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Background: Pancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells., Method: A spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and characterized by electron microscopy, immunohistochemistry and real-time RT-PCR. In order to facilitate the cell type-specific crosstalk analysis by real-time RT-PCR, we developed a novel in vitro 3D co-culture model, where the participating cell types were from different species, human and mouse, respectively. Using species-specific PCR primers, we were able to investigate the crosstalk between stromal and cancer cells without previous cell separation and sorting., Results: We found clear evidence for mutual influence, such as increased proliferation and a shift towards a more mesenchymal phenotype in cancer cells and an activation of pancreatic stellate cells towards the myofibroblast phenotype. Using a heterospecies approach, which we coined virtual sorting, confirmed the findings we made initially in the human-human spheroids., Conclusions: We developed and characterized different easy to set up 3D models to investigate the crosstalk between cancer and stroma cells for pancreatic cancer.

Background: To date, there still is a lack of specific acute pancreatitis markers and specifically an early marker that can reliably predict disease severity. The inflammatory response in acute pancreatitis is mediated in part through oxidative stress and calcineurin-NFAT (Nuclear Factor of Activated T-cells) signaling, which is inducing its own negative regulator, regulator of calcineurin 1 (RCAN1). Caerulein induction is a commonly used in vivo model of experimental acute pancreatitis. Caerulein induces CN-NFAT signaling, reactive oxygen species and inflammation., Methods: To screen for potential markers of acute pancreatitis, we used the caerulein model of experimental acute pancreatitis (AP) in C57Bl/6 J mice. Pancreata from treated and control mice were used for expression profiling. Promising gene candidates were validated in cell culture experiments using primary murine acinar cells and rat AR42J cells. These candidates were then further tested for their usefulness as biomarkers in mouse and human plasma., Results: We identified a number of novel genes, including Regulator of calcineurin 1 (Rcan1) and Sestrin 2 (Sesn2) and demonstrated that they are induced by oxidative stress, by stimulation with H 2 O 2 and by inhibiting caerulein stimulated expression with the antioxidant N-acetylcysteine. We found Rcan1 protein to be significantly elevated in AP-induced mouse plasma as well as in plasma from AP patients., Conclusion: We demonstrated that Rcan1 is regulated by oxidative stress and identified RCAN1 as a potential diagnostic marker of AP., (Copyright © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Cancer stem cells (CSCs) play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC). A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME) on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM) proteins on PDAC CSCs. We quantified the effect of ECM proteins, β1-integrin, and focal adhesion kinase (FAK) on clonogenic PDAC growth and migration in vitro and tumor initiation, growth, and metastasis in vivo in nude mice using shRNA and overexpression constructs as well as small molecule FAK inhibitors. Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth in vitro and in vivo. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.

Background: The HMGA2 protein has experimentally been linked to EMT and cancer stemness. Recent studies imply that tumour-stroma interactions regulate these features and thereby contribute to tumour aggressiveness., Methods: We analysed 253 cases of pancreatic ductal adenocarcinoma (PDAC) and 155 cases of ampullary adenocarcinoma (AAC) for HMGA2 expression by IHC. The data were correlated with stroma abundance and supplemented by experimental studies., Results: HMGA2 acts as an independent prognostic marker associated with a significantly shorter overall survival in both tumour types. Overall, HMGA2-positivity was more frequent in patients with PDAC than with AAC. The HMGA2 status in tumour cells significantly correlated with the abundance of PDGFRβ-defined stroma cells. In vivo co-injection of Panc-1 cancer cells with pancreatic stellate cells increased tumour growth in a manner associated with increased HMGA2 expression. Furthermore, in vitro treatment of Panc-1 with conditioned media from PDGF-BB-activated stellate cells increased their ability to form tumour spheroids., Conclusions: This study identifies HMGA2 expression in tumour cells as an independent prognostic marker in PDAC and AAC. Correlative data analysis gives novel tissue-based evidence for a heterotypic cross-talk with stroma cells as a possible mechanism for HMGA2 induction, which is further supported by experimental models.

Purpose: In preclinical cancer studies, non-invasive functional imaging has become an important tool to assess tumor development and therapeutic effects. Tumor hypoxia is closely associated with tumor aggressiveness and is therefore a key parameter to be monitored. Recently, photoacoustic (PA) imaging with inherently co-registered high-frequency ultrasound (US) has reached preclinical applicability, allowing parallel collection of anatomical and functional information. Dual-wavelength PA imaging can be used to quantify tissue oxygen saturation based on the absorbance spectrum differences between hemoglobin and deoxyhemoglobin., Experimental Design: A new bi-modal PA/US system for small animal imaging was employed to test feasibility and reliability of dual-wavelength PA for measuring relative tissue oxygenation. Murine models of pancreatic and colon cancer were imaged, and differences in tissue oxygenation were compared to immunohistochemistry for hypoxia in the corresponding tissue regions., Results: Functional studies proved feasibility and reliability of oxygenation detection in murine tissue in vivo. Tumor models exhibited different levels of hypoxia in localized regions, which positively correlated with immunohistochemical staining for hypoxia. Contrast-enhanced imaging yielded complementary information on tissue perfusion using the same system., Conclusion: Bimodal PA/US imaging can be utilized to reliably detect hypoxic tumor regions in murine tumor models, thus providing the possibility to collect anatomical and functional information on tumor growth and treatment response live in longitudinal preclinical studies.