Isolation of Quiescent Human Pancreatic Stellate Cells: A Promising in vitro Tool for Studies of Human Pancreatic Stellate Cell Biology

doi:10.1159/000260900

Pancreatology

Volume 10, Issue 4, October 2010, Pages 434-443

https://doi.org/10.1159/000260900 Get rights and content

Abstract

Background: Pancreatic stellate cells (PSCs) play a critical role in pancreatic fibrosis. To date, human PSC biology has been studied using cancer- or inflammation-associated (preactivated) PSCs, but an in vitro model of quiescent normal human PSCs (NhPSCs) has been lacking. Aims: To (i) isolate and characterize quiescent NhPSCs, and (ii) evaluate the response of culture-activated NhPSCs to cytokines and LPS. Methods: Quiescent NhPSCs were isolated from normal pancreatic tissue using density gradient centrifugation. PSC markers, glial fibrillary acidic protein (GFAP), desmin, α-smooth muscle actin (αSMA) and the lipopolysaccharide (LPS) receptors TLR4 and CD14 were identified by immunoblotting and immunocytochemistry. The effect of plateletderived growth factor (PDGF), transforming growth factor β (TGFβ) and LPS on NhPSC activation was also assessed. Results: Freshly isolated NhPSCs displayed a polygonal appearance with refringent cytoplasmic lipid droplets. Culture-activated NhPSCs expressed GFAP, desmin, αSMA, TLR4 and CD14, and were responsive to PDGF, TGFβ and LPS. Conclusion: Isolated NhPSCs expressed the same markers as rat PSCs and human cancer-associated PSCs and responded to PDGF and TGFβ similarly to rat PSCs. NhPSC preparations provide a useful in vitro tool to study the biology of PSCs in their physiological, non-activated state.

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A major barrier to successful pancreatic cancer (PC) treatment is the surrounding stroma, which secretes growth factors/cytokines that promote PC progression. Wnt and tenascin C (TnC) are key ligands secreted by stromal pancreatic stellate cells (PSCs) that then act on PC cells in a paracrine manner to activate the oncogenic β-catenin and YAP/TAZ signaling pathways. Therefore, therapies targeting oncogenic Wnt/TnC cross talk between PC cells and PSCs constitute a promising new therapeutic approach for PC treatment. The metastasis suppressor N-myc downstream-regulated gene-1 (NDRG1) inhibits tumor progression and metastasis in numerous cancers, including PC. We demonstrate herein that targeting NDRG1 using the clinically trialed anticancer agent di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) inhibited Wnt/TnC-mediated interactions between PC cells and the surrounding PSCs. Mechanistically, NDRG1 and DpC markedly inhibit secretion of Wnt3a and TnC by PSCs, while also attenuating Wnt/β-catenin and YAP/TAZ activation and downstream signaling in PC cells. This antioncogenic activity was mediated by direct inhibition of β-catenin and YAP/TAZ nuclear localization and by increasing the Wnt inhibitor, DKK1. Expression of NDRG1 also inhibited transforming growth factor (TGF)-β secretion by PC cells, a key mechanism by which PC cells activate PSCs. Using an in vivo orthotopic PC mouse model, we show DpC downregulated β-catenin, TnC, and YAP/TAZ, while potently increasing NDRG1 expression in PC tumors. We conclude that NDRG1 and DpC inhibit Wnt/TnC-mediated interactions between PC cells and PSCs. These results further illuminate the antioncogenic mechanism of NDRG1 and the potential of targeting this metastasis suppressor to overcome the oncogenic effects of the PC-PSC interaction.
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Citation Excerpt :
Inducible PKN2 (iPKN2) knockout (KO) MEFs were isolated from Rosa26CreERT2PKN2fl/fl C57BL/6 mice as previously described (Quetier et al., 2016). Inducible PKN2KO mouse PSCs were isolated from the pancreas of a male Rosa26CreERT2PKN2fl/fl mouse using a variation of the density centrifugation method (Apte et al., 1998; Bachem et al., 1998; Vonlaufen et al., 2010). Briefly, the pancreas was digested using Collagenase P, 0.1% DNase1 in GBSS for 30 mins at 37°C.
In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2^KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.
Pharmacologic Normalization of Pancreatic Cancer-Associated Fibroblast Secretome Impairs Prometastatic Cross-Talk With Macrophages
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KPC cells were derived from PDA tumor tissue obtained from LSL-KrasG12D/+; LSL-Trp53R172H/+; p48-Cre+/- mice (C57BL/6 background, a generous gift from Professor D. Saur, Technische University of Munich). PSCs were isolated from mouse pancreata, either WT or KOsst1 (a generous gift from Dr C. Viollet, INSERM U1266, Institute of Psychiatry and Neuroscience of Paris)20 (C57BL/6J background), as described.43 In brief, pancreata were minced and digested for 30 minutes at 37°C in a dissociation buffer containing 0.05% collagenase P (Sigma-Aldrich, Lyon, France), 0.1% DNase I (Sigma-Aldrich), and 0.02% Pronase (Sigma-Aldrich) in Grey’s balanced salt solution (GBSS; Sigma-Aldrich).
Cancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein–coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors.
Large-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database.
Proteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms.
We propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies.
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Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma. Cancer-associated fibroblasts (CAFs) have been shown to display a high degree of interconvertible states including quiescent, inflammatory, and myofibroblastic phenotypes; however, the mechanisms by which this plasticity is achieved are poorly understood. Here, we aim to elucidate the role of CAF plasticity and its impact on PDAC biology.
To investigate the role of mesenchymal plasticity in PDAC progression, we generated a PDAC mouse model in which CAF plasticity is modulated by genetic depletion of the transcription factor Prrx1. Primary pancreatic fibroblasts from this mouse model were further characterized by functional in vitro assays. To characterize the impact of CAFs on tumor differentiation and response to chemotherapy, various coculture experiments were performed. In vivo, tumors were characterized by morphology, extracellular matrix composition, and tumor dissemination and metastasis.
Our in vivo findings showed that Prrx1-deficient CAFs remain constitutively activated. Importantly, this CAF phenotype determines tumor differentiation and disrupts systemic tumor dissemination. Mechanistically, coculture experiments of tumor organoids and CAFs showed that CAFs shape the epithelial-to-mesenchymal phenotype and confer gemcitabine resistance of PDAC cells induced by CAF-derived hepatocyte growth factor. Furthermore, gene expression analysis showed that patients with pancreatic cancer with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC.
Here, we define that the Prrx1 transcription factor is critical for tuning CAF activation, allowing a dynamic switch between a dormant and an activated state. This work shows that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.
A modified in vitro tool for isolation and characterization of rat quiescent islet stellate cells
2019, Experimental Cell Research
Citation Excerpt :
However, these methods have two main limitations: 1) pure qISCs cannot be obtained; 2) the characteristics of the isolated ISCs may be affected by islet-secreted hormones. Studies about quiescent stellate cells appear stagnant because of many shortcomings in the current PSC and ISC isolation methods, limiting the yield and viability of quiescent stellate cells [18–20]. Therefore, the successful isolation of primary cells remains essential for the study of ISCs.
Islet stellate cells (ISCs) play a critical role in islet fibrosis, contributing to the progression of pancreatic diseases. Previous studies have focused on fibrosis-associated activated ISCs obtained by standard islet explant techniques. However, in vitro models of quiescent ISCs (qISCs) are lacking. This study aims to develop a method to isolate qISCs and analyze their phenotype during activation.
Immunofluorescence staining was applied to localize ISCs in normal human, rat, and mouse islets. qISCs were isolated from rat islets using density gradient centrifugation (DGC) method. qRT-PCR, immunoblotting, proliferation, and migration assays were employed for their characterization.
Desmin-positive ISCs were detected in normal human, rat, and mouse islets. Freshly isolated qISCs, obtained by density gradient centrifugation, displayed a polygonal appearance with refringent cytoplasmic lipid droplets and expressed transcriptional markers indicating a low activation/quiescent state. With increasing culture time, the marker expression pattern changed, reflecting ISC activation. qISCs contained more lipid droplets and exhibited lower proliferation and migration abilities compared to spindle-shaped ISCs obtained by traditional explant techniques.
This study describes a new method for efficient isolation of qISCs from rat islets, representing a useful in vitro tool to study the biology of ISCs in more physiological conditions.
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¹: Prof. Minoti V. Apte South Western Sydney Clinical School, Faculty of Medicine University of New South Wales, Room 505, Level 5, Wallace Wurth Building Sydney, N.S.W. 2052 (Australia) Tel. +61 2 9385 8273, Fax +61 2 9385 1389

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Isolation of Quiescent Human Pancreatic Stellate Cells: A Promising in vitro Tool for Studies of Human Pancreatic Stellate Cell Biology

Abstract

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Biochem Pharmacol

Am J Pathol

Neoplasia

Periacinar stellate shaped cells in rat pancreas — identification, isolation, and culture

Gut

Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis

Gut