Influence of multiomics on tumor microenvironment remodeling in locally advanced rectal cancer following neoadjuvant chemoradiotherapy plus immunotherapy.

230 Background: Immune checkpoint inhibitors (ICIs) have limited efficacy in microsatellite-stable (MSS) or mismatch repair–proficient (pMMR) locally advanced rectal cancer (LARC). Combining ICIs with neoadjuvant chemoradiotherapy (nCRT) shows promise, but predictive biomarkers are lacking. The tumor microenvironment (TME) remodels during therapy, influencing immune responses. Methods: We retrospectively analyzed 30 MSS/pMMR LARC patients treated with nCRT plus PD-1 blockade. Major pathological response (MPR) was defined as ≤10% residual tumor cells. Matched pre- and post-treatment samples underwent immunohistochemistry (IHC), multiplex immunofluorescence (mIF), bulk RNA sequencing, and single-cell RNA sequencing (scRNA-seq) to characterize immune and stromal changes. Results: Before treatment, the densities of CD8⁺ T cells and CD138⁺ plasma cells in both tumor parenchyma and stroma were higher in the MPR tumors compared with the non-MPR group. After treatment, MPR tumors showed increased CD3⁺, CD8⁺ T cells, B cells, plasma cells, and NK cells in stroma, and elevated CD8⁺ T cells and plasma cells in tumor parenchyma. FOXP3⁺ regulatory T cells (Tregs) were significantly more abundant post-treatment in non-MPR tumors. Paired analysis revealed increased plasma cells and decreased Tregs post-treatment in MPR tumors, while non-MPR tumors had increased Tregs. Bulk RNA-seq showed upregulation of epithelial–mesenchymal transition (EMT) and cancer-associated fibroblast (CAF) genes in MPR tumors, while Wnt and TGF-β signaling pathways were enriched in non-MPR tumors. scRNA-seq corroborated these findings, revealing expanded plasma cell populations and reduced Treg abundance in MPR tumors. In contrast, non-MPR tumors maintained FOXP3⁺ Tregs exhibiting an activated immunosuppressive phenotype co-expressing CTLA4 and TIGIT. Importantly, FOXP3⁺ Tregs significantly decreased after treatment only in MPR tumors. Conclusions: Response to nCRT plus PD-1 blockade in MSS/pMMR LARC is characterized by cytotoxic and humoral immune activation and Treg depletion. Resistance involves sustained immunosuppressive Tregs and Wnt/TGF-β pathway activation. Targeting Tregs may improve treatment efficacy, and multiomic biomarkers can aid patient stratification.