Single-Nucleus and In Situ RNA–Sequencing Reveal Cell Topographies in the Human Pancreas

Abstract

BACKGROUND & AIMS: Molecular evidence of cellular heterogeneity in the human exocrine pancreas has not been yet established because of the local concentration and cascade of hydrolytic enzymes that can rapidly degrade cells and RNA upon pancreatic resection. We sought to better understand the heterogeneity and cellular composition of the pancreas in neonates and adults in healthy and diseased conditions using single-cell-sequencing approaches. METHODS: We innovated single-nucleus RNA-sequencing protocols and profiled more than 120,000 cells from pancreata of adult and neonatal human donors. We validated the single-nucleus findings using RNA fluorescence in situ hybridization, in situ sequencing, and computational approaches. RESULTS: We created the first comprehensive atlas of human pancreas cells, to our knowledge Q9 , including epithelial and nonepithelial constituents, and uncovered 3 distinct acinar cell types, with possible implications for homeostatic and inflammatory processes of the pancreas. The comparison with neonatal single-nucleus-sequencing data

Endocrine Pancreas Development and Dysfunction Through the Lens of Single-Cell RNA-Sequencing

“…The two essential components for PDAC development are: (1) acquisition of an oncogenic mutation by acinar or ductal cells, with KRAS oncogene counting for 90% of cases; (2) inflammation within microenvironment which drives early lesion development. Multiple scRNA-Seq studies in PDAC mouse models and human tumor samples shed light on tumor cell identity and heterogeneity, as well as tumor microenvironment (TME), including role of cancer associated fibroblasts (CAFs) and immune landscape, and cross-talk between tumor and TME ( Ting et al, 2014 ; Bernard et al, 2019 ; Elyada et al, 2019 ; Hosein et al, 2019 ; Ligorio et al, 2019 ; Peng et al, 2019 ; Dominguez et al, 2020 ; Hwang et al, 2020 ; Lee et al, 2020 ; Schlesinger et al, 2020 ; Steele et al, 2020 ; Zhou et al, 2021 ). These studies showed that intra-tumor heterogeneity identified by scRNA-Seq is reliable as a prognostic marker and can be used for personalized treatment choice.…”

“…PDAC can develop when control over a physiological regeneration of exocrine pancreas fails ( Storz, 2017 ). The regeneration is driven by a reversible process of acinar cells dedifferentiation into duct-like exocrine progenitors (acinar-to-ductal metaplasia, ADM), and the transitional, plastic populations were observed in adult human pancreata and PDAC samples by scRNA-Seq ( Qadir et al, 2020 ; Zhou et al, 2021 ) and single-nucleus RNA-Seq (sNuc-Seq) ( Tosti et al, 2021 ). The irreversible dedifferentiation and unrestrained proliferation of these progenitors leads to pancreatic intraepithelial neoplastic lesions (PanIN) and eventually PDAC ( Storz, 2017 ).…”

“…Regenerating gene protein (REG) family-enriched acinar cells were identified as promoting ADM and PanIN growth ( Gironella et al, 2013 ; Liu et al, 2015 ). A small proportion of REG+ population was identified by scRNA-Seq also in non-disease pancreata ( Muraro et al, 2016 ; Tosti et al, 2021 ), and enriched in low-grade lesions ( Bernard et al, 2019 ) and PDAC ( Schlesinger et al, 2020 ). A recent sNuc-Seq study identified REG+ cells as the only exocrine cluster in a pancreatitis sample, whereas they were minority in healthy pancreas and not present in neonatal pancreas ( Tosti et al, 2021 ).…”

“…A small proportion of REG+ population was identified by scRNA-Seq also in non-disease pancreata ( Muraro et al, 2016 ; Tosti et al, 2021 ), and enriched in low-grade lesions ( Bernard et al, 2019 ) and PDAC ( Schlesinger et al, 2020 ). A recent sNuc-Seq study identified REG+ cells as the only exocrine cluster in a pancreatitis sample, whereas they were minority in healthy pancreas and not present in neonatal pancreas ( Tosti et al, 2021 ). In situ sequencing revealed that these cells reside near macrophages, possibly modulating immune response ( Tosti et al, 2021 ).…”

“…A recent sNuc-Seq study identified REG+ cells as the only exocrine cluster in a pancreatitis sample, whereas they were minority in healthy pancreas and not present in neonatal pancreas ( Tosti et al, 2021 ). In situ sequencing revealed that these cells reside near macrophages, possibly modulating immune response ( Tosti et al, 2021 ). Moreover, research on PDAC, pre-metastatic PanIN and intraductal papillary-mucinous neoplasms, identified transition of KRT19+ ductal populations along tumor development, where MUC5AC-rich clusters are present in benign lesions, and MUC5B+ metaplastic populations marks pancreatitis and PDAC ( Bernard et al, 2019 ; Peng et al, 2019 ; Schlesinger et al, 2020 ; Tosti et al, 2021 ).…”