Investigating Immunoregulatory Mechanisms And Targeted Interventions In The Tumor Microenvironment Of Alpha Fetoprotein-positive Hepatocellular Carcinoma At Single-cell Resolution

Background and ObjectiveHepatocellular carcinoma（HCC）ranks among the top five malignancies worldwide in terms of incidence and mortality rates.It is particularly prevalent in China,accounting for about half of the global cases.Domestic data indicate that the five-year survival rate for Chinese patients with HCC is only 12%,posing a severe threat to public health.Alpha-fetoprotein（AFP）,a tumor marker,is widely used in diagnosing HCC.Early diagnosis of AFP-positive HCC（AFP≥20 ng/m L）can be achieved through elevated AFP levels combined with typical radiological features.Studies from domestic and international sources have shown that AFP-positive HCC exhibits more malignant biological behavior than AFP-negative HCC（AFP<20 ng/m L）,as evidenced by greater invasiveness,lower differentiation,later stage at diagnosis,and poorer overall survival.Despite the relatively lower malignancy of AFP-negative HCC,about 30%of HCC patients still face a poor prognosis due to the lack of early diagnostic markers.Research on serum AFP has primarily focused on its performance in predicting survival outcomes,evaluating treatment responses,and combining diagnosis with other biomarkers.However,detailed cytological and molecular analyses based on preoperative AFP levels are still limited to distinguish between AFP-negative and AFP-positive HCC.Literature indicates differences between AFP-negative and positive HCCs,including CTNNB1 gene mutations,enrichment in metabolic and chemical carcinogenesis pathways,and activation of immune and cell adhesion pathways.This suggests potential underlying patterns distinct to the two HCC phenotypes,yet precise treatment strategies for these two types of HCC have not been reported.The occurrence and development of tumors are closely related to the tumor microenvironment（TME）,which includes various tumor-infiltrating immune cells,stromal cells,etc.These components interact with tumor cells to jointly regulate tumor growth and metastasis.In recent years,tumor immunotherapy targeting the TME has become a new research focus.However,exploration of phenotypic mechanisms is limited by traditional sequencing methods,whose results only represent the"overall characterization"of tissues and may miss low-abundance information,leading to an insufficient understanding of the cellular composition and molecular features of the TME.Therefore,single-cell RNA sequencing（sc RNA-seq）technology has been introduced into the field of liver cancer research.This technology can reveal functional characteristics at the single-cell level,allowing for a more comprehensive analysis of tumor heterogeneity.The application of sc RNA-seq technology has opened new perspectives,enabling a deeper understanding of the states and interactions of different cells within the liver cancer TME,such as a key subgroup focused on in this study—tumor-associated macrophage（TAM）expressing osteopontin（encoded by SPP1）,hereinafter referred to as SPP1⁺TAM.This study aims to deeply explore the cellular composition and molecular features of the liver cancer TME by integrating sc RNA-seq with whole-genome and bulk transcriptomics,combined with traditional biological experimental methods,thereby providing new strategies for the precision treatment of liver cancer.Specific research objectives include:（1）Using sc RNA-seq technology to map the comprehensive cellular atlas of AFP-negative and positive HCC;（2）Exploring the cellular composition and biological characteristics of two different phenotypic HCCs;（3）Validating the regulatory function of the SPP1⁺TAM subpopulation in the immunosuppressive environment;（4）Elucidating the interaction mechanisms between SPP1⁺TAM and key subpopulations in the TME and exploring combined immunotherapy.Methods1.Public Database and Clinical Cohort Review:Patients with HCC were stratified based on serum AFP levels to evaluate pathological characteristics and clinical outcomes across subgroups.2.Whole-genome and sc RNA-seq Analysis:Tumor and adjacent normal tissue were collected from HCC patients for comprehensive genomic and cellular ecology analysis within the TME of different HCC phenotypes.3.Malignant Cell Identification and Biological Characteristics Analysis:Utilizing the infer CNV algorithm,malignant cells were identified,and their features were analyzed through differential gene expression,pathway enrichment analysis,and validation with multiplex immunohistochemistry（m IHC）.4.Analysis of T and NK Cells:Subdivision and comparative analysis of T/NK cells were conducted,employing pseudo-time analysis based on Monocle2,differential gene comparison,and T cell clonal expansion analysis to uncover differentiation pathways and characteristics.5.Large-sample Validation of sc RNA-seq Data:Cytometry by time of flight（Cy TOF）and m IHC were used for large-scale clinical validation of T cell immune characteristics identified through sc RNA-seq.6.Study on the Impact of AFP knockdown:The effects of Afp on tumor growth were investigated using Afp knockdown cell line,CCK8 proliferation assays,and subcutaneous xenograft models.7.Role of AFP in Regulating the Immune Microenvironment:Using subcutaneous tumor models,high-pressure tail vein plasmid injection models,and DEN combined with CCl₄models alongside Afp knockdown cell line or adenovirus overexpression of Afp to investigate its regulatory effects on the immune microenvironment.8.Analysis and Validation of Myeloid and Stromal Cell Subgroups:Comparative assessment of subpopulation abundance through cellular dimensionality reduction annotation,elucidating the distribution tendencies among subgroups,and leveraging m IHC technology to confirm the presence of SPP1⁺TAM and specific endothelial cell subpopulations in HCC,followed by clinical sample expansion validation.9.Functional Study of SPP1⁺TAM:Investigating the phagocytic capacity,lymphocyte recruitment ability,and angiogenesis-promoting ability of SPP1⁺TAM through gene set scoring,coupled with experimental validation using Escherichia coli phagocytosis assays and co-culture experiments with HUVECs.10.Intercellular Communication Analysis:Using tools like Cellphone DB and Niche Net to explore interactions and activation signals between SPP1⁺TAM and other subgroups within the TME.11.SPP1⁺TAM and CD44 Interactions Study:Validating the interactions of SPP1⁺TAM with tumor cells and T cells through CD44 using co-culture and co-inoculation models,flow cytometry,and m IHC staining.12.Therapeutic Strategies Targeting the SPP1-CD44 Axis:Exploring the targeted therapy against the SPP1-CD44 axis using monoclonal antibodies against SPP1 or CD44 alone or in combination with PD-1 inhibitors.13.Verification of Stromal-derived SPP1⁺TAM Activation Signals:Investigating the effects of IL-6 and TGF-β1 and their inhibitors on the activation of SPP1⁺TAM.14.Detection of IL-6 and TGF-β1 levels in clinical samples:Using enzyme-linked immunosorbent assay（ELISA）to measure IL-6 and TGF-β1 expression levels in clinical HCC samples and compare between subgroups.ResultsPart I1.AFP-positive HCC patients exhibit worse clinical outcomes than AFP-negative patients,characterized by more aggressive pathological features,lower differentiation,higher clinical staging,and a shorter median survival period.2.Whole-genome sequencing analysis revealed high-frequency mutations in genes such as TP53 and TTN in both types of HCC.Still,these mutations could not definitively differentiate AFP-negative from AFP-positive HCC.3.Data from sc RNA-seq showed a higher proportion of cells in the G2/M phase in AFP-positive HCC,indicating a more active proliferation state.Part II4.In AFP-positive HCC,liver cancer cells upregulated antigen processing and presentation functions,with HLA-DRA molecules being highly expressed in clinical samples,whereas AFP-negative HCC liver cancer cells upregulated genes predominantly enriched in metabolic-related pathways.5.AFP-positive HCC exhibited an increase in exhausted,inhibitory T cell subgroups and elevated expression levels of immune checkpoint-related genes.The differentiation of CD8⁺T cells tended more toward terminal differentiation,with clonal expansion primarily focused on CD8-Exhausted.6.Expanded validation of clinical samples was conducted using Cy TOF and m IHC technologies to corroborate the T cell immune landscape discovered via sc RNA-seq.Furthermore,at the protein level,it was confirmed that T cell subpopulations in AFP-positive HCC exhibit a state of heightened suppression and exhaustion.7.AFP modulated the TME by affecting T cell function,thereby promoting tumor growth.Part III8.The presence of a high abundance of SPP1⁺TAM and tumor-specific endothelial cells in HCC was confirmed via m IHC,with a broader distribution of SPP1⁺TAM in AFP-positive HCC.9.The phagocytic capability and lymphocyte chemotaxis of SPP1⁺TAM were attenuated,whereas their capacity to augment tumor angiogenesis was significantly amplified.10.SPP1⁺TAM engaged in intricate interactions with diverse components of the TME via the CD44 receptor,exhibiting intensified SPP1-CD44 signaling pathways in AFP-positive HCC.11.SPP1⁺TAM were capable of elevating CD44 expression on tumor cell surfaces and facilitating the transition of T cells to an exhausted phenotype,with in vivo assays further evidencing their role in promoting tumorigenic attributes.12.Intervention strategies targeting the SPP1-CD44 axis can effectively inhibit tumor growth,and the combined use with PD-1 inhibitors can minimize tumor burden.13.SPP1⁺TAM were activated by stromal-derived interleukin-6（IL-6）and transforming growth factor beta 1（TGF-β1）.14.Concentrations of IL-6 and TGF-β1 were elevated in AFP-positive HCC specimens relative to those observed in AFP-negative samples.ConclusionThis study,integrating multi-omics sequencing technologies with traditional biological methods,deeply analyzed the TME of AFP-negative and positive HCC,detailing the molecular mechanisms leading to differences in biological characteristics and clinical prognosis between them.The research revealed a highly suppressed and exhausted state of T cells in AFP-positive HCC,and key interactions between cell subgroups centered on SPP1⁺TAM.SPP1⁺TAM,through the CD44 receptor,played a role in promoting tumor stemness,inhibiting T cell proliferation and efficacy,thereby driving the development of HCC.These findings not only deepen the understanding of the complex TME of HCC but also provide a theoretical basis for the precision treatment of HCC patients based on AFP stratification and the development of new treatment strategies targeting the SPP1-CD44 axis.