Phenotypic plasticity of human intestinal stem cells in regeneration and cancer

Phenotypic plasticity is the ability of cells to switch from one identity to another without alterations in genotype. In the healthy intestine, plasticity occurs in response to stem cell loss and facilitates tissue regeneration, while in cancer it contributes to therapeutic resistance, metastasis, and immune evasion. Human organoid models have emerged as a powerful platform for studying phenotypic plasticity in vitro. To study plasticity in the human intestine, this thesis employed a combination of CRISPR/Cas9 genome editing and organoid models. In particular, we used in-trans paired nicking (ITPN) to seamlessly integrate fluorescent knock-in reporters while minimizing off-target effects. In normal human colon organoids, we developed a novel cell type-specific ablation tool based on the diphtheria toxin receptor (DTR), which had not previously been feasible in a human context. Following the selective elimination of LGR5⁺ stem cells, surviving colonocytes respond by reprogramming to the stem cell state. Transcriptomic profiling of this regenerative response revealed the upregulation of injury-induced gene programs and identified a role for retinoic acid (RA), a ligand secreted by stem cells, as a suppressor of plasticity during homeostasis. These findings indicate that stem cells actively repress regenerative plasticity in their progeny by producing RA. To study plasticity in early colorectal cancer (CRC), we established a biobank of early-stage CRC organoids and performed spatial transcriptomic analyses of patient tumor tissues. We identified high-relapse cells specifically at the invasive fronts, where they co-localize with trophocyte-like fibroblasts. These fibroblasts interact with tumor cells to drive the expression of metastatic transcription programs. We validated this interaction using organoid-fibroblast co-cultures and showed that it is mediated by TGF-β and prostaglandins. In conclusion, the human intestinal epithelium displays high levels of phenotypic plasticity in regeneration and cancer. In the healthy intestine, regenerative plasticity is normally suppressed by the stem cells, while in early-stage colorectal cancer, fibroblasts at the invasive front promote metastatic cellular programs in the tumor. These insights into the molecular mechanisms of cell fate plasticity will be instrumental to modulate regenerative processes in therapeutic interventions.