Integrated Spatial Multi-Omics Analysis Reveals Novel Immunoinflammatory CD8+ T Cells Associated with Tertiary Lymphoid Structure and Immunotherapy Response in Colorectal Cancer

Abstract Background: Based on the spatial distribution of immune cells, tumors are classified into three immunophenotypes: immune-inflamed, immune-excluded and immune-desert, among which the immune-inflamed phenotype exhibits favorable prognosis, high immunotherapy response rate, and abundant CD8+ T cells infiltration. However, there is no existing study elucidating the distinctive features of CD8+ T cells under the background of immune-inflamed in colorectal cancer (CRC). Methods: Bulk RNA sequencing (bulk RNA-seq), single cell RNA sequencing (scRNA-seq), spatial transcriptomics RNA sequencing (stRNA-seq) and pathomics were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Integrated spatial multi-omics, combined with immunofluorescence (IF) revealed immune-inflamed related CD8+ CXCL13+ proliferative T cells as a novel component in tertiary lymphoid structure (TLS). A prognostic model related to CD8+ CXCL13+ proliferative T cell was established, with verifications performed internally and externally. Results: Our research distinguished three immunophenotypes in CRC and revealed immune-inflamed related CD8+ CXCL13+ proliferative T cells were characterized by exhaustion features, proliferative ability and effector functions. Remarkably, CD8+ CXCL13+ proliferative T cells were predominantly located within TLS, thus likely marking TLS and contributing to TLS formation. The specifically decreased ETS1 transcriptional activity in immune-inflamed related CD8+ T cells and ETS1 mRNA peaking at terminal differentiation indicated ETS1's key role in developing exhaustion. Finally, the CD8+T related prognostic model can serve as a powerful prognostic index to optimize risk stratification and provide new insights for individualized immunotherapy treatment of CRC. Conclusion: These findings revealed that immune-inflamed related CD8+ CXCL13+ proliferative T cells, predominantly located within TLS, might serve as potential biomarkers for TLS presence, immunotherapy response, and prognosis in CRC. Besides, targeting the ETS1-terminal exhaustion axis was highlighted for enhancing the efficacy of immunotherapy for CRC. Our research provides new insights into the determinants affecting TLS and ICI responsiveness.