Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer deaths worldwide1. Studies in human tissues and in mouse models have suggested that for many cancers, stem cells sustain early mutations driving tumour development2,3. For the pancreas, however, mechanisms underlying cellular renewal and initiation of PDAC remain unresolved. Here, using lineage tracing from the endogenous telomerase reverse transcriptase (Tert) locus, we identify a rare TERT-positive subpopulation of pancreatic acinar cells dispersed throughout the exocrine compartment. During homeostasis, these TERThigh acinar cells renew the pancreas by forming expanding clones of acinar cells, whereas randomly marked acinar cells do not form these clones. Specific expression of mutant Kras in TERThigh acinar cells accelerates acinar clone formation and causes transdifferentiation to ductal pre-invasive pancreatic intraepithelial neoplasms by upregulating Ras–MAPK signalling and activating the downstream kinase ERK (phospho-ERK). In resected human pancreatic neoplasms, we find that foci of phospho-ERK-positive acinar cells are common and frequently contain activating KRAS mutations, suggesting that these acinar regions represent an early cancer precursor lesion. These data support a model in which rare TERThigh acinar cells may sustain KRAS mutations, driving acinar cell expansion and creating a field of aberrant cells initiating pancreatic tumorigenesis.
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npj Regenerative Medicine Open Access 11 February 2022
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Custom Python scripts used in this manuscript are available at (https://github.com/cmroake/KRAS_scripts).
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This work was supported by grants from the NIH (AG056575, CA244114 and CA197563; S.E.A.), the Emerson Collective (S.E.A.), anonymous support (S.E.A.), the DFG (NE 2006/1-1; P.N.), California TRDRP (25FT-0011; P.N.) and the California Institute for Regenerative Medicine (R.J.L.). We thank members of the Artandi laboratory for critical comments and P. Chu from the Stanford Human Pathology/Histology Service Center for technical assistance, and acknowledge the Stanford Functional Genomics Facility and Stanford Shared FACS Facility.
The authors declare no competing interests.
Peer review information Nature thanks Laura Wood and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Identification of a TERT-high subpopulation in acinar cells and beta cells but not duct cells using lineage tracing.
a, TertCreERT2/+; Rosa26LSL-tdTomato/+ mouse, which responds to tamoxifen-induced Cre-mediated activation of the tdTomato reporter allele. b, Immunohistochemistry analysis of Tomato in a TertCreERT2/+; Rosa26LSL-tdTomato/+ (TertCreER) pancreas after oil and tamoxifen injection (n=4 mice for oil ctrl; n=11 mice for tamoxifen). Mo, month. c, d, Co-immunofluorescence stains 10 days after single dose of tamoxifen (5mg) in TertCreER pancreas (n=4 mice). Tomato (red, c, d), DAPI (blue, c, d), Insulin (grey, c, d) Sox9 (green, c) and Amylase (green, d) are shown. e, f, Single-molecule RNA FISH for Tert mRNA (e) on FACS-purified bulk acinar cells (e, f) from TertCreER mice 10d after Tamoxifen (5mg) injection (n=4 mice). g, Quantification of Tomato-positive cells in TertCreER mice in the acinar cell compartment 10 days and within pancreatic ductal cells 10 days and 12 months post tamoxifen activation (n=5 mice). h, qRT-PCR for indicated genes in FACS-sorted TomNeg (white bars) and TomPos (grey bars) acinar cells. i, j, Immunofluorescence analysis of a TertCreER pancreas 10 days after a single dose of 5mg tamoxifen (n=5 mice). Tomato (red), Dclk1 (i, green) or BMI1 (j, green) and DAPI (blue) are shown. Scale bar, 100µm (b), 50µm (e, f) and 25µm (c, d, i, j). All data are mean± SD. P values calculated by two-sided Mann-Whitney test (h).
Extended Data Fig. 2 Randomly marked acinar cells show limited clonogenicity and TERT-high acinar cells renew to yield TERT-low cells.
a, Schematic of lineage tracing in TertCreER mice treated with a single dose of tamoxifen on postnatal day 60 and analysis at indicated time points. Mo, month. b–d, Immunohistochemistry for Tomato in Ptf1aCreER mice 10d post tamoxifen (5mg (b), 0.5mg (c) and 0.05mg (d)) injection (n=4 mice). e, Distribution of Tomato-positive acinar cells per clone for each time point in Ptf1aCreER mice (each color represents one mouse; n=4 for each time point) f, TertCreER mice are injected with Tamoxifen (5mg) on postnatal day 60 and put on BrdU water (1mg/ml) for 30 days. g, Immunofluorescence analysis of a TertCreER pancreas after 30 days of BrdU water. Tomato (red), BrdU (green) and DAPI (blue) are shown. TERTHigh Tomato/BrdU double positive cells (green arrow) and TERTLow BrdU positive cells (orange arrow) shown. h, Quantification of TERTLow and TERTHigh BrdU+ cells (n=5 mice) (mean ± SD). i–k, Single-molecule RNA FISH for Tert on FACS-purified bulk (i), TERTHigh (j) and TERTLow derived (k) acinar cells of TertCreER mice(n=3). Scale bars, 25µm (g), 50µm (i–k), 100 µm (b–d). P values calculated by two-sided Mann-Whitney test (h).
Extended Data Fig. 3 During injury, TERTHigh acinar show enhanced repopulation compared with randomly marked acinar cells.
a, Schema of cerulein-induced pancreatitis. b, Immunofluorescence analysis of TertCreER mice 30 days after cerulein injections. Tomato (red), Ki67 (green) and DAPI (blue) are shown. TERTHigh, Ki67+ positive cells (green arrow) and TERTLow, Ki67+ cells (orange arrows) are shown. c, Quantification of TERTHigh, Ki67+ and TERTLow, Ki67+ cells at indicated time points (n=5 mice) (mean ± SD). d, Schematic of L-Arginine induced pancreatitis. e, Immunofluorescence analysis of TertCreER mice 30 days after L-Arginine induced pancreatitis. f, Quantification of Tomato-positive cells per clone with and without injury (n=5 mice) (mean ± SD). Scale bars, 25µm (d), 250 µm (e). P values calculated by two-sided Mann-Whitney test (c, f).
Extended Data Fig. 4 Enhanced PanIN formation from TERT-High acinar cells compared with sparsely labeled randomly marked acinar cells.
a, Schema for cerulein-induced pancreatitis. b, Immunohistochemistry for Tomato in Ptf1aCreER; Kras mice and TertCreER; Kras mice one month after cerulein-induced pancreatitis (AP). c, Quantification of number of clones per 20x high-power field (HPF) in Ptf1aCreER; Kras mice (n=7) and TertCreER; Kras mice (n=7) one month after cerulein-induced pancreatitis (AP) (mean ± SD). d, e, Representative metaplastic area (d, dashed line) and PanIN lesion (e, black arrow) in H&E staining of TertCreER; Kras mice one-month post injury (AP) (n=9 mice). f, g, Representative H&E staining (f) and Immunohistochemistry for Tomato (g) of Ptf1aCreER; Krasmice one-month post injury (AP) with high dose tamoxifen (5mg) (n=9 mice). h–k, Immunofluorescence analysis of a TertCreER (h, i) and TertCreER; Kras (j, k) mice one month after sham (h, j) or cerulein (i, k) treatment. Acinar cell expansion (red arrows) and PanINs (white arrow) are shown. Scale bar 100µm (a, c–f) and 50µm (g–j).
Extended Data Fig. 5 MEK inhibitor prevents PanIN formation from TERT-High cells expressing mutant Kras.
a, Schema of cerulein-induced pancreatitis with MEKi treatment (Trametinib). b, c, H&E (b) and Tomato (c) staining of TertCreER; Kras mice one month after cerulein induced pancreatitis with (Trametinib) and without (Vehicle) MEKi treatment. d, Quantification of Tomato-positive cells per clone with (n=4 mice) and without (n=3 mice) MEK inhibition (mean ± SD). Scale bars, 100µm.
pERK 1/2 staining of human pancreas cancer specimen using indicated antibodies and IgG control (n=7 for #4376, n=44 for #4370 and n=3 for IgG control. Scale bars, 100µm.
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Neuhöfer, P., Roake, C.M., Kim, S.J. et al. Acinar cell clonal expansion in pancreas homeostasis and carcinogenesis. Nature 597, 715–719 (2021). https://doi.org/10.1038/s41586-021-03916-2
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