Identification of Ppy‐lineage cells as a novel origin of pancreatic ductal adenocarcinoma

The Ppy gene encodes pancreatic polypeptide (PP) secreted by PP‐ or γ‐cells, which are a subtype of endocrine cells localised mainly in the islet periphery. For a detailed characterisation of PP cells, we aimed to establish PP cell lines. To this end, we generated a mouse model harbouring the SV40 large T antigen (TAg) in the Rosa26 locus, which is expressed upon Ppy‐promoter‐mediated Cre–loxP recombination. Whereas Insulin1‐CreERT‐mediated TAg expression in beta cells resulted in insulinoma, surprisingly, Ppy‐Cre‐mediated TAg expression resulted in the malignant transformation of Ppy‐lineage cells. These mice showed distorted islet structural integrity at 5 days of age compared with normal islets. CK19+ duct‐like lesions contiguous with the islets were observed at 2 weeks of age, and mice developed aggressive pancreatic ductal adenocarcinoma (PDAC) at 4 weeks of age, suggesting that PDAC can originate from the islet/endocrine pancreas. This was unexpected as PDAC is believed to originate from the exocrine pancreas. RNA‐sequencing analysis of Ppy‐lineage islet cells from 7‐day‐old TAg+ mice showed a downregulation and an upregulation of endocrine and exocrine genes, respectively, in addition to the upregulation of genes and pathways associated with PDAC. These results suggest that the expression of an oncogene in Ppy‐lineage cells induces a switch from endocrine cell fate to PDAC. Our findings demonstrate that Ppy‐lineage cells may be an origin of PDAC and may provide novel insights into the pathogenesis of pancreatic cancer, as well as possible therapeutic strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Introduction
Pancreatic gamma (γ)-cells, also known as pancreatic polypeptide (PP) cells, are a small population of endocrine cells in the islet periphery.They secrete PP, encoded by the Ppy gene.Although a previous report showed that embryos lacking Ppy-expressing cells also lacked beta and delta cells, a recent study demonstrated that mice lacking Ppy-expressing cells had a reduction in beta, alpha, and delta cells.These findings highlight the crucial role of Ppy-expressing cells in the differentiation of other islet cells through a possible endocrine or paracrine effect on other cells or cell-lineage relationships [1,2].Our group and others have recently shown that pancreatic islet cells comprise two lineages, namely, Ppy-lineage cells and non-Ppy-lineage cells [2,3].The plasticity and regenerative potentials of Ppy-lineage beta cells were shown in these previous reports, as these cells occupy the islets following induced beta-cell ablation in an experimental diabetes mouse model.Ppy-lineage cells are reprogrammed to produce insulin in this model [2,3].These reports suggest that Ppy-lineage cells play a significant role in islet regeneration or may have progenitor cell-like properties and may be susceptible to malignant transformation, as tissue regeneration has been linked with cancer [4][5][6][7][8].PP cells have been shown to exist in islets, as well as to be interspersed within the exocrine parenchyma [9] as single or small clusters of endocrine cells.About 65% of human PDAC lesions occur in the head of the pancreas [10], which is a region abundant in PP cells in both mice [9] and humans [10][11][12].Additionally, some cytochrome P450 isoenzymes, which are carcinogen-metabolising enzymes, exist in areas where PP cells are abundant in the murine pancreas and are exclusively expressed in PP cells in humans [13][14][15].The susceptibility of the head region of the human pancreas to inflammation and malignant transformation may be owing to its abundance in PP cells, which have high expression levels of carcinogen-activating cytochrome P450 monooxygenase, and xenobiotic-metabolising enzymes, while being deficient in detoxifying glutathione S-transferase enzymes [15].
The incidence and mortality rate of PDAC are on the increase, making it one of the most lethal malignancies in recent times [16].PDAC lesions have a high occurrence of mutations that lead to abnormal activation of the Kirsten rat sarcoma viral oncogene homologue (KRAS), a small RAS family GTP-binding protein that mediates signalling pathways involved in cellular proliferation, differentiation, and survival, including the retinoblastoma-E2 factor (Rb-E2F) pathway, in addition to abnormalities in the murine double minute 2-tumour protein (MDM2-p53) pathway [17][18][19], with 50% of human PDAC tumours having TP53 mutations [18,20].TAg is known to interact with the Rb family proteins and Trp53 in these pathways.TAg disrupts the cell cycle, leading to the abnormal proliferation of cells and suppression of the normal apoptotic gatekeeper function of Trp53 [21,22].Thus, in this study, we generated and characterised a mouse model that develops PDAC from islet and extra-islet Ppy-lineage cells within a few weeks from birth.Our results further corroborate that PDAC can originate from the endocrine pancreas.

Mouse experiments
The protocol for animal experiments in this study was approved by the Animal Care and Ethical Committee of Gunma University (Approval Number 21-061).The FRT-TAg-FRT fragment was integrated into a plasmid DNA construct containing CAG-EGFP-loxP flanked (a gift from Professor Satoru Takahashi, Tsukuba University, Ibaraki, Japan) by seamless cloning to prepare the target DNA plasmid.The target DNA plasmid was microinjected into fertilised oocytes of C57BL/6 mice together with Cas9-expressing vector, and a guide RNA to recombine the inserted sequences into the Rosa26 locus.Surviving embryos were transferred into the oviducts of pseudo-pregnant females to obtain candidate knock-in mice.Offspring with the correct recombination were selected by genomic PCR [23].
The founder mice were crossed with wild-type mice, and genomic DNA was extracted from the tail snips of offspring.Correct integration of the target knock-in sequence as well as its random integration into loci other than the Rosa26 locus was detected using PCR-specific primers (see supplementary material, Table S1).Progenies with correct integration of the knock-in sequence and no random integration were selected for analysis.Ppy Cre/+ knock-in mice were previously described and established, by replacing the protein-coding region of Ppy in exon 2 with nuclear translocation signal (NLS)-Cre [24].Ins1-CreERT has also been described previously [25].

Histological, immunofluorescence (IF), immunohistochemistry (IHC), and quantification analyses
Histological, IF, and IHC analyses were performed according to standard protocols.Primary antibodies were used for IF and IHC analyses (supplementary materials, Table S2).Primary antibodies for IF were detected using goat-or donkey-raised secondary antibodies conjugated with fluorescein.Nuclei were counterstained with DAPI (Dojindo, Kumamoto, Japan).An FV1000 confocal microscope (Olympus, Tokyo, Japan) was used to capture fluorescence images.Quantification of cells was performed by manually counting cells positive for antibody binding and red fluorescent protein (tdTomato) in five to ten sections per pancreas of five mice using Adobe Photoshop 2021 (Adobe, San José, CA, USA).
Pancreatic tissues were harvested from 4-week-old PRTT mice, trimmed, and washed in 0.9% NaCl three times, and were subsequently cut into fragments of about 2 Â 2 Â 2 mm and placed in 0.9% NaCl for immediate transplantation.Islets and tissues were transplanted under the skin of athymic nude mice (Japan SLC, Hamamatsu, Japan) under inhaled isoflurane anaesthesia (Narcobit-E type 11; Natsume Seisakusho, Osaka, Japan).

RNA-sequencing (RNA-seq) analysis
Pancreatic islets cells were isolated from 7-day-old Ppy Cre/+ ;Rosa26 tdTomato/+ (control) and PRTT mice to obtain Ppy-lineage cells (n = 3 mice per group), respectively.Islet cells were sorted for tdTomato fluorescence using FACS (fluorescence-activated cell sorting) flow cytometry, as described previously [29].Total RNA isolation, cDNA synthesis, and library preparation were performed using a SMART-Seq ® HT Kit (R400748; Takara Bio Inc., Tokyo, Japan).All samples had a peak size of 596 base pairs and were sequenced on NextSeq 500 (Illumina, San Diego, CA, USA).After trimming with fastp (version 0.21.0), read counts were obtained using STAR (spliced transcripts alignment to a reference) version 2.5.3a(https://github.com/alexdobin/STAR)with RSEM (RNA-seq by expectation-maximisation) version 1.3.3(https://deweylab.github.io/RSEM/)and were mapped to the Mus musculus genome assembly (Genome Reference Consortium GRCm38, UCSC version mm10).At least 47 million (M) uniquely mapped reads were obtained for each sample.Differentially expressed genes (DEGs) between two groups (control and PRTT) were detected using the TCC-iDEGES-edgeR pipeline on R (https://www.R-project.org/;4.2.2) packages TCC (1.30.0) and edgeR (3.32.1).Genes with adjusted p values (q values) less than 0.01 were defined as significant DEGs.A volcano plot was created using GraphPad Prism 9.1.1(GraphPad Software, San Diego, CA, USA), and the clusterProfiler (3.18.1) package of R was used for gene ontology (GO) analysis.Heatmaps of the expression levels of DEGs were generated using Heatmapper (http://www.heatmapper.ca/)with the following settings: Clustering method, Average linkage; and Distance measurement method, Pearson [30].An Ingenuity Pathway Analysis (IPA) Match CL license was purchased from QIAGEN for the use of all features and tools in the IPA software (QIAGEN Inc., https:// www.qiagenbioinformatics.com/products).RNA-seq datasets generated in this study have been deposited in the Gene Expression Omnibus and are accessible through GEO Series accession number GSE245435.

Results
The initial aim of this study was to isolate and characterise PP cell lines.To achieve this, a mouse model harbouring the TAg flanked by loxP sites at the Rosa26 locus (Figure 1A) was generated.In the Rosa26-CAG-EGFP-TAg (Rosa26 LSL-TAg/+ ) mouse model, TAg is not expressed until a loxP-flanked cassette encoding EGFP and a transcription termination signal is excised by Cre-loxP recombination.Once recombined, the TAg is expressed under the control of the CAG promoter (Figure 1A) in the Rosa26 locus.Heterozygous Rosa26 LSL-TAg/+ mice were crossed with Ppy Cre/+ [24] and Ins1-CreERT [25] transgenic Cre driver mice to generate mice expressing TAg in PP cells (Figure 1A) and beta cells, respectively.Mouse Ins1-CreERT enables the expression of TAg in beta cells (Ins1 CreERT/+ ; Rosa26 LSL-TAg/+ ), and hence the comparison of tumours formed by other pancreatic endocrine cells.Cre-loxP recombination of the Rosa26 locus in the progenies of this crossing was assessed by allele-specific PCR genotyping.Mice with the Ppy Cre and TAg heterozygous alleles, Ppy Cre/+ ;Rosa26 LSL-TAg/+ (PRT), and Ppy Cre/+ (control) were born at the expected frequency and used for analysis.

Ins1-driven TAg expression results in insulinoma
The expression of TAg in beta cells has been previously reported to induce beta-cell tumours [31].TAg + beta cells were observed as early as P1 (supplementary material, Figure S1A, arrowheads), and insulinomas were observed in the pancreata of 9-week-old MIP1 CreERT/+ ;Rosa26 LSL-TAg/+ mice (supplementary material, Figure S1B,C).TAg expression in beta cells was independent of tamoxifen administration, probably due to leakage of Cre-mediated recombination, which has been described in previous reports [32][33][34].In line with our presumption, IF analysis detected TAg + cells in islet tumours.Tumours had reduced immunoreactivity to insulin compared with control islets (supplementary material, Figure S1C).This suggests that TAg expression induces changes in beta-cell characteristics.

Macroscopic morphology of Ppy-driven pancreatic tumours
To induce Cre-mediated TAg expression in Ppy-lineage cells, heterozygous Rosa26 LSL-TAg/+ knock-in mice were crossed with Ppy Cre/Cre transgenic mice (Figure 1A).Mice harbouring the TAg in Ppy-lineage cells, i.e.PRT mice (Figure 1A), had lower body weights and blood glucose levels at 4 weeks than control littermates (supplementary material, Figure S2A,B).Unexpectedly, most PRT mice died within a period of 4-6 weeks after birth, with a mean and median survival age of 4.7 weeks (Figure 1B).Mice were sacrificed at 4 weeks old, and pancreata were subjected to macroscopic and histological analyses.Pancreata developed neoplasms with complete penetrance (31 out of 31 animals), characterised by multilocular lesions (Figure 1C, arrow).To our surprise, histological analysis showed the presence of multiple pancreatic lesions and PDAC in PRT mice at 4 weeks of age (supplementary material, Figure S3 and Figure 1D,b).IHC analysis detected TAg expression in the nuclei of the PDAC lesions (Figure 1D,c).

Ppy-lineage cells develop lesions from the islet periphery, which rapidly progress into PDAC
To identify the cells of origin of PDAC and other neoplastic lesions in the pancreata of PRT mice, histological and IHC analyses of control and PRT mice were performed at embryonic days E14.5 and E15.5, and postnatal days P5, P7, P10, P14, P20, and P28 of development.TAg was detected as early as E15.5 using a reporter mouse that has tdTomato fluorescence in addition to TAg expression in Ppy-lineage cells (PRTT) (supplementary material, Figure S4A).An increase in Ki67 + Ppy-lineage cells was observed at E15.5 in PRTT mice compared with control mice (supplementary material, Figure S4B), suggesting the increased proliferation of Ppy-lineage cells.Loss of the structural integrity of islets (Figure 2A) characterised by impairment of epithelial cadherin (E-cadherin) was observed by IF analysis at P5 (Figure 2B).Rapid proliferation of Ppy-lineage cells within the islets and islet periphery was observed at P10 (Figure 2C).Transformed cells had reduced endocrine signals (Figure 2D) and complete loss of PP signals (Figure 2E) at P10. TAg + (Figure 3A,b) but cytokeratin Ppy-lineage cell-derived mouse pancreatic ductal adenocarcinoma model 19 À (CK19 À ) abnormal protrusions (Figure 2A, yellow box; Figure 3A,b,f, bracket), which are absent in control mice, were observed in the periphery of the islets of PRT mice at P10.At P14, abnormal contiguous duct-like structures (Figure 3A,c) that were TAg + and CK19 + (a ductal cell marker highly expressed in PDAC tissue) were found to originate from the islet periphery (Figure 3A,c,g and supplementary material, Figure S5A).This consecutive analysis suggests that lesions observed at P20 (Figure 3A,d,h) in PRT mice appear to be invasive PDAC originating from Ppy-lineage cells in the pancreatic islets.Furthermore, abnormal duct-like structures with variable MUC5AC (a PanIN-specific marker) signals were also observed in P20 PRT mouse islets (Figure 3B,b, arrowheads, and supplementary material, Figures S5B,C,b).These intra-islet PanINlike lesions had moderate nuclear atypia but did not express cytoplasmic mucin (Figure 3B,d), as shown by     Ppy-lineage cell-derived mouse pancreatic ductal adenocarcinoma model 435

Heterogeneity of Ppy-lineage cells
To further ascertain the characteristics of the cells of origin of lesions in this mouse model, we performed a lineage tracing analysis using 7-day-old reporter mice with tdTomato fluorescence in Ppy-lineage cells (Figure 5A).Ppy gene expression was detected by tdTomato fluorescence within the islets and islet periphery (Figure 5C).IF analysis also revealed the presence of Ppy-lineage (tdTomato + ) cells embedded within the exocrine parenchyma, either as single cells or as small clusters of cells (Figure 5D-F).Most of the tdTomato + cells were positive for synaptophysin, an endocrine cell marker (Figure 5D), whereas a small fraction of cells positive for amylase, an acinar cell marker (Figure 5E), and CK19, a ductal cell marker (Figure 5F), was also identified.Next, we performed quantification analysis of tdTomato + cells and observed that in 7-day-old mice, 89.8% of tdTomato + (Ppy-lineage) cells were endocrine cells (synaptophysin + ), 6.9% were acinarlike cells (amylase + ), and 1.1% were ductal-like cells (CK19 + ) (Figure 5B).This further corroborates previous reports [2,3] highlighting the heterogeneity of Ppy-lineage cells.

Transplanted pancreatic tissue and islets from PRTT mice develop PDAC in athymic mice
To confirm the tumour-forming ability of Ppy-lineage islet cells, and to investigate whether the neoplasms observed in 4-week-old PRTT mice were PDAC lesions,

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OB Pereye et al we isolated and transplanted islets from PRTT juvenile mice and 4-week-old tissue allografts to athymic mice (Figure 6A and supplementary material, Figure S6A).PRTT pancreatic allografts were able to proliferate and develop tumours in the subcutaneous tissues of athymic mice (n = 2 for islets, n = 4 for tissue) (Figure 6B,C,b,c and supplementary material, Figure S6B,C,a-d).
Of the three athymic mice transplanted with islets isolated from P7-P10 PRTT mice (average of 250 islets per transplant site), two developed PDAC tumours 40 days after a single transplant (Figure 6A-C,b,c,e), suggesting that juvenile PRTT mouse islet cells possess PDAC-forming ability.Eleven out of 12 sites (four tissue fragments per site) in athymic mice (n = 4) transplanted with pancreatic tissue from 4-week-old PRTT mice developed tumours 4 weeks after the first transplantation (supplementary material, Figure S6A).All tissue allografts harvested and transplanted into a second group of athymic mice (n = 4) developed PDAC 4 weeks after the second transplantation (supplementary material, Figure S6A-C,a-d).Tumours formed from all allografts increased in size over time, formed ductal structures similar to intralobular and interlobular ducts (Figure 6C,b,c,e and supplementary material, Figure S6C,a-d), were TAg + (supplementary material, Figure S6C,b) and CK19 + (Figure 6C,e and supplementary material, Figure S6C,c), and had tdTomato fluorescence (Figure 6C,c and supplementary material, Figure S6C,d), indicating that the lesions formed were from Ppy-lineage cells.Ppy-lineage cell-derived mouse pancreatic ductal adenocarcinoma model 437

TAg expression altered the gene expression profile of islet-derived Ppy-lineage cells
Finally, to investigate the gene expression profiles of Ppy-lineage islet-derived PDAC-forming cells, we performed RNA-seq.Islets were isolated from 7-day-old Ppy Cre/+ ;Rosa26 tdTomato/+ (control) and PRTT mice.tdTomato + cells were separated from other islet cells in each group using FACS (supplementary material, Figures S7A,B and S8A).We profiled a total of 100 tdTomato + cells, pooled from three mice for each group.RNA-seq analysis compared the gene expression profiles of 23,412 genes between the two groups.A total of 3,098 genes showed expression variations between PRTT and control mice (Àlog 10 adjusted p value cut-off: 2) (supplementary material, Figure S8B).The expression of genes, such as Cdk1, Pbk, Ccnb1, Cenpe, and Mki67 (supplementary material, Figure S8B, red dots), which are upregulated in cancer cells, was significantly increased in PRTT.IPA identified the Rb family and E2f family transcription factors and Trp53 as upstream regulators of these genes (supplementary material, Table S3).In addition, gene ontology analysis demonstrated the enrichment of gene sets involved in cell proliferation, such as chromosome organisation, organelle fission, and nuclear division (supplementary material, Figure S8C).Conversely, gene sets involved in hormone secretion, such as hormone transport, protein secretion, and the establishment of protein localization to extracellular regions, were downregulated in PRTT (supplementary material, Figure S8D).Furthermore, our group previously performed single-cell analysis of mouse pancreatic islets cells and identified PP-cell signature genes (log 2 FC ≥ 0.06) [2].We compared the expression patterns of these genes between control and PRTT mice and found that the expression of 17 PP-cell signature genes including Tspan8 was downregulated in PRTT (supplementary material, Figure S8E).The expression of previously identified endocrine cell-associated genes from the PanglaoDB database [38] showed that genes encoding hormones, such as Ppy, Pyy, Gcg, Sst, and Iapp, in addition to Mafb and Arx were downregulated.However, the expression of Isl1, Neurod1, Npy, and Slc38a5 was upregulated in PRTT compared with control (supplementary material, Figure S8F).Furthermore, acinar cell-specific genes, including Prss1, Prss2, Cpa1, Amy2b, and Amy2a5 (supplementary material, Figure S9A), and ductal cell-related genes such as Krt19 and Krt20, as well as Pdx1 and Muc1 (supplementary material, Figure S9B), were upregulated following TAg expression.These results suggest that TAg expression in Ppy-lineage cells induces a downregulation of genes that are characteristic to endocrine and PP cells, and an upregulation of acinar and ductal cell-associated genes.Moreover, PDACassociated genes, such as Brca1, Brca2, Cdkn2, Mcm5, Stmn1, Snai2, and Id1 (supplementary material, Figure S8B), were differentially expressed in PRTT.These findings indicate that the expression of oncogenes in Ppy-lineage cells attenuates their endocrine cell characteristics and induces a switch to an exocrine cell fate and PDAC.

Discussion
In this report, we have described a mouse model that develops PDAC from Ppy-lineage cells of the endocrine pancreas.Several studies have demonstrated the cells of origin of PDAC, and it is widely accepted that the precursor lesions originate from acinar or ductal cells.However, the view that PDAC originates solely from the exocrine pancreas is uncertain, as most of the animal models used in studying PDAC target the pancreatic progenitor cells [39][40][41][42] or the exocrine pancreas [39,43,44].Pancreatic islets are rich in capillaries, nutrients, and growth factors [45][46][47], making them a favourable niche for the development and growth of cancers, as tumour progression is tightly linked to enriched microenvironments [48].To date, studies on pancreatic carcinogenesis that target the endocrine pancreas have been performed exclusively on beta cells, and pancreatic endocrine cells are considered to be refractory to oncogenic injury [49,50].However, studies have suggested that malignant transformation in the pancreas may originate from within the islet cells [51][52][53][54][55]. Despite these reports, mouse models that specifically target progenitor cells of the endocrine pancreas or the developing endocrine pancreas are yet to be established, and hence the endocrine origin of PDAC remains uncertain.In this study, we showed that whereas the expression of TAg in beta cells of the developing pancreas results in insulinoma, intriguingly, Ppy-lineage cells with TAg expression result in a different path of tumourigenesis, and a switch from endocrine cell fate towards exocrine-like characteristics and PDAC.KRAS mutations and abnormalities in the Rb-E2F and MDM2-p53 pathways, which are common in human PDAC, have been recapitulated in genetically engineered mice (GEMs) used in previous PDAC studies [17][18][19][20].Our TAg-expressing mouse model has similar dysregulation of these pathways [21,22].Whereas the direct link and clinical significance of the role of SV40 large T in human cancer are yet to be established, viruses such as JC and BK polyomaviruses, which have a similar TAg to SV40, are considered to be possible human carcinogens [56].
Our study demonstrates that PDAC can originate directly from Ppy-lineage cells within the islets, which is a rather different origin from the already established acinar and ductal origins identified using GEMs of PDAC.Tumourigenesis in the islet is marked with the loss of E-cadherin, a cell-cell adhesion molecule, which is accompanied with the loss of the structural integrity of islets, as early as P5.Malignant transformation has been associated with the loss of adherens junctions, which are crucial for cell-cell adhesion mediated by E-cadherin [57].Snai2, which belongs to the Snail family of zinc transcription factors and is known to repress E-cadherin transcription as well as mediate epithelial-mesenchymal transition [58], an event associated with tumour development, progression, and metastasis [59], was also upregulated (supplementary material, Figure S8B).Kras mutations were not detected in PRTT mice at P7, suggesting that at this stage, Kras activity might not be a key driver of Ppy-lineage cell-derived neoplasms.However, pERK + lesions, which occur when Kras is active [60], were seen in 4-week-old mice (Figure 3C,b).This suggests that Kras activation might occur at an advanced stage of Ppy-lineage cell-derived PDAC.
Studies have shown that Ppy-lineage cells are heterogeneous, and they have been suggested to be plastic with regenerative ability [2,3].We confirmed the heterogeneous characteristics of Ppy-lineage cells (Figure 5), and these features may make them susceptible to injury and malignancy, as tissue regeneration has been linked with cancer.IF analysis revealed that most Ppy-lineagelabelled cells show endocrine characteristics, as indicated by their positivity for synaptophysin (89.8%) (Figure 5B).We have lines of evidence that a small fraction of Ppy-lineage-labelled cells is also amylase + (6.93%) and CK19 + (1.08%) (Figure 5B), confirming the heterogeneity of Ppy-lineage cells.Hence, we cannot categorically say that Ppy-lineage cell-derived PDACs exclusively originate from endocrine cells.The Cre used in this study was designed to reproduce endogenous Ppy gene expression, with the start codon of Cre knocked into the exact coding region of PP on the Ppy locus, and the 5'-and 3'-regulatory regions maintained as much as possible [24].Thus, the possibility that Ppy-Cre is ectopically expressed in exocrine cells, and from which PRT mouse PDAC originates, is unlikely.Furthermore, metaanalysis of the gene expression profile datasets from 1,200 PDAC patients demonstrated a tumour-specific subtype of PDAC with PPY gene expression [61].This further corroborates the presence of Ppy-lineage cell-derived PDACs.RNA-seq analysis of Ppy-lineage islet cells from 7-day-old PRTT mice showed a downregulation of endocrine genes (supplementary material, Figure S8F), including PP-cell signature genes (supplementary material, Figure S8E), as well as an upregulation of exocrine genes (supplementary material, Figure S9) and PDAC-associated genes (supplementary material, Figure S8B).This suggests a reprogramming of Ppy-lineage cells, and a switch from endocrine cell fate to exocrine-like fate and PDAC upon transformation.These gene expression profiles and histological changes over time in PRTT islets, in addition to PRTT islet allografts with PDAC-forming ability (Figure 6), collectively indicate that Ppy-lineage cells of the endocrine pancreas are an origin of PDAC.
Identifying the cells of origin of lesions in PDAC diagnosis is usually difficult.Using a mouse model, this study demonstrated the developmental stages of PDAC originating from Ppy-lineage cells, to ascertain the endocrine origin of its preneoplastic lesions, and the changes involved in the malignant transformation of endocrinederived PDAC.While a subtype of human PDAC with PPY gene expression has been identified, further studies are needed to ascertain the pathways involved in the tumourigenesis of Ppy-lineage cell-derived PDAC.This may provide novel insights into the pathogenesis of pancreatic cancer and the development of new therapeutic strategies.

Figure 1 .
Figure 1.TAg expression in Ppy-lineage cells results in aggressive PDAC 4 weeks after birth.(A) Genetic construct of Ppy Cre/+ ;Rosa26 LSL-TAg/+ (PRT) mice.(B) Kaplan-Meier analysis of per cent survival of control and PRT mice.The red line indicates the death of PRT mice (days are approximated and clustered into weeks).(C) Gross anatomy of the control pancreas and multilocular lesions (indicated by yellow arrow) formed in the pancreas of PRT mice.(D) H&E staining of the ventral pancreas from 4-week-old Ppy Cre/+ (control) (a) and PRT mice (b).(c) Immunohistochemistry (IHC) with TAg (brown) of representative pancreatic sections from 4-week-old PRT mice.Scale bars: 50 μm (D,a-c).

Figure 3 .
Figure 3. Characterisation of a Ppy-lineage cell-derived tumour.IHC analysis of serial sections of pancreatic tissue of PRT mice at P5, P10, P14, and P20 stages of development with (A) TAg (a-d) and CK19 (e-h).(a) Enlarged image of the inset is shown, with arrowheads indicating TAg + cells.(b, f) Brackets indicate abnormal protrusions from islets at P10. (B) IHC staining for MUC5AC (brown) in pancreatic sections of (a) 18-week-old KPC mice and (b) P20 PRT mice (arrowheads indicate islets).Alcian blue staining of (c) KPC mice and (d) P20 PRT mice (arrowheads indicate islets).(C) IHC staining for pERK in pancreatic sections from 4-week-old control (a) and PRT male mice (b).Scale bars: 50 μm (A,a-c,e-g); 100 μm (A,d,h; B,a-d; C,a,b)].
434 OB Pereye et al Transformed Ppy-lineage extra-islet cells develop into pancreatic lesions with the histological progression of PDAC Besides Ppy-lineage cell-derived lesions in the islet periphery, we also identified TAg + extra-islet lesions (Figure 4A,a-d; Figure 4B,c-f).TAg signals were observed in single and small clusters of extra-islets cells interspersed within the exocrine parenchyma of PRT pancreata at E18.5 and P5 (Figure 4B,a,b).Notably, these clusters of Ppy-lineage cells interspersed within the pancreatic acinar cells do not have similar structural integrity to the normal acinus (Figure 4B,a,b, arrowheads).It is worth noting that TAg signals were not observed in cells within normal acini.To ascertain the fate of these cells upon TAg expression, we performed morphological analyses at different developmental stages.Analysis of tissue sections demonstrated acinarto-ductal-like metaplasia in extra-islet cells as early as P7 (Figure 4A,a), with tissue showing varying degrees of nuclear atypia with tumour progression (Figure 4A,a-d, enlarged image).Representative images of TAg expression detected in the nuclei of the extra-islet cells at different developmental stages (E18.5,P5, P7, P10, P14, and P20) are shown in the IHC analysis (Figure 4B,a-f).It is noteworthy that whereas CK19 signals were observed in preneoplastic lesions derived from extra-islet Ppy-lineage cells at P10 (supplementary material, Figure S5A), CK19 was not detected in the lesions originating from the islet periphery until P14 (Figure 3A,e-g).This suggests that the intra-and extra-islet Ppy-lineage cells probably have different characteristics.However, both intra-and extra-islet lesions were not stained with Alcian blue (Figure 3B,d).