scholarly journals Organotypic Culture of Acinar Cells for the Study of Pancreatic Cancer Initiation

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2606
Author(s):  
Carlotta Paoli ◽  
Alessandro Carrer
Keyword(s):  
Pancreatic Cancer ◽  
Ex Vivo ◽  
Acinar Cells ◽  
Lineage Tracing ◽  
Metabolic Reprogramming ◽  
Pancreatic Acinar Cells ◽  
Ductal Adenocarcinoma ◽  
Molecular Features ◽  
The Impact

The carcinogenesis of pancreatic ductal adenocarcinoma (PDA) progresses according to multi-step evolution, whereby the disease acquires increasingly aggressive pathological features. On the other hand, disease inception is poorly investigated. Decoding the cascade of events that leads to oncogenic transformation is crucial to design strategies for early diagnosis as well as to tackle tumor onset. Lineage-tracing experiments demonstrated that pancreatic cancerous lesions originate from acinar cells, a highly specialized cell type in the pancreatic epithelium. Primary acinar cells can survive in vitro as organoid-like 3D spheroids, which can transdifferentiate into cells with a clear ductal morphology in response to different cell- and non-cell-autonomous stimuli. This event, termed acinar-to-ductal metaplasia, recapitulates the histological and molecular features of disease initiation. Here, we will discuss the isolation and culture of primary pancreatic acinar cells, providing a historical and technical perspective. The impact of pancreatic cancer research will also be debated. In particular, we will dissect the roles of transcriptional, epigenetic, and metabolic reprogramming for tumor initiation and we will show how that can be modeled using ex vivo acinar cell cultures. Finally, mechanisms of PDA initiation described using organotypical cultures will be reviewed.

scholarly journals Organotypic Culture of Acinar Cells for the Study of Pancreatic Cancer Initiation

2020 ◽  
Author(s):  
Alessandro Carrer ◽  
Carlotta Paoli
Keyword(s):  
Pancreatic Cancer ◽  
Ex Vivo ◽  
Acinar Cells ◽  
Lineage Tracing ◽  
Pancreatic Acinar Cells ◽  
Design Strategies ◽  
Tumor Onset ◽  
Molecular Features ◽  
Primary Mouse

Multi-step evolution characterizes the carcinogenesis of GI malignancies, where the disease progresses through the accrual of genetic mutations, co-option of multiple host cellular factors and the acquisition of increasingly aggressive pathological features. Ensuing cancer complexity and plasticity are challenges for therapeutic approaches. On the other hand, disease inception is often triggered by a defined oncogenic event, which leads to escalating effects on gene expression, cellular metabolism, chromatin organization and ultimately cell identity. Decoding this molecular cascade of events is crucial to design strategies for early diagnosis as well as to tackle tumor onset. To this aim, the in vitro culture of non-transformed epithelial cells represents a major technical challenge. Genetic studies, combined with lineage-tracing experiments demonstrated that pancreatic cancerous lesions originate from acinar cells, a specialized cell type in the pancreatic epithelium, following mutations of the KRAS oncogene. Ex vivo culture of pancreatic acinar cells has allowed the identification of early mutant KRAS-driven alterations. Primary acinar cells survive in vitro in organoid-like 3D spheroids, which can recapitulate histological and molecular features of disease initiation. Here, we will discuss the isolation and culture of primary mouse pancreatic acinar cells, providing and historical and technical perspective. Impact for pancreatic cancer research will also be debated; in particular we will dissect the role of KRAS signaling in driving oncogenic transformation and highlight differences with canonical 2D culture of established cancer cell lines.

scholarly journals Legumain is activated in macrophages during pancreatitis

2016 ◽  
Vol 311 (3) ◽  
pp. G548-G560 ◽  
Author(s):  
Laura E. Edgington-Mitchell ◽  
Thomas Wartmann ◽  
Alicia K. Fleming ◽  
Vasilena Gocheva ◽  
Wouter A. van der Linden ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Ex Vivo ◽  
Inflammatory Diseases ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Dependent Manner ◽  
Molecule Inhibitor ◽  
Acinar To Ductal Metaplasia ◽  
Lysosomal Cysteine Protease ◽  
Ex Vivo Imaging

Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68+ macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.

scholarly journals Nardilysin inhibits pancreatitis and suppresses pancreatic ductal adenocarcinoma initiation in mice

Gut ◽  
2018 ◽  
Vol 68 (5) ◽  
pp. 882-892 ◽  
Author(s):  
Kozo Ikuta ◽  
Akihisa Fukuda ◽  
Satoshi Ogawa ◽  
Kenji Masuo ◽  
Norihiro Goto ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Acinar Cells ◽  
Global Gene Expression ◽  
Pancreatic Acinar Cells ◽  
Ductal Adenocarcinoma ◽  
Ectodomain Shedding ◽  
Pancreatic Development ◽  
Transcriptional Coregulator ◽  
The Impact ◽  
Intraepithelial Lesions

ObjectiveNardilysin (NRDC), a zinc peptidase, exhibits multiple localisation-dependent functions including as an enhancer of ectodomain shedding in the extracellular space and a transcriptional coregulator in the nucleus. In this study, we investigated its functional role in exocrine pancreatic development, homeostasis and the formation of pancreatic ductal adenocarcinoma (PDA).DesignWe analysed Ptf1a-Cre; Nrdcflox/flox mice to investigate the impact of Nrdc deletion. Pancreatic acinar cells were isolated from Nrdcflox/flox mice and infected with adenovirus expressing Cre recombinase to examine the impact of Nrdc inactivation. Global gene expression in Nrdc-cKO pancreas was analysed compared with wild-type pancreas by microarray analysis. We also analysed Ptf1a-Cre; KrasG12D; Nrdcflox/flox mice to investigate the impact of Nrdc deletion in the context of oncogenic Kras. A total of 51 human samples of pancreatic intraepithelial lesions (PanIN) and PDA were examined by immunohistochemistry for NRDC.ResultsWe found that pancreatic deletion of Nrdc leads to spontaneous chronic pancreatitis concomitant with acinar-to-ductal conversion, increased apoptosis and atrophic pancreas in mice. Acinar-to-ductal conversion was observed mainly through a non-cell autonomous mechanism, and the expression of several chemokines was significantly increased in Nrdc-null pancreatic acinar cells. Furthermore, pancreatic deletion of Nrdc dramatically accelerated KrasG12D-driven PanIN and subsequent PDA formation in mice. These data demonstrate a previously unappreciated anti-inflammatory and tumour suppressive functions of Nrdc in the pancreas in mice. Finally, absence of NRDC expression was observed in a subset of human PanIN and PDA.ConclusionNrdc inhibits pancreatitis and suppresses PDA initiation in mice.

Generation of insulin-secreting cells from pancreatic acinar cells of animal models of type 1 diabetes

2007 ◽  
Vol 292 (1) ◽  
pp. E158-E165 ◽  
Author(s):  
Masaaki Okuno ◽  
Kohtaro Minami ◽  
Akinori Okumachi ◽  
Kazumasa Miyawaki ◽  
Norihide Yokoi ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Animal Models ◽  
Cell Lineage ◽  
Acinar Cells ◽  
Lineage Tracing ◽  
Pancreatic Acinar Cells ◽  
Diabetic State ◽  
Insulin Secreting Cells

We recently found that pancreatic acinar cells isolated from normal adult mouse can transdifferentiate into insulin-secreting cells in vitro. Using two different animal models of type 1 diabetes, we show here that insulin-secreting cells can also be generated from pancreatic acinar cells of rodents in the diabetic state with absolute insulin deficiency. When pancreatic acinar cells of streptozotocin-treated mice were cultured in suspension in the presence of epidermal growth factor and nicotinamide under low-serum condition, expressions of insulin genes gradually increased. In addition, expressions of other pancreatic hormones, including glucagon, somatostatin, and pancreatic polypeptide, were also induced. Analysis by the Cre/loxP-based direct cell lineage tracing system revealed that these newly made cells originated from amylase-expressing pancreatic acinar cells. Insulin secretion from the newly made cells was significantly stimulated by high glucose and other secretagogues. In addition, insulin-secreting cells were generated from pancreatic acinar cells of Komeda diabetes-prone rats, another animal model of type 1 diabetes. The present study demonstrates that insulin-secreting cells can be generated by transdifferentiation from pancreatic acinar cells of rodents in the diabetic state and further suggests that pancreatic acinar cells represent a potential source of autologous transplantable insulin-secreting cells for treatment of type 1 diabetes.

scholarly journals Occult polyclonality of preclinical pancreatic cancer models drives in vitro evolution.

2021 ◽  
Author(s):  
Maria E Monberg ◽  
Heather Geiger ◽  
Roshan Sharma ◽  
Jaewon J Lee ◽  
Alexander Semaan ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
Ex Vivo ◽  
Growth Models ◽  
Ductal Adenocarcinoma ◽  
Pdac Cell ◽  
Sequencing Technologies ◽  
The Impact

Intratumoral heterogeneity (ITH) is a hallmark of cancer. The advent of single-cell technologies has helped uncover ITH in a high-throughput manner in different cancers across varied contexts. Here we apply single-cell sequencing technologies to reveal striking ITH in assumptively oligoclonal pancreatic ductal adenocarcinoma (PDAC) cell lines. Our findings reveal a high degree of both genomic and transcriptomic heterogeneity in established and globally utilized PDAC cell lines, custodial variation induced by growing apparently identical PDAC cell lines in different laboratories, and profound transcriptomic shifts in transitioning from 2D to 3D spheroid growth models. Our findings also call into question the validity of widely available immortalized, non-transformed pancreatic lines as contemporaneous control lines in experiments. Further, while patient-derived organoid (PDOs) are known to reflect the cognate in vivo biology of the parental tumor, we identify transcriptomic shifts during ex vivo passage that might hamper their predictive abilities over time. The impact of these findings on rigor and reproducibility of experimental data generated using established preclinical PDAC models between and across laboratories is uncertain, but a matter of concern.

scholarly journals Raloxifene inhibits pancreatic adenocarcinoma growth by interfering with ERβ and IL-6/gp130/STAT3 signaling

2020 ◽  
Author(s):  
Ioannis Pozios ◽  
Nina N. Seel ◽  
Nina A. Hering ◽  
Lisa Hartmann ◽  
Verena Liu ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Tumor Growth ◽  
Therapeutic Option ◽  
Tumor Xenograft ◽  
Ductal Adenocarcinoma ◽  
Stat3 Signaling ◽  
Stat3 Phosphorylation ◽  
The Impact

Abstract Purpose Currently, the exact role of estrogen receptor (ER) signaling in pancreatic cancer is unknown. Recently, we showed that expression of phosphorylated ERβ correlates with a poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). Here, we hypothesized that raloxifene, a FDA-approved selective ER modulator (SERM), may suppress PDAC tumor growth by interfering with ERβ signaling. To test this hypothesis, we studied the impact of raloxifene on interleukin-6/glycoprotein-130/signal transducer and activator of transcription-3 (IL-6/gp130/STAT3) signaling. Methods Human PDAC cell lines were exposed to raloxifene after which growth inhibition was assessed using a BrdU assay. ER knockdown was performed using siRNAs specific for ERα and ERβ. The effects of raloxifene on IL-6 expression and STAT3 phosphorylation in PDAC cells were assessed by ELISA and Western blotting, respectively. In addition, raloxifene was administered to an orthotopic PDAC tumor xenograft mouse model, after which tumor growth was monitored and immunohistochemistry was performed. Results Raloxifene inhibited the in vitro growth of PDAC cells, and this effect was reversed by siRNA-mediated knockdown of ERβ, but not of ERα, indicating ER isotype-specific signaling. We also found that treatment with raloxifene inhibited the release of IL-6 and suppressed the phosphorylation of STAT3Y705 in PDAC cells. In vivo, we found that orthotopic PDAC tumor growth, lymph node and liver metastases as well as Ki-67 expression were reduced in mice treated with raloxifene. Conclusions Inhibition of ERβ and the IL-6/gp130/STAT3 signaling pathway by raloxifene leads to potent reduction of PDAC growth in vitro and in vivo. Our results suggest that ERβ signaling and IL-6/gp130 interaction may serve as promising drug targets for pancreatic cancer and that raloxifene may serve as an attractive therapeutic option for PDAC patients expressing the ERβ isotype.

scholarly journals Acinar phenotype is preserved in human exocrine pancreas cells cultured at low temperature: implications for lineage-tracing of β-cell neogenesis

2016 ◽  
Vol 36 (3) ◽  
Author(s):  
Josué K. Mfopou ◽  
Isabelle Houbracken ◽  
Elke Wauters ◽  
Iris Mathijs ◽  
Imane Song ◽  
...  
Keyword(s):  
Low Temperature ◽  
Acinar Cells ◽  
Lineage Tracing ◽  
Pancreatic Acinar Cells ◽  
Cell Labelling ◽  
Β Cell ◽  
Adenovirus Vectors ◽  
Temperature Exposure ◽  
Low Temperature Exposure

In vitro cultured pancreatic acinar cells rapidly differentiate. Low temperature exposure prevents this process and improves the efficiency of acinar cell labelling with adenovirus vectors. This may help in tracing β-cell neogenesis from human pancreatic acinar cells.

scholarly journals Cholesterol Activates Cyclic AMP Signaling in Metaplastic Acinar Cells

Metabolites ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 141
Author(s):  
Francesca Grisan ◽  
Martina Spacci ◽  
Carlotta Paoli ◽  
Andrea Costamagna ◽  
Marco Fantuz ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Ex Vivo ◽  
Cholesterol Biosynthesis ◽  
Spatial Dynamics ◽  
Acinar Cells ◽  
Adenosine Monophosphate ◽  
Pancreatic Acinar Cells ◽  
Pancreatic Carcinogenesis ◽  
Transformed Cell Lines ◽  
The Impact

Cholesterol is a non-essential metabolite that exerts both structural and signaling functions. However, cholesterol biosynthesis is elevated, and actively supports, pancreatic carcinogenesis. Our previous work showed that statins block the reprogramming of mutant KRAS-expressing acinar cells, that spontaneously undergo a metaplastic event termed acinar-to-ductal metaplasia (ADM) to initiate carcinogenesis. Here we tested the impact of cholesterol supplementation on isolated primary wild-type acinar cells and observed enhanced ductal transdifferentiation, associated with generation of the second messenger cyclic adenosine monophosphate (cAMP) and the induction of downstream protein kinase A (PKA). Inhibition of PKA suppresses cholesterol-induced ADM ex vivo. Live imaging using fluorescent biosensors dissected the temporal and spatial dynamics of PKA activation upon cholesterol addition and showed uneven activation both in the cytosol and on the outer mitochondrial membrane of primary pancreatic acinar cells. The ability of cholesterol to activate cAMP signaling is lost in tumor cells. Qualitative examination of multiple normal and transformed cell lines supports the notion that the cAMP/PKA axis plays different roles during multi-step pancreatic carcinogenesis. Collectively, our findings describe the impact of cholesterol availability on the cyclic AMP/PKA axis and plasticity of pancreatic acinar cells.

Effects of oncogenic K-ras on pancreatic acinar cells In vitro

2001 ◽  
Vol 120 (5) ◽  
pp. A722-A722
Author(s):  
Y BI ◽  
C LOGSDON
Keyword(s):  
Acinar Cells ◽  
Pancreatic Acinar Cells

TNF-α Suppresses Autophagic Flux in Acinar Cells in IgG4-Related Sialadenitis

2019 ◽  
Vol 98 (12) ◽  
pp. 1386-1396 ◽  
Author(s):  
X. Hong ◽  
S.N. Min ◽  
Y.Y. Zhang ◽  
Y.T. Lin ◽  
F. Wang ◽  
...  
Keyword(s):  
Acinar Cell ◽  
Cell Injury ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Autophagic Flux ◽  
C6 Cells ◽  
Tnf Α ◽  
Α Level

IgG4-related sialadenitis (IgG4-RS) is a newly recognized immune-mediated systemic fibroinflammatory disease that affects salivary glands and leads to hyposalivation. Tumor necrosis factor–α (TNF-α) is a critical proinflammatory cytokine involved in several salivary gland disorders, but its role and mechanism regarding acinar cell injury in IgG4-RS are unknown. Here, we found that TNF-α level was significantly increased in serum and submandibular gland (SMG) of patients and that serum TNF-α level was negatively correlated with saliva flow rate. Ultrastructural observations of IgG4-RS SMGs revealed accumulation of large autophagic vacuoles, as well as dense fibrous bundles, decreased secretory granules, widened intercellular spaces, swollen mitochondria, and expanded endoplasmic reticulum. Expression levels of LC3 and p62 were both increased in patients’ SMGs. TNF-α treatment led to elevated levels of LC3II and p62 in both SMG-C6 cells and cultured human SMG tissues but did not further increase their levels when combined with bafilomycin A1 treatment. Moreover, transfection of Ad-mCherry-GFP-LC3B in SMG-C6 cells confirmed the suppression of autophagic flux after TNF-α treatment. Immunofluorescence imaging revealed that costaining of LC3 and the lysosomal marker LAMP2 was significantly decreased in patients, TNF-α–treated SMG-C6 cells, and cultured human SMGs, indicating a reduction in autophagosome-lysosome fusion. Furthermore, the ratio of pro/mature cathepsin D was elevated in vivo, ex vivo, and in vitro. TNF-α also appeared to induce abnormal acidification of lysosomes in acinar cells, as assessed by lysosomal pH and LysoTracker DND-26 fluorescence intensity. In addition, TNF-α treatment induced transcription factor EB (TFEB) redistribution in SMG-C6 cells, which was consistent with the changes observed in IgG4-RS patients. TNF-α increased the phosphorylation of extracellular signal–regulated kinase (ERK) 1/2, and inhibition of ERK1/2 by U0126 reversed TNF-α–induced TFEB redistribution, lysosomal dysfunction, and autophagic flux suppression. These findings suggest that TNF-α is a key cytokine related to acinar cell injury in IgG4-RS through ERK1/2-mediated autophagic flux suppression.

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