Tumor stroma-immune interactions shape the immunosuppressive microenvironment and predict response to neoadjuvant...

The tumor microenvironment, particularly the tumor stroma, plays a critical role in tumor progression, immune evasion, and therapeutic resistance. However, its interaction with the immune landscape in rectal cancer (RC) remains incompletely understood. This study aimed to comprehensively characterize the stromal-immune ecosystem associated with the tumor stroma ratio (TSR) in RC and to evaluate its clinical and therapeutic relevance.

We analyzed a multicenter cohort of 498 patients with treatment-naïve RC in whom TSR was assessed on H&E-stained sections. Integrative multi-omics analyses were performed, including bulk RNA sequencing (n=118) and single-cell RNA/T-cell receptor (TCR) sequencing (n=10). Key findings were validated by immunohistochemistry (n=114) and multiplex immunofluorescence (n=20). Survival analyses and statistical comparisons were conducted to evaluate clinical associations and treatment responses.

High TSR was an independent predictor of unfavorable disease-free survival and cancer-specific survival and was associated with aggressive clinicopathological features. Single-cell analyses revealed that TSR-high tumors exhibited a profoundly immunosuppressive microenvironment, characterized by clonally expanded terminally exhausted CD8⁺ T cells (CD8⁺ Tex-CXCL13) and activated CD4⁺ regulatory T cells (CD4⁺ Treg-TNFRSF4). Two LRRC15⁺ cancer-associated fibroblast (CAF) subsets (mCAF-CTHRC1 and mCAF-FAP) were enriched in TSR-high tumors. Among them, mCAF-CTHRC1 was associated with increased Treg abundance and activation features, with predicted interactions with CD4⁺ Treg-TNFRSF4 cells through the LGALS9-CD44 signaling axis. In addition, SPP1-expressing monocytes (Mon-SPP1) and malignant epithelial cells were prominent in TSR-high tumors and showed a predicted SPP1-CD44 interaction with T-cell subsets, suggesting potential involvement in immunosuppressive stromal-immune interactions. In patients receiving neoadjuvant therapy, pretreatment TSR-low tumors showed improved pathological response and survival outcomes compared with TSR-high tumors. In the neoadjuvant chemoradiotherapy plus immunotherapy cohort, TSR-low tumors were associated with a significantly higher major pathological response rate, whereas pathological complete response showed a non-significant trend in the same direction.

High TSR identifies a clinically aggressive subtype of RC characterized by a profoundly immunosuppressive stromal-immune ecosystem enriched for exhausted T cells, immunosuppressive CAF programs, and SPP1-associated stromal-myeloid interactions. These findings highlight LGALS9-, LRRC15-, and SPP1-related stromal-immune pathways as candidate stromal-immune therapeutic vulnerabilities that warrant further mechanistic and preclinical validation.