The acidic tumor microenvironment (TME) favors cancer aggressiveness via incompletely understood pathways. Here, we asked whether adaptation to environmental acidosis (pH 6.5) selects for human pancreatic cancer stem cell (CSC) properties. RNA sequencing (RNA-seq) of acid-adapted (AA) Panc-1 cells revealed CSC pathway enrichment and upregulation of CSC markers. AA Panc-1 cells exhibited classical CSC characteristics including increased aldehyde dehydrogenase (ALDH) activity and b-catenin activity. Panc-1, PaTu8988s, and MiaPaCa-2 cells all exhibited increased pancreatosphere-forming efficiency after acid adaptation but differed in CSC marker expression and did not exhibit typical flow cytometric CSC populations. However, single-nucleus sequencing revealed the acid adaptation-induced emergence of Panc-1 cell subpopulations with clear CSC characteristics. In orthotopic mouse tumors, AA Panc-1 cells exhibited enhanced aggressiveness, liver and lung metastasis, compared to controls. Collectively, our work suggests that acid adaptation enriches for pancreatic CSC phenotypes with unusual traits via several trajectories, providing new insight into how acidic microenvironments favor cancer aggressiveness.

Background & Aims: Epithelial tumors generally resemble the cellular architecture of their tissue of origin. However, this link remains largely unexplored in the pancreas. Methods: Using Nanostring GeoMx DSP®, Resolve Molecular Cartography® and Nanostring CosMx®, and integration with single cell RNAseq datasets, we mapped the human pancreatic ductal epithelium in non-neoplastic pancreas, and compared it to pancreatic cancer subtypes. Results: Groups of Keratin-5+ cells among the Pan-Cytokeratin+ cells in the duct have a gene signature reminiscent of stem cells and (supra)basal cells from other tissues. In spatial analysis at single cell resolution, the pancreatic duct manifests as a stratified epithelium comprising a basal and four luminal populations; In large ducts, KRT5+ basal (BAS) cells express ∆Np63 while KRT5+ luminal (LUM)-B cells reside supra-basally and are distinct from the common KRT5- LUM-A cells. LUM-C and LUM-D cells pertain to intercalated ducts and ductal glands, respectively. LUM-A and -C cells express gel-forming mucins while LUM-B cells have membrane-bound MUC4 and MUC16. In cancer, BAS and LUM-B signatures associate with the basal-like pancreatic ductal adenocarcinoma (PDAC) and correlate with lower survival but exhibit a mixed spatial pattern with a diffracted gene signature. In contrast, adenosquamous cancers of the pancreas (ASCP) preserve the normal spatially unmixed identity of LUM-B cells and BAS cells that is regulated by ∆Np63. Next to ∆Np63, conserved drug targets were identified for both populations. Conclusion: This study offers a refined pancreatic tumor classification based on the native ductal architecture, with better preservation of the LUM-B and BAS cell population identity in ASCP than in PDAC, including the conserved drug targets. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND & AIMS:Acinar-to-ductal metaplasia (ADM) is crucial in the development of pancreatic ductal adenocarcinoma. However, our understanding of the induction and resolution of ADM remains limited. We conducted comparative transcriptome analyses to identify conserved mechanisms of ADM in mouse and human. METHODS:We identified Sox4 among the top up-regulated genes. We validated the analysis by RNA in situ hybridization. We performed experiments in mice with acinar-specific deletion of Sox4 (Ptf1a: CreER; Rosa26-LSL-YFPLSL-YFP; Sox4fl/fl) with and without an activating mutation in Kras (KrasLSL-G12D/+). Mice were given caerulein to induce pancreatitis. We performed phenotypic analysis by immunohistochemistry, tissue decellularization, and single-cell RNA sequencing. RESULTS:We demonstrated that Sox4 is reactivated in ADM and pancreatic intraepithelial neoplasias. Contrary to findings in other tissues, Sox4 actually counteracts cellular dedifferentiation and helps maintain tissue homeostasis. Moreover, our investigations unveiled the indispensable role of Sox4 in the specification of mucin-producing cells and tuft-like cells from acinar cells. We identified Sox4-dependent non-cell-autonomous mechanisms regulating the stromal reaction during disease progression. Notably, Sox4-inferred targets are activated upon KRAS inactivation and tumor regression. CONCLUSIONS:Our results indicate that our transcriptome analysis can be used to investigate conserved mechanisms of tissue injury. We demonstrate that Sox4 restrains acinar dedifferentiation and is necessary for the specification of acinar-derived metaplastic cells in pancreatic injury and cancer initiation and is activated upon Kras ablation and tumor regression in mice. By uncovering novel potential strategies to promote tissue homeostasis, our findings offer new avenues for preventing the development of pancreatic ductal adenocarcinoma.

Background and Aims:The regenerative capacity of the pancreas diminishes with age. Understanding acinar cell responses to injury and the resolution of regenerative processes is crucial for tissue homeostasis. However, knowledge about the impact of aging on these processes remains limited. Methods:To investigate the influence of aging on pancreas regeneration, we established a cohort of young (7-14 weeks) and old (18 months) C57bl/6 mice. Experimental pancreatitis was induced using caerulein, and pancreas samples were collected at various time points after induction, covering acute damage response, inflammation, peak proliferation, and inflammation resolution. Our analysis involved immunohistochemistry, quantitative imaging, and gene expression analyses. Results:Our study revealed a significant decline in the regenerative capacity of the pancreas in old mice. Despite similar morphology and transcriptional profiles between the pancreas of young and old mice under homeostasis, the aged pancreas is primed to generate an exacerbated proinflammatory reaction in response to injury. Specifically, we observed notable upregulation of Junb expression in acinar cells and aberrant myofibroblast activation in the aged pancreas. Conclusion:The response of acinar cells to injury in the pancreas of aged mice is characterized by an increased susceptibility to inflammation and stromal reactions. Our findings uncover a pre-existing proinflammatory state in aged acinar cells, offering insights into potential strategies to prevent the onset of pancreatic insufficiency and the development of inflammatory conditions. These insights hold implications for preventing conditions such as chronic pancreatitis and pancreatic ductal adenocarcinoma.

Cellular plasticity underpins heterogeneity in embryogenic progenitor cells and cancer cells. The transcription factor deltaNp63 (ΔNp63) has been implicated in regulating cellular plasticity in several epithelial tissues. Despite a recently established role in steering plasticity of pancreatic cancer, ΔNp63 remains unstudied in pancreatic development. Using murine single-cell sequencing data and RNA and protein in situ stainings, we assessed the spatio-temporal expression of Trp63 and ?NP63 in the embryonic pancreas. ΔNP63 demonstrates a transient and spatially restricted expression in the multipotent pancreatic progenitor (MPP) compartment delineating pro-exocrine progenitor cells. Lineage tracing of TP63+ cells marks a subset of MPPs and descendant exocrine acinar and centro-acinar/terminal duct cells. Lack of ΔNP63 in knock-out mice leads to hypotrophic exocrine acini with reduced levels of differentiation markers. In summary, ΔNp63 confers heterogeneity within the MPP compartment, supporting exocrine cell development. These new insights in developmental plasticity have potential implications for pancreatic regeneration and cancer.

Document describing important findings related to the main manuscript.

Document describing important mouse lines, treatment protocols, antibodies and primers used in the present study.

Gene alterations play a prominent role in driving cancer initiation and progression. Yet, mutations on oncogenes (those genes that promote tumorigenesis) only transform cells under certain cellular contexts. The mechanisms controlling neoplastic transformation (oncogenic competence) are poorly understood in pancreatic ductal adenocarcinoma (PDAC). Our laboratory investigates the interplay of mutations on the Kirsten Rat Sarcoma oncogene (Kras), developmental transcriptional programs, and the tissue microenvironment PDAC initiation. Our data demonstrate that Sox4 is necessary for the specification of cellular states in preinvasive cancer lesions and regulates the characteristics and cellular compositions of the tumor microenvironment in an autochthonous genetic model of PDAC. The pancreas has a remarkable ability to regenerate and recover from acute pancreatitis. In this process, acinar cells repress the expression of genes associated with acinar function and transiently activate a gene program that resembles pancreas progenitors of development. This mechanism, defined as cellular plasticity, alleviates tissue damage, stimulates acinar proliferation, and is necessary for pancreas regeneration. However, it makes the acinar cells competent to malignant transformation mediated by Kras. To investigate SOX4 function in pancreas regeneration and cancer, we used the KCacinar mouse model (Ptf1a-CreER; Kras G12DLSL). After injury, we observed a transient four-fold increase in the expression of SOX4, which correlates with the morphological and molecular evidence of cellular plasticity. KCacinar mice rapidly develop mucinous pancreatic intraepithelial neoplasias (PanINs) after caerulein-induced pancreatitis. Histological analysis KCAcinar Sox4-depleted pancreas (KCSAcinar) reveals a distinct cystic lesion lined by cuboidal or flattened epithelium with large irregular and hyperchromatic nuclei and absence of mucin-producing cells. Mice lacking Sox4 showed a significant reduction in the number of tuft cells. Furthermore, we observed an extensive reduction of the tumor stroma surrounding epithelial acinar-derived lesions in KCSAcinar compared to KC littermates. Next, we perform gene expression analysis of lesions 21 days after the induction of pancreatitis. Principal component analysis clusters the samples according to the genotype. Differential gene expression validated our histopathological analysis and showed a significant reduction in the expression of mucins and tuft cell markers. Cumulative, our data suggest that Sox4 is necessary for the specification of cellular states in the precursor lesions of PDAC, and that the cellular state of the tumor cell of origin determines the characteristics and cellular composition of the tumor microenvironment. Our work will continue to unravel the function of Sox4 in cancer initiation and the interaction of signaling pathways activated in pancreas regeneration with Kras mutations. Citation Format: Jonathan Baldan, Juan Camacho Roda, Charlotte Vestrup Rift, Jane Preuss Hasselby, Patrick Jacquemin, Ilse Rooman, Véronique Lefebvre, Luis Arnes. Sox4-dependent acinar cell plasticity in pancreatic regeneration and cancer initiation [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B032.

Abstract Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations. Significance: This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.

Maintenance of the pancreatic acinar cell phenotype suppresses tumor formation. Hence, repetitive acute or chronic pancreatitis, stress conditions in which the acinar cells dedifferentiate, predispose for cancer formation in the pancreas. Dedifferentiated acinar cells acquire a large panel of duct cell-specific markers. However, it remains unclear to what extent dedifferentiated acini differ from native duct cells and which genes are uniquely regulating acinar cell dedifferentiation. Moreover, most studies have been performed on mice since the availability of human cells is scarce. Here, we applied a non-genetic lineage tracing method of human pancreatic exocrine acinar and duct cells that allowed cell-type-specific gene expression profiling by RNA sequencing. Subsequent to this discovery analysis, one transcription factor that was unique for dedifferentiated acinar cells was functionally characterized. RNA sequencing analysis showed that human dedifferentiated acinar cells expressed genes in “Pathways of cancer” with a prominence of MECOM (EVI-1), a transcription factor that was not expressed by duct cells. During mouse embryonic development, pre-acinar cells also transiently expressed MECOM and in the adult mouse pancreas, MECOM was re-expressed when mice were subjected to acute and chronic pancreatitis, conditions in which acinar cells dedifferentiate. In human cells and in mice, MECOM expression correlated with and was directly regulated by SOX9. Mouse acinar cells that, by genetic manipulation, lose the ability to upregulate MECOM showed impaired cell adhesion, more prominent acinar cell death, and suppressed acinar cell dedifferentiation by limited ERK signaling. In conclusion, we transcriptionally profiled the two major human pancreatic exocrine cell types, acinar and duct cells, during experimental stress conditions. We provide insights that in dedifferentiated acinar cells, cancer pathways are upregulated in which MECOM is a critical regulator that suppresses acinar cell death by permitting cellular dedifferentiation.

Acute pancreatitis is a transient and local inflammation of the pancreas characterized by immune cell infiltration, fibrosis, and edema.1Habtezion A. et al.Gastroenterology. 2019; 156: 1941-1950Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar It mainly affects acinar cells, causing acinar metaplasia, and thereby constitutes a favorable environment for the development of pancreatic cancer in human beings and mouse models.2Guerra C. et al.Cancer Cell. 2007; 11: 291-302Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar Despite the significant involvement of redox-dependent mechanisms in pancreatitis (eg, mitogen-activated protein kinase signaling, autophagy, disulfide stress, calcium signaling), supplementation with generic antioxidants is therapeutically unsuccessful,3Perez S. et al.Redox Biol. 2015; 5: 1-14Crossref PubMed Scopus (62) Google Scholar highlighting the need to identify specific targets amenable to pharmacologic therapy. To identify redox targets relevant to pancreatitis, we first compared the transcriptional landscape of Fluorescence-activated cell sorting (FACS)-sorted acinar cells from control and cerulein-treated mice (cerulein is a pancreatitis-inducing compound). We identified an increased expression of activators of the peroxiredoxin pathway such as peroxiredoxin-1 (Prdx1), sulfiredoxin (Srxn1), and thioredoxin (Txn1) (Supplementary Figure 1A). Among the typical 2-cystein family members, mouse and human peroxiredoxin-1 protein (PRX-I), -II, -III, and -IV, only the expression of PRX-I was selectively induced in metaplastic acinar cells, at advanced stages of acute pancreatitis (Figure 1A and Supplementary Figure 1B-G). Accordingly, in primary human acinar cells cultured under conditions that mimic pancreatitis-induced metaplasia, we found substantially higher levels of PRX-I in metaplastic cells (days 3–4) compared with normal acini (day 0) (Figure 1B and Supplementary Figure 1E and F). PRX-I has been shown to interact with inflammatory factors, such as nuclear factor κB (NF-κB) and macrophage migration inhibitory factor, suggesting its involvement in the pathophysiology of pancreatitis.4Bertoldi M. Protein Pept Lett. 2016; 23: 69-77Crossref PubMed Scopus (15) Google Scholar To investigate the role of PRX-I in pancreatitis, we genetically ablated its expression using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) (Supplementary Figure 2A–D). This constitutive inactivation recapitulates the clinical context in which a drug administrated to patients would inhibit its target in all cell types. Although a previous report showed that long-term constitutive PRX-I deletion causes anemia and a shortened lifespan,5Neumann C.A. et al.Nature. 2003; 424: 561-565Crossref PubMed Scopus (612) Google Scholar we did not observe any pancreas-specific anomaly in Prdx1-/- mice (age, 3 mo). Prdx1-/- mice were born at the expected Mendelian frequency, showed normal postnatal development, and were fertile. Next, we analyzed the histology of pancreata from Prdx1+/+, Prdx1+/-, and Prdx1-/- mice treated with cerulein in early and late acute settings (Supplementary Figure 2E and F). At early acute pancreatitis time points, Prdx1+/+ and Prdx1+/- pancreata (considered together as controls [Ctrl]) showed a slight increase in PRX-I expression (Supplementary Figure 3A and B). The extent of edema and immune infiltration observed in Ctrl pancreata was not affected in Prdx1-/- mice; the low PRX-I expression, at early pancreatitis, probably explains the minimal effects observed after its genetic ablation (Supplementary Figure 3C and D). Interestingly, at late acute pancreatitis, PRX-I expression was strongly increased in metaplastic acini (Supplementary Figure 3E and F). At this time point, pancreata from Prdx1-/- mice showed a well-preserved architecture with a significantly 2-fold higher number of normal acini and a 3-fold reduction in metaplastic area compared with Ctrl (Figure 1C and D and Supplementary Figure 4A and B). CD45-positive immune cell infiltration and collagen deposit both were decreased significantly by 2-fold in Prdx1-/- compared with Ctrl mice (Figure 1C and D). PRX-I usually is described as an antioxidant enzyme with high catalytic efficiency.6Ogusucu R. et al.Free Radic Biol Med. 2007; 42: 326-334Crossref PubMed Scopus (153) Google Scholar Interestingly, the content of protein carbonyls and 4-hydroxynonenal (4-HNE)-protein adducts was comparable in pancreata from Ctrl and Prdx1-/- mice (Figure 1E). This suggested that the antioxidant function of PRX-I is not playing a predominant role in pancreatitis, which prompted us to search for additional roles of PRX-I. Previous reports have shown that PRX-I can be secreted from cultured cells in response to inflammatory stimuli and can bind to Toll-like receptor 4 to activate NF-κB–mediated production of proinflammatory cytokines.7Mullen L. et al.Mol Med. 2015; 21: 98-108Crossref PubMed Scopus (68) Google Scholar, 8Riddell J.R. et al.J Immunol. 2010; 184: 1022-1030Crossref PubMed Scopus (161) Google Scholar, 9Liu D.L. et al.Int Immunopharmacol. 2016; 41: 82-89Crossref PubMed Scopus (48) Google Scholar Accordingly, we detected PRX-I in the culture medium of primary mouse acinar cells undergoing metaplasia, highlighting their ability to release PRX-I (Figure 2A). Strikingly, primary mouse acinar cells treated with recombinant PRX-I protein released significantly more proinflammatory cytokines interleukin 6 and tumor necrosis factor-α compared with untreated cells (Figure 2B). In line with this result, Prdx1-/- pancreata showed a reduced expression of interleukin 6 and tumor necrosis factor-α, in the interstitial space between acinar cells, compared with their Ctrl counterparts (Figure 2C and D). Similarly, the expression and nuclear translocation of signal transducer and activator of transcription 3 and NF-κB (subunit p65), 2 transcriptional factors controlling the expression of proinflammatory cytokines, were decreased by 2- to 3-fold in Prdx1-/- pancreata (Figure 2C and D and Supplementary Figure 4A). Thus, our findings show that a mechanism linking the secretion of PRX-I to the production of proinflammatory cytokines may operate in vivo. In summary, we discovered that the ablation of PRX-I reduces the severity of inflammation and related acinar-to-ductal metaplasia (Supplementary Figure 4C). Our results support PRX-I as a potential therapeutic target to reduce pancreatic inflammation and related damage. The authors thank Mourad El Kaddouri, Jean-Nicolas Lodewyckx, Freddy Abrassart, and Nicolas Dauguet for technical help. Transcript profiling: GSE163254. Download .pdf (.1 MB) Help with pdf files Supplementary Data 1 Download .pdf (.81 MB) Help with pdf files Supplementary Data 2

Human pancreatic exocrine cells were cultured in 3D suspension and formed pancreatospheres composed of acinar-derived and duct-like cells. We investigated, up to 6 days, the fate of human pancreatic acinar cells using fluorescein-conjugated Ulex Europaeus Agglutinin 1 lectin, a previously published acinar-specific non-genetic lineage tracing strategy. At day 4, fluorescence-activated cell sort for the intracellularly incorporated FITC-conjugated UEA1 lectin and the duct-specific CA1 9.9 surface marker, distinguished acinar-derived cells ( UEA1 + CA1 9.9 − ) from duct-like cells ( UEA1 − CA1 9.9 + ) and acinar-to-duct-like transdifferentiated cells ( UEA1 + CA1 9 . 9 + ). mRNA expression analysis of the acinar-derived ( UEA1 + CA19 . 9 − ) and duct-like ( UEA1 - CA19 . 9 + ) cell fractions with concomitant immunocytochemical analysis of the pancreatospheres revealed acquisition of an embryonic signature in the UEA1 + CA19 . 9 − acinar-derived cells characterized by de novo expression of SOX9 and CD142 , robust expression of PDX1 and surface expression of GP2 . The colocalisation of CD142, a multipotent pancreatic progenitor surface marker, PDX1, SOX9 and GP2 is reminiscent of a cellular state present during human embryonic development. Addition of TGF-beta signalling inhibitor Alk5iII, induced a 28-fold increased KI67 -labeling in pancreatospheres, more pronounced in the CD142 + GP2 + acinar-derived cells. These findings with human cells underscore the remarkable plasticity of pancreatic exocrine acinar cells, previously described in rodents, and could find applications in the field of regenerative medicine.

The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into β-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of β-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-β-cell transdifferentiation.