LAUSR

The response to tumor-initiating inflammatory and genetic insults can vary among morphologically indistinguishable cells, suggesting as yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, premalignant, and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identified heterogeneous cell states that are primed for diverse, late-emerging neoplastic fates and linked these to chromatin remodeling at cell-cell communication loci. Using an inference approach, we revealed signaling gene modules and tissue-level cross-talk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that was functionally validated in mice. Our results uncover a neoplasia-specific tissue-remodeling program that may be exploited for pancreatic cancer interception. Description Editor’s summary The development of pancreatic cancer involves a complex interplay between pancreatic lesions and the surrounding tissue microenvironment. Burdziak et al. performed an in-depth analysis of the evolution of pancreatic tumors. Using several model systems, including newly developed computational methods, tumor progression stages leading to pancreatic adenocarcinoma were predicted based upon communication gene modules. Tissue remodeling was traced from a healthy pancreas to an inflamed state, the premalignant stage, and then to full-blown malignancy and distant metastasis. Epigenetic plasticity in early progenitor–like epithelial cells primed pancreatic cells for neoplastic transformation. Plasticity was associated with the remodeling of accessible chromatin close to cell-cell signaling genes and an interleukin-33–mediated inflammatory feedback loop between epithelial and immune cells. —Priscilla N. Kelly Single-cell analysis reveals that enhanced epigenetic plasticity drives pro-neoplastic cross-talk in early pancreatic cancer. INTRODUCTION Virtually all cancers begin with genetic alterations in healthy cells, but mounting evidence suggests that nongenetic events such as environmental signaling play a crucial role in unleashing tumorigenesis. In the pancreas, epithelial cells harboring an activating mutation in the Kras proto-oncogene can remain phenotypically normal until an inflammatory event that drives cellular plasticity and tissue remodeling. The inflammation-driven molecular, cellular, and tissue changes that precede and direct tumor formation remain poorly understood. RATIONALE Understanding tumorigenesis requires a high-resolution view of events spanning cancer progression. We leveraged genetically engineered mouse models (GEMMs), single-cell genomics (RNA sequencing and assay for transposase-accessible chromatin sequencing), 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 and functional approaches to characterize epigenetic plasticity and to infer cell-cell communication impacts on tissue remodeling. RESULTS Our 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 diverse Kras-mutant cell populations are defined by distinct chromatin accessibility patterns and undergo inflammation-driven cell fate transitions that precede preneoplastic and premalignant lesion formation. Furthermore, a subset of early Kras-mutant cell states exhibit marked similarity to either benign or malignant fates that emerge weeks to months later; for instance, Kras-mutant Nestin-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. 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 coexpression patterns, with distinct cell subpopulations each expressing 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 endows specific plastic epithelial populations with greater capability for cross-talk, including numerous communication routes with immune populations. As one example, we identified a feedback loop between inflammation-driven Kras-mutant epithelial and immune cell states involving interleukin-33 (IL-33), which was previously implicated in pancreatic tumorigenesis. Using a new GEMM that enables spatiotemporally controlled suppression of epithelial Il33 expression during mutant Kras-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 toward typical neoplasia. CONCLUSION Multimodal 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 that Kras-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 toward neoplastic lesions. A lens into epigenetic plasticity and its consequences on tumorigenesis. A genetically engineered mouse model enabled a longitudinal view of cell states in early pancreatic cancer progression, revealing plasticity endowed through epigenetic priming. Plastic cell states express distinct sets of communication genes involved in tumorigenesis-specific communication, as visualized by highly multiplexed imaging. Mouse illustration was created with BioRender (https://biorender.com/).