Tissue architecture and immune niches govern ctDNA release in colorectal cancer

Kuehberger, S.; Sallinger, K.; Mueller, C.-T.; Escriva-Conde, M.; Marco-Salas, S.; Andaloro, S.; Beichler, C.; Graf, R.; Pankratz, K.; Enzi, J.; Binder, S.; Scheiber, M.; Bonstingl, L.; Blatterer, J.; Uranitsch, S.; Moitzi, G.; Schmoelzer, H.; Hauser, H.; Strohmeyer, K.; Nilsson, M.; Lax, S.; Syrnioti, A.; Oehler, R.; Hoefler, G.; Aigner, F.; El-Heliebi, A.; Heitzer, E.

doi:10.1101/2025.11.28.691139

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bioRxiv · 10.1101/2025.11.28.691139

Tissue architecture and immune niches govern ctDNA release in colorectal cancer

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Abstract

Circulating tumor DNA (ctDNA) is central to liquid biopsy-based cancer detection, yet its release into the bloodstream varies widely and remains poorly understood. To define the tissue-level determinants of ctDNA shedding in colorectal cancer (CRC), we integrated tumor-informed plasma sequencing with detailed histopathology, immunophenotyping, spatial transcriptomics, and in situ mutation detection in resectable stages (I-III). ctDNA detectability increased with tumor burden, and high ctDNA shedders exhibited a distinct architectural and microenvironmental phenotype characterized by expanded necrotic pseudolumina, frequent epithelial barrier disruption, and dense myeloid infiltration. Spatial profiling revealed stress-associated malignant programs and a myeloid-rich immune-luminal niche. In situ sequencing confirmed plasma-detected mutations within pseudoluminal debris, identifying these structures as focal reservoirs of shed DNA. These findings provide a mechanistic framework linking tissue architecture, immune remodelling, and spatially organized cell death to ctDNA release with implications for refining liquid biopsy applications.

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Kuehberger, S., Sallinger, K., Mueller, C.-T., Escriva-Conde, M., Marco-Salas, S., Andaloro, S., Beichler, C., Graf, R., Pankratz, K., Enzi, J., Binder, S., Scheiber, M., Bonstingl, L., Blatterer, J., Uranitsch, S., Moitzi, G., Schmoelzer, H., Hauser, H., Strohmeyer, K., Nilsson, M., Lax, S., Syrnioti, A., Oehler, R., Hoefler, G., Aigner, F., El-Heliebi, A., Heitzer, E.. 2025-12-01. Tissue architecture and immune niches govern ctDNA release in colorectal cancer. https://doi.org/10.1101/2025.11.28.691139

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