Tertiary lymphoid structure-associated chemokine programs define an immune-organized tumor microenvironment with prognostic relevance in hepatocellular carcinoma

Abstract

BACKGROUND

Hepatocellular carcinoma (HCC) exhibits marked heterogeneity in the tumor immune microenvironment, leading to variable outcomes and immunotherapy responses. Tertiary lymphoid structures (TLSs) are organized immune architectures associated with anti-tumor immunity, but their transcriptomic programs and biomarker potential remain unclear.

AIM

To systematically characterize TLS-associated chemokine programs in HCC by integrating bulk transcriptomic data, single-cell RNA sequencing (scRNA-seq), and histologically defined TLS status across multiple independent cohorts, and to evaluate their relationship with immune organization and clinical outcomes.

METHODS

Transcriptomic TLS signatures were derived from histologically annotated HCC samples in the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort using differential expression and gene set enrichment analyses. The 12-chemokine TLS signature was further evaluated. scRNA-seq data from GSE149614 were analyzed to localize TLS-associated chemokine and receptor expression across cellular compartments. A TLS-related prognostic model was constructed using least absolute shrinkage and selection operator Cox regression in the TCGA-LIHC cohort and externally validated in the International Cancer Genome Consortium-Liver Cancer-RIKEN-Japan (ICGC-LIRI-JP) cohort. Prognostic value was assessed using survival analysis and time-dependent C-index analysis.

RESULTS

TLS-associated chemokines were dysregulated in tumors. CCL2, CCL3, CCL4, CCL19, CCL21, and CXCL13 were downregulated, whereas CCL8, CCL18, CXCL9, CXCL10, and CXCL11 were upregulated. Higher expression of CCL2, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, and CXCL10 was associated with longer OS, and most chemokines with favorable recurrence-free survival, progression-free survival and disease-specific survival. Single-cell analysis showed that these chemokines were mainly expressed by T cells, B cells, myeloid cells, and fibroblasts, with T cells as the dominant cell type in CXCL and CCL signaling crosstalk. Histologically TLS-positive tumors had better overall survival (OS), higher 12-chemokine scores, lower tumor purity, greater immune infiltration, and increased checkpoint expression. A seven-gene model (CCR7, ICOS, SEMA3C, VTCN1, IRF4, MS4A1, ADAM28) identified high-risk patients with poorer OS in TCGA-LIHC [hazard ratio (HR) = 1.716, P = 0.005] and ICGC-LIRI-JP (HR = 2.12, P = 0.035).

CONCLUSION

TLS-associated chemokine programs are associated with an organized immune state in HCC and provide a biologically informative framework for immune-informed prognostic stratification.

Keywords: Hepatocellular carcinoma; Tertiary lymphoid structures; Tumor immune microenvironment; Prognostic signature; Transcriptomics

Core Tip: Tertiary lymphoid structures (TLSs) are organized immune aggregates and their transcriptional programs remain poorly defined in hepatocellular carcinoma (HCC). Using multiple public cohorts, we anchored a 12-chemokine TLS signature to histology-annotated TLS status, localized chemokine and receptor expression using single-cell transcriptomic profiling, and derived a seven-gene prognostic score. The score stratified overall survival in a training cohort and an independent validation cohort. This study provides a scalable, histology-anchored transcriptomic framework for immune stratification in HCC.