Tissue architecture and immune niches govern ctDNA release in colorectal cancer

Circulating tumor DNA (ctDNA) is central to liquid biopsy-based cancer detection, yet the biological basis of its highly variable release into the bloodstream remains poorly understood. To define the tissue-level determinants of ctDNA shedding in colorectal cancer (CRC), we integrated tumor-informed plasma sequencing in a cohort of 59 patients in resectable stage I-III disease with detailed histopathology and immunophenotyping ( n = 49), spatial transcriptomics ( n = 4), and in situ mutation detection ( n = 6). ctDNA detectability increased with tumor burden, and ctDNA shedders exhibited a distinct architectural and microenvironmental phenotype characterized by expanded pseudolumina containing necrotic debris, frequent epithelial barrier disruption, and dense myeloid infiltration in lumina and stroma. Spatial profiling revealed stress-associated malignant programs and a myeloid-rich immune-luminal niche. In situ mutation detection localized plasma-identified mutations to necrotic material within pseudoluminal structures, establishing these compartments as potential focal reservoirs of shed DNA. These findings provide a mechanistic framework linking tissue architecture, immune remodeling, and spatially organized cell death to ctDNA release with implications for refining liquid biopsy applications.