Chantal Allgöwer, Medhanie Mulaw, James Nagai, Sandra Wiedenmann, Elena Anne Ringel, Benedetta Ferrara, Lorenzo Piemonti, Tengku Ibrahim Maulana, Luisa T. Ferreira, Adam Flinders, Claudia Teufel, Leon Reichardt, Paul Lopatta, Dharini Srinivasan, Anton Lahusen, Thomas Seufferlein, Nadine Therese Gaisa, Annika Beck, Jessica Lindenmayer, Michael K. Melzer, Eleni Zimmer, Élodie Roger, Sandra Heller, J.-Mathias Löhr, Stefan Liebau, Peter Loskill, Yuan-Na Lin, Paolo Riccardo Camisa, Claus Jorgensen, Stefano Crippa, Matthias Meier, Meike Hohwieler, Ivan G. Costa, Markus Breunig, Alexander Kleger
BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) are highly lethal and aggressive with oncogenic KRAS being the main oncogenic driver of the disease. PDACs have been extensively profiled at advanced stages, and in advanced disease the tumor microenvironment is a major determinant that critically shapes patient outcomes. Since the molecular events occurring prior to invasive growth remain poorly understood, we aimed to investigate changes in the precancerous epithelium and its surrounding niche. METHODS: We acquired time-resolved, single-cell transcriptomic (scRNAseq), and accessible-chromatin data from human pluripotent stem cell-derived pancreatic duct-like organoids (PDLO) inducibly expressing KRASG12D and from various niche cells. RESULTS: Analysis of the pure epithelium already revealed key signatures of matrix remodeling and inflammation-related signaling upon few days of KRASG12D expression. Machine learning captured KRASG12D-dependent transcriptomic classifiers with high prediction accuracy and niche preparatory relevance. Various co-culture approaches followed by scRNAseq and functional validation, including T-cell microfluidics, demonstrated that the KRASG12D-induced PDLO-secretome activates pancreatic stellate cells (PaSCs) and protects precancerous organoids from T cell infiltration. Additional, in silico approaches reconstructed a virtual pancreatic (pre)cancerous space to profile cell–cell interactions between PDLOs and niche cells. TNFα emerged as a top-ranked ligand and was functionally validated to mediate T-cell shielding and PaSC activation. Cyst fluid from 80 prospectively sampled Intraductal Papillary Mucinous Neoplasm (IPMNs) –well-known cystic PDAC precursor lesions– showed a stepwise TNFα rise across LGD (low-grade), HGD (high-grade), and IC (invasive cancer). CONCLUSION: Our study reveals that oncogenic KRAS orchestrates niche-preparatory programs that precede PDAC formation and highlight a T cell exclusion program governed by epithelial-derived TNFα.