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. 2022 Apr 27:13:865452.
doi: 10.3389/fphys.2022.865452. eCollection 2022.

Enteroendocrine Cell Formation Is an Early Event in Pancreatic Tumorigenesis

Leah R Caplan  1 , Vera Vavinskaya  2 , David G Gelikman  1   3 , Nidhi Jyotsana  1 , Vincent Q Trinh  4 , Kenneth P Olive  5 , Marcus C B Tan  4   6   7 , Kathleen E DelGiorno  1   6   7   8
Affiliations

Enteroendocrine Cell Formation Is an Early Event in Pancreatic Tumorigenesis

Leah R Caplan et al. Front Physiol. .

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of only 11%, due, in part, to late diagnosis, making the need to understand early events in tumorigenesis critical. Acinar-to-ductal metaplasia (ADM), when not resolved, is a PDAC precursor. Recently, we showed that ADM is constituted by a heterogenous population of cells, including hormone-producing enteroendocrine cells (EECs: gamma, delta, epsilon, and enterochromaffin cells). In this study, we employed histopathological techniques to identify and quantify the abundance of EEC subtypes throughout pancreatic tumorigenesis in mouse models and human disease. We found that EECs are most abundant in ADM and significantly decrease with lesion progression. Co-immunofluorescence identifies distinct lineages and bihormonal populations. Evaluation of EEC abundance in mice lacking Pou2f3 demonstrates that the tuft cell master regulator transcription factor is not required for EEC formation. We compared these data to human neoplasia and PDAC and observed similar trends. Lastly, we confirm that EECs are a normal cellular compartment within the murine and human pancreatic ductal trees. Altogether, these data identify EECs as a cellular compartment of the normal pancreas, which expands early in tumorigenesis and is largely lost with disease progression.

Keywords: enteroendocrine cells; ghrelin; pancreas; pancreatic polypeptide (PP); serotonin; somatostatin.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Enteroendocrine cells as a cellular compartment of the normal murine pancreas. Immunohistochemistry for synaptophysin (brown) in the (A) small and (B) large ducts of the normal pancreas, as well as in (C) the pancreatobiliary duct. I, islet. Scale bar, 50 μm.
FIGURE 2
FIGURE 2
Characterization of enteroendocrine cell subtypes in the normal murine pancreas. Co-immunofluorescence for DAPI (blue), γ Actin (cyan) and (A) insulin (Ins, magenta) and synaptophysin (Syp, white), (B) glucagon (Gcg, magenta) and synaptophysin (Syp, white), (C) serotonin (5-HT, magenta) and PP (white), and (D) ghrelin (GHRL, magenta) and SST (white) in ducts of pancreas, including the pancreatobiliary duct, or small intestines. Scale bar, 50 μm.
FIGURE 3
FIGURE 3
Enteroendocrine cell abundance decreases throughout pancreatic tumorigenesis. Quantification of EECs as the percentage of synaptophysin (SYP) positive cells per lesion in metaplastic ducts (meta) through increasing grades of PanIN (1A-3) in (A) 6- or 12-month old KC mice or (B) KPC mice in various stages of disease progression. *p < 0.05; ***p < 0.005; ****p < 0.001. (C) Representative SYP IHC of lesions of increasing grade from either KC or KPC mice. I, islet. (D) IHC for SYP+ or SYP-adenocarcinoma in KPC mice. Scale bar, 50 μm.
FIGURE 4
FIGURE 4
Delta and gamma cell abundance throughout pancreatic tumorigenesis. Quantification of (A) delta cells (SST+) or (B) gamma cells (PP+) in lesions from KC or KPC mice. *p < 0.05; **p < 0.01, ***p < 0.005; ****p < 0.001. (C) Representative SST or PP IHC of lesions of increasing grade in KPC mice. (D) IHC for SST or PP in adenocarcinoma in KPC mice. Arrows, positive cells. Scale bar, 50 μm.
FIGURE 5
FIGURE 5
Enterochromaffin and epsilon cell abundance throughout pancreatic tumorigenesis. Quantification of (A) enterochromaffin cells (5-HT+) or (B) epsilon cells (GHRL+) in lesions from KC or KPC mice. *p < 0.05; **p < 0.01, ***p < 0.005; ****p < 0.001. (C) Representative 5-HT or GHRL IHC of lesions of increasing grade in KPC mice. (D) IHC for 5-HT or GHRL in adenocarcinoma in KPC mice. Arrows, positive cells. Scale bar, 50 μm.
FIGURE 6
FIGURE 6
EEC subtypes identified by distinct and overlapping hormone signatures. Co-immunofluorescence and quantification of (A) 5-HT and ghrelin (0.67 and 2.33% co-expression, respectively), (B) 5-HT and SST (0.33 and 0% co-expression, respectively), (C) 5-HT and PP (0.67 and 0.67% co-expression, respectively), (D) ghrelin and SST (1 and 0.67% co-expression, respectively), (E) PP and ghrelin (0 and 0.33% co-expression, respectively), and (F) PP and SST (47.7 and 81% co-expression, respectively). Scale bar, 50 μm.
FIGURE 7
FIGURE 7
POU2F3 is not required for EEC formation but affects abundance. (A) UMAP and (B) Violin plot of Pou2f3 expression in an injury-induced pancreatic metaplasia dataset demonstrating expression in tuft cells as well as the predicted tuft/EEC common progenitor population. Modified from Ma et al. (C) IHC for POU2F3 or (D) COX1 or SYP in 6-month-old KC or KPouC pancreata. Scale bar, 50 μm. (E) Quantification of pan-EEC marker synaptophysin (SYP) and EEC subtype markers (F) SST, delta cells, (G) PP, gamma cells, (H) GHRL, epsilon cells, and (I) 5-HT, enterochromaffin cells, in the pancreata of 6- and 12-month-old KC or KPouC mice. **p < 0.01; ****p < 0.001.
FIGURE 8
FIGURE 8
Enteroendocrine cells as a cellular compartment of the normal human pancreas. Synaptophysin IHC of (A) large, pathologically normal ducts in human pancreata. Scale bar, 100 μm. (B) Synaptophysin IHC of pancreata from four human donors determined to have normal islet function. Images are representative of the head, body, and tail of the same pancreata. Scale bars, 50 μm for main images, 25 μm for inserts.
FIGURE 9
FIGURE 9
Endocrine/Enteroendocrine cell subtypes in the ducts of the human pancreas. Co-immunofluorescence for hormones (A) INS (white) and SYP (magenta), (B) GCG (white) and SYP (magenta), (C) GHRL (white) and SST (magenta), or (D) PP (white) and 5-HT (magenta), and γ Actin (cyan) and DAPI (blue). Scale bar, 50 μm.
FIGURE 10
FIGURE 10
Enteroendocrine cell subtype dynamics throughout human pancreatic tumorigenesis. Co-immunofluorescence for (A) GHRL (magenta), (B) SST (white), (C) 5-HT (magenta), or (D) PP (white) and γactin (cyan), and DAPI (blue) in human ADM lesions. (E) Co-immunofluorescence for SST (white), 5-HT (magenta), or PP (white) and γactin (cyan), and DAPI (blue) in human adenocarcinoma. Scale bar, 50 μm. (F) Table depicting the number of human lesions harboring at least one hormone+ cell for each EEC subtype and organized by lesion grade. The number of EEC-containing lesions were totaled per hormone, and the relative distribution (%) of EEC-containing graded lesions are displayed (11 patients); red = highest % of total; green = lowest % of total.
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