EGF and EREG regulate early oncoprotein-mediated tissue transformation in intestinal organoids.

Somatic mutations, coupled with abnormal extracellular signaling cues, promote tumor development. The abundance and affinity of EGF-family ligands for EGFR influence tissue development and play a central role in organoid culture by differentially regulating the balance between crypt proliferation and villus differentiation in mouse small intestinal organoids (mSIOs). To test how medium ligand composition affects early oncogenic transformation, we generated a Ptprg KO organoid line by CRISPR/Cas9 and two previously described organoid lines harboring synthetic bio-orthogonal expression systems for KRASG12V and β-Ctn4A, activated by the membrane-diffusible ligands TMP or TMP-Cl. Measurements of crypt and villus region area showed that acute oncogenic activity did not alter organoid growth in media containing 50 ng/mL EGF; however, organoids grown without EGF consistently showed differential growth of the crypt region. Furthermore, EREG and sub-saturating EGF (0.5-5 ng/mL) treatments differentially affected crypt/villus development in each organoid line. Finally, we measured organoid morphology and found that crypt domain spatial distribution is independent of crypt area and requires EGF or EREG, highlighting the role of both proliferation and crypt organization in oncogenic tissue transformation.