IntroductionVirtually all cancers begin with genetic alteration in healthy cells, yet mounting evidence suggests that non-genetic events such as environmental signaling play a crucial role in unleashing tumorigenesis. In the pancreas, epithelial cells harboring an activating mutation in theKrasproto-oncogene can remain phenotypically normal until an inflammatory event, which drives cellular plasticity and tissue remodeling. The inflammation-driven molecular, cellular, and tissue changes that precede and direct tumor formation remain poorly understood.RationaleUnderstanding tumorigenesis requires a high-resolution view of events spanning cancer progression. We leveraged genetically engineered mouse models (GEMMs), single-cell genomic (RNA-seq and ATAC-seq) and imaging technologies to measure pancreatic epithelial cell-states across physiological, premalignant, and malignant stages. To analyze this rich and complex dataset, we developed computational approaches to characterize epigenetic plasticity and to infer cell-cell communication impacts on tissue remodeling.ResultsOur data revealed that early in tumorigenesis,Kras-mutant cells are capable of acquiring multiple highly reproducible cell-states that are undetectable in normal or regenerating pancreata. Several such states align with experimentally validated cells-of-origin of neoplastic lesions, some of which display a high degree of plasticity upon inflammatory insult. These diverseKras-mutant cell populations are defined by distinct chromatin accessibility patterns and undergo inflammation-driven cell fate transitions that precede pre-neoplastic and premalignant lesion formation. Furthermore, a subset of earlyKras-mutant cell-states exhibit marked similarity to either the benign or malignant fates that emerge weeks to months later; for instance,Kras-mutantNestin-positive progenitor-like cells display accessible chromatin near genes active in malignant tumors.We defined and quantified epigenetic plasticity as the diversity in transcriptional phenotypes that is enabled or restricted by a given epigenetic accessibility landscape. Intriguingly, these plastic cell-states are enriched for open chromatin near cell-cell communication genes encoding ligands and cell-surface receptors, suggesting an increased propensity to communicate with the microenvironment. Given the rapid remodeling of both the epithelial and immune compartments during inflammation, we hypothesize that this epigenetically enabled communication is a major driver of tumorigenesis. We found that the premalignant epithelium displays extraordinary modularity with respect to communication gene co-expression patterns; distinct cell subpopulations each express a unique set of receptors and ligands that define the nature of incoming and outgoing signals that they can receive and send.Through the development of Calligraphy, an algorithm that utilizes this receptor-ligand modularity to robustly infer the cell-cell communication underlying tissue remodeling, we showed that the enhanced signaling repertoire of early neoplastic tissue specifically endows plastic epithelial populations with greater capability for crosstalk, including numerous communication routes with immune populations. As one example, we identified a feedback loop between inflammation-driven epithelial and immune cell-states involving IL-33, previously implicated in pancreatic tumorigenesis. Using a new GEMM that enables spatiotemporally controlled suppression of epithelialIl33expression duringKras-initiated neoplasia, we functionally demonstrated that the loop initiated by epithelial IL-33 directs exit from a highly plastic inflammation-induced epithelial state, enabling progression towards typical neoplasia.ConclusionMultimodal single-cell profiling of tumorigenesis in mouse models identified the cellular and tissue determinants of pancreatic cancer initiation, and a rigorous quantification of plasticity enabled the discovery of plasticity-associated gene programs. We found thatKras-mutant subpopulations markedly increase epigenetic plasticity upon inflammation, reshaping their communication potential with immune cells, and establishing aberrant cell-cell communication loops that drive their progression towards neoplastic lesions.