Novel Acinar Metaplastic States Uncovered in Exocrine Pancreas Disease
We used single-cell RNA sequencing to profile mouse pancreas across models of acute pancreatitis (AP), recurrent acute pancreatitis (RAP), chronic pancreatitis (CP), and oncogenic Kras-driven acute pancreatitis (K-AP, abbreviated APK). We captured both whole-pancreas cell suspensions and FACS-enriched mKate2+ epithelial populations from defined timepoints spanning early injury, recurrent damage, chronic inflammation, and Kras-driven precursor lesion formation. These data characterize acinar and epithelial plasticity and associated microenvironmental remodeling across pancreatitis and Kras-mediated disease initiation. BACKGROUND & AIMS: In response to injury, pancreatic acinar cells undergo acinar-to-ductal metaplasia (ADM), marked by loss of acinar identity and acquisition of ductal features. While ADM can resolve to support tissue repair, it may also persist and serve as a precursor to pancreatic cancer. Whether diverse pancreatic stressors drive a shared or context-specific ADM program remains unclear. We sought to comprehensively define metaplastic responses to clinically relevant exocrine pancreas diseases known to increase cancer risk. METHODS: We profiled ADM and the surrounding microenvironment across mouse models of exocrine disease—including acute, recurrent, and chronic pancreatitis, as well as in the setting of oncogenic Kras—capturing over 300,000 single cells. To enable high-quality transcriptomic profiling in enzyme-rich tissue, we leveraged FixNCut, a method that preserves RNA integrity in the exocrine pancreas. Findings were validated in human pancreas tissue using CosMx spatial transcriptomics. RESULTS: We identify a conserved acinar response across disease contexts that gives rise to previously unrecognized distinct metaplastic states, including a “gateway” ADM population that precedes more advanced metaplastic states marked by complete loss of acinar identity. In pancreatic intraepithelial neoplasia (PanIN) precancerous lesions, we detect classical-like and basal-like states, suggesting that pancreatic cancer subtypes are specified much earlier than previously appreciated. In Kras-mutant tissue, we identify a second wave of inflammation and the emergence of an immunosuppressive niche, coinciding with PanIN formation. CONCLUSIONS: Our findings define a conserved program of acinar plasticity across exocrine pancreas diseases. We further link unresolved ADM to immune remodeling during precursor lesion formation and observe the emergence of pancreatic cancer subtypes in early PanIN lesions. Overall design: Pancreatitis was induced in male mice at 8–9 weeks of age by intraperitoneal caerulein injection. For acute pancreatitis (AP), male C57BL/6 mice were injected with caerulein (0.1 µg/g diluted in saline) on two consecutive days once every hour for 8 hours. For recurrent acute pancreatitis (RAP), this 2-day injection protocol was repeated weekly for 6 weeks, and RAP samples (2x, 4x, 6x) were collected 3 days after the final injection. Kras(G12D) mice were generated by crossing Ptf1a-CreERT/+, CAGs-LSL-rtTA-IRES-mKate2/+, and LSL-KrasG12D alleles. One week before AP induction, KrasG12D mice (K-AP) received tamoxifen (0.25 mg/g in corn oil) by oral gavage for 3 consecutive days to activate Kras and mKate2. Chronic pancreatitis (CP) was induced in Ptf1a-CreERT/+, CAGs-LSL-rtTA-IRES-mKate2/+ mice using a 4-week caerulein protocol (0.25 µg/g, twice per day, 5 days per week). Pancreas tissue was collected at multiple timepoints after AP or K-AP (14 hr, 72 hr, 7 day, 14 day, 6 weeks, 12 weeks) and 72hr after the last injection for  CP or RAP protocols. Single-cell suspensions were prepared from whole pancreas, and for selected CP and K-AP timepoints, mKate2+ cells were enriched by FACS to obtain epithelial-only datasets before single-cell library preparation.