Comparative Analysis of Tumor and Peri-tumor Immune Microenvironments in Hepatocellular Carcinoma

목차 (Table of Contents)

• ABSTRACT 1
• CONTENTS 4
• LIST OF TABLES 8
• LIST OF FIGURES 8
• ABBREVIATIONS 12
• INTRODUCTION 13
• MATERIALS AND METHODS 21
• 1. Study Population
• 2. Human Liver tissue single cell preparation
• 3. Peripheral Blood Mononuclear Cell isolation and cryopreservation
• 4. A Patient-Derived Tumor Spheroid Platform for co-culture with patient derived CD8 T cell
• 5. Isolation of Spheroids and Co-culture with Patient-Derived CD8 T cells
• 6. Cell Stimulation and Culture for Functional Assays
• 7. Patient-derived CD45 selection & CD8 T cell isolation
• 8. Flow cytometry analysis of Immune cell
• 9. RNA Extraction
• 10. cDNA synthesis
• 11. Real-Time Quantitative PCR
• 12. Data acquisition and preprocessing
• 13. Integration, Normalization and scaling
• 14. Dimensional reduction and clustering
• 15. Cell type annotation
• 16. DEG analysis
• 17. Visualization and comparative analysis
• 18. Cell-cell communication analysis
• 19. Statistical analysis
• RESULTS 41
• 1. Study Design and Experimental Workflow
• 2. Cellular Composition of the HCC Microenvironment via scRNA-seq
• 3. Identification and Marker Expression of Myeloid Cell Subsets
• 4. Frequency of Dendritic Cells Across Tissue Compartments
• 5. Functional Status of Dendritic Cells
• 6. Accumulation of Macrophages within the Tumor Tissue
• 7. Immunosuppressive Phenotype of Intra-tumoral Macrophages
• 8. Redistribution of T cell and NK cell Subsets in the TME
• 9. Functional Gene Signatures Across T cell and NK cell Subsets
• 10. Numerical Decline of NK Cells in Tumor Tissue
• 11. Impaired Cytotoxic Function of NK Cells
• 12. Transcriptional Pathway Analysis of Tumor-Infiltrating NK Cells
• 13. Altered CD4/CD8 T cell Ratio in Tumor Tissue
• 14. Functional Impairment of Tumor-Infiltrating CD4 T cells
• 15. Functional Impairment of Tumor-Infiltrating CD8 T cells
• 16. Downregulation of T cell Effector Genes in Tumor Tissue
• 17. Altered Metabolic and Protein Synthesis Pathways in Tumor Infiltrating CD4 T cells
• 18. Suppressed Metabolic Pathways and Activated Signaling in Tumor-Infiltrating CD8 T cells
• 19. Transcriptional Analysis of Inhibitory Receptor Genes in T cells
• 20. PD-1 Protein Expression on T cells and its Correlation with Clinical Parameters
• 21. Cell-Cell Interaction Network within the Tumor Microenvironment
• 22. Ex vivo Expansion and Functional Potential of Patient-Derived CD8 T cells
• 23. Reactivation of CD8 T cells upon Co-culture with Tumor Spheroids
• 24. Identification of Tumor-Reactive T cells using Activation Markers 4-1BB and CD107a
• 25. Complete Gating Strategy for Flow cytometry Analysis
• DISCUSSION 102
• CONCLUSION 106
• REFERENCES 110