NF-κB-active tumors with matrix CAFs and suppressive immunity as key resistance mechanisms to chemoradiation in rectal cancer.

Most patients with intermediate-to-high-risk locally advanced rectal cancer (LARC) undergo neoadjuvant chemoradiotherapy (NCRT), but reliable predictive biomarkers and well-defined resistance mechanisms remain lacking. We sought biomarkers and molecular mechanisms of NCRT resistance in LARC. We analyzed paired pre-NCRT and post-NCRT biopsies from 26 patients with intermediate-to-high-risk LARC using single-cell RNA sequencing and spatial transcriptomics. Suboptimal responders showed pre-NCRT activation of NF-κB, Wnt, Notch, VEGF, fatty acid, and glutamate pathways, whereas optimal responders exhibited higher cell-cycle and oxidative phosphorylation activity in tumor cells. We experimentally validated that NF-κB inhibition increased chemoradiation sensitivity in colorectal cancer cells. Matrix cancer-associated fibroblasts (mCAFs), characterized by transforming growth factor-β/extracellular matrix programs, preferentially interacted with NF-κB-high tumor cells and immunosuppressive populations in suboptimal responders, whereas optimal responders exhibited cytotoxic CD8⁺ T cell expansion and conventional dendritic cell-2 enrichment. Spatial mapping revealed mCAF-dense, extracellular matrix-rich niches colocalized with NF-κB-rich tumor-adjacent regions in suboptimal cases. Synchronous rectal and sigmoid tumors from one patient shared suboptimal response with NF-κB activation and mCAF enrichment, suggesting the resistance arises from adaptive epithelial-stromal programs rather than site-specific genetics. Collectively, integrated multicompartment signatures, rather than a single molecular profile, define an NCRT-resistant ecosystem in which tumor NF-κB activation, mCAF enrichment, and immune suppression converge to sustain non-response. In line with this framework, combined NF-κB and oxidative phosphorylation scores stratified response status with robust performance (area under the curve = 0.875) and highlighted resistance-associated therapeutic targets.