Single-cell profiling of the tumor microenvironment in pMMR/MSS rectal cancer treated with neoadjuvant PD-1 inhibitor combined with FOLFOXIRI and radiotherapy: Insights from the PANFORTE trial.

3647 Background: Programmed cell death-1 (PD-1) inhibitor-based combination therapies have emerged as a promising neoadjuvant strategy for locally advanced rectal cancer (LARC). However, the underlying tumor microenvironment (TME) dynamics during multimodal chemoimmunoradiotherapy remain incompletely understood. This study leveraged single-cell RNA sequencing to systematically map the TME remodeling induced by neoadjuvant PD-1 inhibitor combined with chemoradiotherapy in the PANFORTE trial (NCT06099951), aiming to identify cellular correlates of therapeutic response and resistance in pMMR/MSS rectal cancer. Methods: Pre- and post-treatment tumor samples from patients with pMMR/MSS rectal cancer enrolled in the PANFORTE trial and treated with neoadjuvant FOLFOXIRI, PD-1 inhibitor, and radiotherapy were subjected to single-cell RNA sequencing. Following stringent quality control, 227,447 high-quality cells from 50 samples were retained and annotated into seven major lineages. Cellular compositional changes across response groups were quantified using the observed-to-expected (Ro/e) index. Fibroblast subpopulations were identified via non-negative matrix factorization (NMF), and pseudotime trajectory analysis of myeloid cells was performed with Monocle 2. Results: Single-cell profiling revealed marked TME remodeling after neoadjuvant therapy. Complete responders (CR) showed significant stromal reduction, while non-responders (NCR) retained higher proportions of myeloid cells, endothelial cells, and fibroblasts. CR tumors exhibited enrichment of effector populations (NK cells, CD8⁺ effector memory T cells), whereas NCR tumors expanded immunosuppressive subsets (regulatory T cells, naïve T cells). Fibroblast subclustering identified inflammatory cancer-associated fibroblasts (iCAFs) as enriched in NCR. Pseudotime analysis demonstrated that myeloid cells in CR differentiated toward M1-like states, while NCR samples were biased toward M2-like programs. Conclusions: This study delineates distinct TME landscapes underlying differential responses to neoadjuvant FOLFOXIRI, PD-1 inhibitor, and radiotherapy in pMMR/MSS rectal cancer. Responders are characterized by cytotoxic immune activation and stromal depletion, whereas non-responders maintain an immunosuppressive milieu rich in iCAFs and M2-like myeloid cells. These findings provide mechanistic insight into treatment resistance and highlight potential cellular targets for enhancing therapeutic efficacy. Clinical trial information: NCT06099951 .