High-definition spatial and single-cell multiomics reveal immunological remodeling and radiation resistance mechanisms in colorectal cancer

Radiation therapy remains central to colorectal cancer care, yet many patients experience incomplete response or recurrence. Understanding why tumors adapt instead of regress requires mapping how radiation reshapes both local and systemic immunity. The ImmunoRad ROBIN initiative addresses this need by integrating high definition spatial and single cell profiling to identify biological processes that sustain radiation resistance.Our cohort included six patients sampled before treatment, after radiation, and at surgery across tumor, adjacent mucosa, and lymph nodes. Using VisiumHD at 2 × 2 μm resolution, we generated more than one and a half million spatial spots, providing a near cellular view of therapy induced remodeling. A dominant pattern emerged at the tumor stroma boundary, where radiation triggered extracellular matrix activation, fibroblast signaling, and wound repair programs. These regions were enriched for M2 like macrophages expressing profibrotic and angiogenic genes. Their expansion suggests that a macrophage dependent repair niche forms at the invasive front and creates conditions that support tumor persistence.These stromal changes aligned with localized epithelial stress responses, showing that adaptation occurs through coordinated tissue level remodeling. Epithelial compartments displayed DNA damage repair, interferon signaling, and partial plasticity, especially near M2 rich zones. Adjacent mucosa showed weaker shifts in antigen presentation and barrier pathways. These patterns indicate that radiation injury extends across tumor and non-tumor tissue, generating new gradients that may influence treatment outcome.To assess systemic adaptation, we performed single cell RNA sequencing on matched blood. Early after radiation, circulating lymphocytes and dendritic cells showed transient interferon signatures. By surgery, these activated populations contracted and were replaced by suppressive myeloid subsets with reduced cytotoxic T cells, suggesting that initial immune activation shifts into a suppressive state. Lymph nodes exhibited disrupted follicular structure, reduced germinal center polarity, and expansion of mantle zones. Integrated analysis showed enrichment of regulatory T cells and exhausted CD8 T cells, consistent with impaired antigen driven immunity.Together, these findings support a model in which radiation triggers acute injury and immune activation that rapidly transitions into macrophage driven repair and systemic immune suppression. This coupled remodeling allows tumor cells to survive therapy and regain growth potential. The ImmunoRad ROBIN framework offers a scalable strategy for decoding treatment induced ecosystem changes and highlights stromal remodeling and lymph node dysfunction as central contributors to radiation resistance in colorectal cancer.