Stromal Prostaglandin is a Dominant Spatial Regulator of Cell-fate Plasticity in Colorectal Cancer

Colorectal cancer (CRC) tumours with high stromal content have a worse outcome, but the mechanisms governing this are unclear. Using high-throughput single-cell perturbation analysis of CRC patient-derived organoids (PDOs) and cancer-associated fibroblasts (CAFs) we find that epithelial cells with high stromal-communication potential are marked by the transcriptional co-repressor DACH1 . To define the causal regulators of stromal-epithelial signalling, we developed a novel CRISPR screening platform to perturb the CAF secretome and measure epithelial stem cell responses at single-cell resolution. Intercellular CRISPR screening and full factorial ligand analysis revealed that stromal Prostaglandin E2 (PGE ₂ ) is a dominant regulator of CRC cell-fate plasticity. Stromal PGE ₂ converts DACH1 ⁺ epithelia from a chemosensitive proliferative colonic stem cell (proCSC) fate into a chemorefractory and prometastatic revival colonic stem cell (revCSC) fate. PGE ₂ -driven epithelial transdetermination is rapid and reversible, providing an acute mechanism for stromal-driven plasticity in CRC tumours. Genetic and pharmacological inhibition of stromal COX2 inhibits epithelial plasticity, trapping CRC epithelia in an anti-metastatic and chemosensitive proCSC fate. PTGS2 ⁺ CAFs support a spatially resolved revCSC to proCSC plasticity gradient in human CRC tumours marked by increasing DACH1 expression. These results reveal that stromal prostaglandin is a dominant spatial regulator of poor-prognosis cell-fates and may explain the benefit of anti-COX therapies in both preventing and treating CRC.