Deep visual proteomics reveals an in vivo-like phenotype of orthotopically transplanted human colon organoids

Intestinal epithelial damage predisposes to disorders like inflammatory bowel disease (IBD), with organoid transplantation emerging as a potential treatment. However, it is not known how well organoids recapitulate in vivo intestinal epithelial cells (IECs). We employed deep visual proteomics (DVP), integrating AI-guided cell classification, laser microdissection, and ultra-high-sensitivity proteomics at the single-cell level to generate an in-depth proteome resource of IECs directly isolated from the human colon and organoids. While in vitro organoids display high proliferation and low functional signatures, xenotransplantation induces a remarkable shift toward an in vivo-like phenotype. We recapitulated this transition by modifying culture conditions. Our data provide a comprehensive spatial proteomics resource and validate xenotransplanted organoids as suitable models for studying human IEC behavior with unprecedented molecular detail and demonstrate their clinical potential for patients with IBD and other intestinal disorders. A record of this paper's transparent peer review process is included in the supplemental information.