Single-cell sequencing to reveal immune features of treatment response to neoadjuvant chemoradiotherapy plus immunotherapy in locally advanced rectal cancer.

229 Background: Neoadjuvant chemoradiotherapy plus immunotherapy (NCRIT) shows promise in treating locally advanced rectal cancer (LARC), but many patients do not respond. The immune and stromal factors influencing response remain unclear. Methods: This retrospective multicenter study included 30 patients with locally advanced rectal cancer (LARC) treated with neoadjuvant chemoradiotherapy combined with immunotherapy (NCRIT). Fresh tumor specimens from 5 patients before NCRIT and 11 patients after NCRIT were subjected to single-cell RNA sequencing (scRNA-seq) and paired T cell receptor (TCR) sequencing to profile immune and stromal cell states and clonotypes. Pre- and post-NCRIT formalin-fixed paraffin-embedded (FFPE) samples from all patients were analyzed by immunohistochemistry (IHC) and multiplex immunofluorescence (mIF). Results: Pre- and post-NCRIT tumors from non-responders showed enrichment of CTLA4⁺CD4⁺ regulatory T cells with activated immunosuppressive function and clonal expansion, while post-NCRIT tumors from responders had expanded CD4⁺IL7R⁺-LTB⁺ helper T cells linked to mature tertiary lymphoid structures. Among CD8⁺ T cells, exhausted CD8⁺GZMB⁺CXCL13⁺ were enriched in post-NCRIT tumors from non-responders; interferon-activated CD8⁺MX1⁺ cells predominated in pre- and post-NCRIT tumors from responders. Cancer-associated fibroblasts (CAFs) with enhanced epithelial–mesenchymal transition (CAF-HILPDA, CAF-DES-RSPO3, CAF-DES-SYNM) were enriched in post-NCRIT tumors from non-responders, while immune-infiltration-associated CAF subsets (CAF-CXCL14, CAF-HHIP) were enriched in post-NCRIT tumors from responders. SPP1⁺ macrophages were also increased in post-NCRIT tumors from non-responders. Integrative analysis revealed HILPDA⁺ CAFs and SPP1⁺ macrophages as interacting partners with spatial proximity, potentially facilitating Treg recruitment and immunosuppression in non-responders. mIF confirmed close spatial proximity between HILPDA⁺ CAFs and SPP1⁺ macrophages. Conclusions: Our integrated single-cell and spatial analysis reveals distinct immune–stromal ecosystems associated with NCRIT response in LARC. Resistance is characterized by a fibroblast–macrophage–Treg axis promoting immunosuppression and EMT, whereas response correlates with expanded cytotoxic T cells and immune-recruiting CAFs. These findings offer mechanistic insights, potential biomarkers for stratification, and therapeutic targets to enhance neoadjuvant efficacy.