver cancer intestinal metastases using markers for FOXP3, CD4, α-SMA, FAP, TGF-β, and Pan-CK (Figure 4A). Quantitative image analysis enabled precise identification and enumeration of total CD4⁺ T cells, FOXP3⁺ cells, and double-positive CD4⁺FOXP3⁺ regulatory T cells (Tregs) within the metastatic lesions (Figure 4B). The densities were 1.15% and 0.78% per mm² for CD4⁺ and FOXP3⁺ cells, respectively, with Tregs (CD4⁺FOXP3⁺) comprising only 0.30% per mm² of the analyzed area.\u003c/p\u003e\n\u003cp\u003eTo contextualize these findings, the Treg proportion in intestinal metastases (determined by mIF) was compared to that in high-frequency metastatic sites (bone, lung, adrenal gland) based on single-cell sequencing data (Figure 4C). The analysis revealed that Treg infiltration in rare intestinal metastases was significantly lower than in high-frequency metastatic lesions. This suggests a distinct immune landscape in rare metastatic sites characterized by reduced Treg presence, which may contribute to their unique biological behavior.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Epigenetic Activation Markers BRD4 and KMT2A Are Upregulated in Rare Intestinal Metastases\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the molecular characteristics of rare metastatic sites, RNA-SCOPE multiplex immunofluorescence staining was performed for the epigenetic transcriptional activation markers BRD4 and KMT2A (Figure 5). Expression levels of both BRD4 and KMT2A were markedly elevated in intestinal metastases compared to bone metastases—a representative high-frequency metastatic site. Merged images show increased nuclear localization and higher abundance of BRD4 and KMT2A in intestinal lesions, whereas expression in bone metastases was substantially lower. Together with prior immune profiling (Figure 4), these findings indicate that intestinal metastases are characterized by reduced Treg infiltration and enhanced epigenetic activation. Upregulation of BRD4 and KMT2A may underlie the unique transcriptional and immunological features of rare metastatic lesions, potentially influencing their biological behavior and therapeutic response.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, single-cell transcriptomic analysis and multiplex immunofluorescence imaging were integrated to systematically characterize the immune and epigenetic landscapes of primary HCC and its metastatic lesions. The results revealed substantial heterogeneity in both immune cell composition and epigenetic regulation across different metastatic sites. Notably, high-frequency metastatic sites such as bone, lung, and adrenal gland exhibited significant enrichment of regulatory T cells (Tregs) and downregulation of epigenetic activation pathways. In contrast, rare intestinal metastases were characterized by reduced Treg infiltration and marked upregulation of the epigenetic activation markers BRD4 and KMT2A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRole of Treg cells in metastatic progression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expansion of Treg cells in high-frequency metastatic lesions highlights their pivotal role in promoting metastatic progression through immune suppression. Tregs are well established for their ability to suppress anti-tumor immune responses, thereby facilitating tumor cell survival and dissemination [17,18]. These findings align with prior reports linking increased Treg abundance to immune evasion and poor prognosis in multiple cancers [18,19]. The pronounced enrichment of Tregs in bone, lung, and adrenal gland metastases suggests that these tissues provide a permissive microenvironment for tumor growth, potentially mediated by enhanced immunosuppressive signaling [20,21]. Conversely, the markedly lower proportion of Tregs in intestinal metastases indicates a divergent immune landscape that may restrict tumor cell colonization or outgrowth at this uncommon site.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEpigenetic regulation and site-specific differences\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntegrated analysis demonstrated consistent downregulation of epigenetic modification pathways in Tregs from high-frequency metastatic lesions, supported by both transcriptomic data and gene set enrichment analyses. This suppression of epigenetic activity may contribute to the stabilization of Treg identity and function within immunosuppressive niches, further enabling metastatic progression [22]. In contrast, rare intestinal metastases displayed strong upregulation of epigenetic activation markers BRD4 and KMT2A, indicative of a more transcriptionally active state [22,23]. The elevated expression and nuclear localization of these markers suggest that heightened epigenetic activation plays a critical role in shaping the unique molecular and immunological profile of intestinal metastases. These observations underscore the importance of site-specific epigenetic regulation in modulating the tumor microenvironment and influencing metastatic behavior [20,23].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUnique features and implications of rare intestinal metastases\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe combination of reduced Treg infiltration and increased epigenetic activation in intestinal metastases reflects a distinct biological state that may account for the rarity and atypical clinical behavior of these lesions [23]. A less immunosuppressive microenvironment could constrain metastatic establishment, while enhanced epigenetic activation might drive alternative transcriptional programs affecting tumor cell survival or therapeutic response [22]. These site-specific disparities emphasize the complexity of metastatic evolution and suggest that rare metastatic lesions may require individualized therapeutic strategies differing from those effective at common metastatic sites [19,24].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical implications and future directions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese findings have important implications for developing precision medicine approaches targeting metastatic HCC. The differential distribution of Tregs and epigenetic activation markers across metastatic sites indicates that immune and epigenetic modulators could be applied in a site-directed manner to enhance treatment efficacy [19,24,25]. For instance, targeting Treg-mediated immunosuppression may be particularly advantageous in managing high-frequency metastatic lesions, whereas inhibitors of epigenetic activation could be explored for rare sites such as intestinal metastases [22]. Future studies should validate these observations in larger patient cohorts and investigate the mechanistic links between immune regulation, epigenetic dynamics, and metastatic tropism [20,21,26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is limited by the relatively small sample size and the absence of functional validation for key molecular findings. Further investigation using in vivo models and mechanistic experiments will be required to fully elucidate the roles of Treg cells and epigenetic regulators in metastatic progression and site-specific adaptation [26].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this study elucidates the immune and epigenetic heterogeneity of metastatic HCC, uncovering distinct regulatory mechanisms that govern metastasis at high-frequency versus rare sites. These findings deepen the mechanistic understanding of metastatic biology and may guide the development of novel, site-specific therapeutic strategies for advanced liver cancer[24,25].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki.Informed consent was obtained from all patients or their legal guardians prior to sample collection and participation in the study. All clinical data were de-identified to protect patient privacy, and all procedures adhered to relevant ethical and regulatory requirements for human research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJing Guo and Hua Li are co-first authors and contributed equally to this work. They were responsible for writing the original draft and editing the manuscript. Minghao Li,Mingjian Yang and Hao Wei undertook clinical data curation, including data collection, collation, organization, and verification. Zhendong Guo performed bioinformatics data analysis. Jianbin Zhuang and Changliang Wu contributed to the conceptualization and study design, and also provided supervision. All authors have read and approved the final manuscript, and agree to be accountable for all aspects of the work ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll single-cell RNA sequencing datasets analyzed in this study are deposited in official national public repositories. Access to these sequencing datasets requires independent official application and approval from the repository authority, and the corresponding authors are not authorized to redistribute the raw sequencing data directly. All other relevant original experimental data, including multiplex immunofluorescence imaging data, RNAscope in situ hybridization results, and associated clinical metadata generated in this work, are available from the corresponding authors (Jianbin Zhuang and Changliang Wu) upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1.Development and promotion of appropriate technology of traditional Chinese medicine in Guangxi Zhuang Autonomous Region，Grant No.：GZSY2025049\u003c/p\u003e\n\u003cp\u003e2.Guangxi Zhuang Autonomous Region Health Commission self-funded research projects，Grant No.:Z-A20230445\u003c/p\u003e\n\u003cp\u003e3.Guangxi International Zhuang Medical Hospital, Hospital-level Project, Grant No.：2023GZYJKT001\u003c/p\u003e\n\u003cp\u003e4.Guangxi Key Laboratory of Medical Genetics and Genomics Research, Open project, Grant No.：GXKMGG202202\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians 71:209-249. https://doi.org/10.3322/caac.21660\u003c/li\u003e\n \u003cli\u003eVillanueva A (2019) Hepatocellular carcinoma. New England Journal of Medicine 380:1450-1462. https://doi.org/10.1056/NEJMra1713263\u003c/li\u003e\n \u003cli\u003eForner A, Reig M, Bruix J (2018) Hepatocellular carcinoma. Lancet 391:1301-1314. https://doi.org/10.1016/S0140-6736(18)30010-2\u003c/li\u003e\n \u003cli\u003eLlovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roessler S, Schwabe RF, Heikenwalder M, El-Serag HB, Peck-Radosavljevic M, Zhu AX, Gores GJ, Finn RS (2021) Hepatocellular carcinoma. Nature Reviews Disease Primers 7:6. https://doi.org/10.1038/s41572-020-00240-3\u003c/li\u003e\n \u003cli\u003eBudhu A, Forgues M, Ye QH, Jia HL, He P, Zanetti KA, Kammula US, Chen Y, Qin LX, Tang ZY, Wang XW (2006) Prediction of venous metastasis, recurrence, and prognosis in hepatocellular carcinoma based on a unique immune response signature of the liver microenvironment. Cancer Cell 10:99-111. https://doi.org/10.1016/j.ccr.2006.06.016\u003c/li\u003e\n \u003cli\u003eKatyal S, Oliver JH III, Peterson MS, Ferris JV, Carr BS, Baron RL (2000) Extrahepatic metastases of hepatocellular carcinoma. Radiology 216:698-703. https://doi.org/10.1148/radiology.216.3.r00se24698\u003c/li\u003e\n \u003cli\u003eNatsuizaka M, Omura T, Akaike T, Kuwata Y, Yamazaki K, Sato T, Kanda T, Arai M, Ishikawa K, Fujita N, Takei Y, Saito H, Ohto M (2005) Clinical features of hepatocellular carcinoma with extrahepatic metastases. World Journal of Gastroenterology 11:4685-4689. https://doi.org/10.3748/wjg.v11.i31.4685\u003c/li\u003e\n \u003cli\u003eImianowski CJ, Chen Q, Workman CJ, Vignali DAA (2025) Regulatory T cells in the tumour microenvironment. Nature Reviews Cancer 25:703\u0026ndash;722. https://doi.org/10.1038/s41568-025-00000-0\u003c/li\u003e\n \u003cli\u003eZheng X, Fang Z, Liu X, Deng S, Zhou P, Wang X, Zhang C, Li Z, Xu X, Liu M, Li J, Wang F, Wang Y, Chen J (2020) Increased regulatory T cells are associated with immune suppression and poor prognosis in hepatocellular carcinoma. Cell and Molecular Immunology 17:807-820. https://doi.org/10.1038/s41423-019-0324-z\u003c/li\u003e\n \u003cli\u003eTogashi Y, Shitara K, Nishikawa H (2019) Regulatory T cells in cancer immunosuppression\u0026mdash;implications for anticancer therapy. Nature Reviews Clinical Oncology 16:356-371. https://doi.org/10.1038/s41571-019-0175-7\u003c/li\u003e\n \u003cli\u003ePlitas G, Konopacki C, Wu K, Bos PD, Morrow M, Putintseva EV, Chudakov DM, Rudensky AY (2016) Regulatory T cells exhibit distinct features in human breast cancer. Cell 165:1123-1135. https://doi.org/10.1016/j.cell.2016.04.056\u003c/li\u003e\n \u003cli\u003eSaito T, Nishikawa H, Yamashita T, Nagano H, Sugiyama M, Nakagawa K, Maeda Y, Okudaira H, Honda M, Kaneko S (2021) Treg heterogeneity and function in cancer: a review. Immunity 54:1047-1063. https://doi.org/10.1016/j.immuni.2021.04.014\u003c/li\u003e\n \u003cli\u003eFlavahan WA, Gaskell E, Bernstein BE (2017) Epigenetic plasticity and the hallmarks of cancer. Science 357:eaal2380. https://doi.org/10.1126/science.aal2380\u003c/li\u003e\n \u003cli\u003eFilippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T, Hickman TT, Felletar I, Philpott M, Munro S, McKeown MR, Wang Y, Christie AL, West N, Cameron MJ, Schwartz B, Heightman TD, La Thangue NB, French CA, Wiest O, Bradner JE, Knapp S (2010) Selective inhibition of BET bromodomains. Nature 468:1067-1073. https://doi.org/10.1038/nature09504\u003c/li\u003e\n \u003cli\u003eLi B, Carey M, Workman JL (2007) The Role of Chromatin during Transcription. Cell 128:707-719. https://doi.org/10.1016/j.cell.2007.01.015\u003c/li\u003e\n \u003cli\u003eDawson MA, Kouzarides T (2012) Cancer epigenetics: from mechanism to therapy. Cell 150:12-27. https://doi.org/10.1016/j.cell.2012.06.013\u003c/li\u003e\n \u003cli\u003eWolf D, Sopper S, Pircher A, Gastl G, Wolf AM (2015) Treg(s) in Cancer: Friends or Foe? J Cell Physiol 230:2598-2605. https://doi.org/10.1002/jcp.25016\u003c/li\u003e\n \u003cli\u003eKoyama S, Akbay EA, Li YY, Herter-Sprie GS, Buczkowski KA, Richards WG, Gandhi L, Red-Horse K, Cortinovis D, Sorrentino L, Wong KK, Dranoff G, Freeman GJ, J\u0026auml;nne PA, Hodi FS, Freeman GJ (2016) Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nature Communications 7:10501. https://doi.org/10.1038/ncomms10501\u003c/li\u003e\n \u003cli\u003eHarding JJ, El Dika I, Abou-Alfa GK (2019) Immunotherapy in hepatocellular carcinoma: current status and future directions. Hepatology 70:1652-1657. https://doi.org/10.1002/hep.30337\u003c/li\u003e\n \u003cli\u003eZhou J, Zhou Y, Yin Y, He Y, Chen L, Liu L, Zhang G, Zhang X, Wang Y, Wang X, Zhang H, Wang Z, Li J, Wang J, Wang X (2022) Crosstalk between tumor microenvironment and cancer cells in hepatocellular carcinoma: the role of exosomes. Cancer Letters 524:36-45. https://doi.org/10.1016/j.canlet.2021.10.035\u003c/li\u003e\n \u003cli\u003eChen DS, Mellman I (2017) Elements of cancer immunity and the cancer\u0026ndash;immune set point. Nature 541:321-330. https://doi.org/10.1038/nature21349\u003c/li\u003e\n \u003cli\u003eAhuja N, Sharma AR, Baylin SB (2016) Epigenetic Therapeutics: A New Weapon in the War Against Cancer. Annu Rev Med 67:73-89. https://doi.org/10.1146/annurev-med-111314-035900\u003c/li\u003e\n \u003cli\u003eSia D, Hoshida Y, Villanueva A, Roayaie S, Ferrer J, Tabak B, Peix J, Sole M, Tovar V, Alsinet C, Cornella H, Klotzle B, Thung S, Fiel MI, Llovet JM (2013) Integrative molecular analysis of intrahepatic cholangiocarcinoma reveals two distinct subgroups and therapeutic opportunities. Gastroenterology 144:829-840. https://doi.org/10.1053/j.gastro.2012.12.039\u003c/li\u003e\n \u003cli\u003eFinn RS, Qin S, Ikeda M, Galle PR, Ducreux M, Kim TY, Kudo M, Breder V, Merle P, Kaseb AO, Li D, Verret W, Xu DZ, Hernandez S, Liu J, Huang C, Mulla S, Wang Y, Lim HY, Zhu AX (2020) Atezolizumab plus bevacizumab in unresectable hepatocellular carcinoma. New England Journal of Medicine 382:1894-1905. https://doi.org/10.1056/NEJMoa1915745\u003c/li\u003e\n \u003cli\u003eSharma P, Allison JP (2015) The future of immune checkpoint therapy. Science 348:56-61. https://doi.org/10.1126/science.aaa8172\u003c/li\u003e\n \u003cli\u003eGupta S, Saha S, Guha S, Saha SK, Saha S, Saha S (2021) Liver transplantation for non-hepatocellular carcinoma malignancies: current status and future prospects. World Journal of Gastroenterology 27:4497-4510. https://doi.org/10.3748/wjg.v27.i27.4497\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hepatocellular carcinoma, Metastasis, Immune microenvironment, Regulatory T cells (Tregs), Epigenetic activation","lastPublishedDoi":"10.21203/rs.3.rs-9566783/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9566783/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Metastatic hepatocellular carcinoma (HCC) exhibits pronounced heterogeneity across anatomical sites, posing significant challenges for effective treatment. In this study, we systematically characterized the immune and epigenetic landscapes of primary HCC tumors and their metastatic lesions, including common metastatic sites—bone, lung, and adrenal gland—and rarer intestinal metastases. Using single-cell RNA sequencing and multiplex immunofluorescence, we identified a marked enrichment of regulatory T cells (Tregs) in frequent metastatic sites, accompanied by downregulation of critical epigenetic activation pathways. In contrast, intestinal metastases exhibited significantly lower Treg infiltration and pronounced upregulation of the epigenetic regulators BRD4 and KMT2A, indicative of a distinct transcriptional activation state. These findings reveal substantial heterogeneity in both immune composition and site-specific epigenetic regulation, suggesting that Treg-mediated immunosuppression and differential epigenetic activity jointly shape the metastatic microenvironment. Our results provide novel insights into the molecular mechanisms underlying metastatic tropism and highlight the potential for developing tailored therapeutic strategies targeting immune and epigenetic modulators in advanced HCC.","manuscriptTitle":"Distinct Epigenetic Modifications of Regulatory T Cells in High-Frequency versus Rare Metastatic Sites of Hepatocellular Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 19:08:03","doi":"10.21203/rs.3.rs-9566783/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T12:55:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T04:15:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T04:15:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-29T13:41:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4eab706c-a35c-4dc0-a84c-98f371a05370","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T12:55:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T04:15:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T04:15:18+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":67251718,"name":"Biological sciences/Cancer"},{"id":67251719,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":67251720,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-05-19T19:08:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 19:08:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9566783","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9566783","identity":"rs-9566783","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}