Deep visual proteomics advances human colon organoid models by revealing a switch to an in vivo-like phenotype upon xenotransplantation

Introduction: Organoid models are essential tools in disease modeling and regenerative medicine, providing a unique platform to closely mimic human tissue architecture and function. However, it is important to assess to which degree these models accurately replicate physiological features. Likewise, addressing safety concerns in organoid transplantation is critical, as cellular misbehavior after transplantation can prevent integration and functionality in clinical settings. This study introduces drastic improvements to our Deep Visual Proteomics (DVP) technology, overcoming limitations in sensitivity and depth, thus providing a colon mucosa atlas and profound insights into organoid cellular behaviors.

Methods: Organoids were cultured under in vitro conditions or transplanted into murine colons to simulate a more dynamic biological environment. Simultaneously, native human colon samples were analyzed to serve as the in vivo benchmark of intestinal epithelial cells (IECs). To characterize 136 samples consisting of ~100 to 500 cell contours, we improved our DVP technology, which integrates high-resolution fluorescence imaging with AI-guided cell segmentation and classification, laser microdissection, and mass spectrometry-based proteomics. We dramatically increased the sensitivity of the proteome acquisition by coupling the Evosep One running the Whisper40 nanoflow gradient on a 15 cm ionopticks Aurora column to the Orbitrap Astral mass spectrometer. This setup enabled us to detect 8,865 unique proteins across samples and ~5000 unique proteins from only ~20 intestinal stem cells isolated from transplanted human colon organoids.

Results: Our improved DVP technology revealed remarkable insights into the proteomic landscape of IECs. We created an in-depth spatial proteome atlas of the human colon mucosa that serves as a critical reference for assessing organoid models and future research on human intestinal biology. Comparing proteomes of IECs in vivo and those grown as organoids in vitro revealed a robust correlation. However, IECs grown in vitro had more proliferative and lower functional signatures compared to their in vivo counterparts. Xenotransplantation of human colon organoids into the murine colon as well as adjusting the culture conditions led to a reversion of the proliferative phenotype to a more in vivo-like state.

Outlook: By increasing the sensitivity of DVP, we are now able to substantially cover single cell-type proteomes in vivo. This enhanced our understanding of IEC behavior in different biological systems and provided a valuable reference for researchers looking to design more accurate in vitro models that better mimic in vivo conditions. The ability of xenotransplanted organoids to revert to an in vivo-like phenotype highlights their potential for use in regenerative medicine. This includes applications in treating diseases such as inflammatory bowel disease by replenishing impaired IECs.