TGFß1 promotes collective invasion of oncogenically transformed intestinal organoids by inducing partial epithelial-mesenchymal transition independently of Snail1 and Zeb1

Cancer cells simultaneously featuring epithelial and mesenchymal traits appear particularly competent to invade and metastasize. However, genetic prerequisites and signaling pathways promoting such partial epithelial-to-mesenchymal transitions (EMT) remain obscure. Here we report that murine intestinal organoids with hyperactive Wnt and MAPK signaling, and mutant Trp53 are purely epithelial and non-invasive in vitro, but initiate a collective invasion program when treated with TGFß1. Invasive organoids reciprocally interact with the extracellular matrix (ECM), and autonomously deposit and remodel components thereof. Although cells at the organoid/ECM interface shed certain epithelial characteristics, invasive organoids largely maintain epithelial gene expression while concomitantly upregulating a mesenchymal program. TGFß1-induced phenotypic changes involve canonical, Smad4-dependent signaling, yet, are unimpeded by knockout of Snai1 and Zeb1 - otherwise key regulators of epithelial-to-mesenchymal transitions. The finding of TGFß1-inducible, Snail1/Zeb1-independent collective invasion of oncogenically transformed intestinal organoids provides novel mechanistic insights and broadens the spectrum of context-dependent manifestations of partial EMT. Overall design: Small intestinal organoids were established from two different C57BL/6N founder animals with the genotype Apc580S/580S, KrasLSL-G12D/+, Trp53LSL-R172H/+, tgVillin-CreERT2. Organoids were labelled according to the identifier of the mouse from which they were derived (animals #931 and #947). By treatment with 4-hydroxy-tamoxifen, recombination was induced to generate Apc-deficient organoids expressing oncogenic KrasG12D and dominant negative Trp53R172H (so called TKA organoids). Time-resolved gene expression analysis by RNA sequencing (RNA-seq) was performed with TKA organoids. For this, organoids were mechanically disrupted and seeded in 3 mg/ml Matrigel. At 40 h after seeding, organoids had re-developed a cystic shape and treatment was started. Organoids were stimulated with 5 ng/ml human TGFß1 for 6, 24, 48, and 72 h, or were harvested at the onset of the experiment (C-0) and after 72 h of cultivation with solvent (C-72) to account for culture-dependent effects. RNA was isolated from organoids and submitted for sequencing. For each of the two TKA organoid lines 931 and 947, RNA-seq data was collected from two independent biological replicates.