These results advance our understanding of Mek/Erk-driven mTOR activation and its downstream targets in PDAC, and provide a mechanistic rationale for effective therapeutic matching for Aldh1a3-positive PDACs.

Objective The initial steps of pancreatic regeneration versus carcinogenesis are insufficiently understood. Although a combination of oncogenic Kras and inflammation has been shown to induce malignancy, molecular networks of early carcinogenesis remain poorly defined. Design We compared early events during inflammation, regeneration and carcinogenesis on histological and transcriptional levels with a high temporal resolution using a well-established mouse model of pancreatitis and of inflammation-accelerated Kras G12D -driven pancreatic ductal adenocarcinoma. Quantitative expression data were analysed and extensively modelled in silico. Results We defined three distinctive phases-termed inflammation, regeneration and refinement-following induction of moderate acute pancreatitis in wild-type mice. These corresponded to different waves of proliferation of mesenchymal, progenitor-like and acinar cells. Pancreas regeneration required a coordinated transition of proliferation between progenitor-like and acinar cells. In mice harbouring an oncogenic Kras mutation and challenged with pancreatitis, there was an extended inflammatory phase and a parallel, continuous proliferation of mesenchymal, progenitor-like and acinar cells. Analysis of high-resolution transcriptional data from wild-type animals revealed that organ regeneration relied on a complex interaction of a gene network that normally governs acinar cell homeostasis, exocrine specification and intercellular signalling. In mice with oncogenic Kras, a specific carcinogenic signature was found, which was preserved in full-blown mouse pancreas cancer. Conclusions These data define a transcriptional signature of early pancreatic carcinogenesis and a molecular network driving formation of preneoplastic lesions, which allows for more targeted biomarker development in order to detect cancer earlier in patients with pancreatitis.

Background & AimsAlthough nearly half of pancreatic ductal adenocarcinoma (PDAC) patients have diabetes mellitus with episodes of hyperglycemia, its tumor microenvironment is hypoglycemic. Thus, it is crucial for PDAC cells to develop adaptive mechanisms dealing with oscillating glucose levels. So far, the biological impact of such glycemic variability on PDAC biology remains unknown.MethodsMurine PDAC cells were cultured in low- and high-glucose medium to investigate the molecular, biochemical, and metabolic influence of glycemic variability on tumor behavior. A set of in vivo functional assays including orthotopic implantation and portal and tail vein injection were used. Results were further confirmed on tissues from PDAC patients.ResultsGlycemic variability has no significant effect on PDAC cell proliferation. Hypoglycemia is associated with local invasion and angiogenesis, whereas hyperglycemia promotes metastatic colonization. Increased metastatic colonization under hyperglycemia is due to increased expression of runt related transcription factor 3 (Runx3), which further activates expression of collagen, type VI, alpha 1 (Col6a1), forming a glycemic pro-metastatic pathway. Through epigenetic machinery, retinoic acid receptor beta (Rarb) expression fluctuates according to glycemic variability, acting as a critical sensor relaying the glycemic signal to Runx3/Col6a1. Moreover, the signal axis of Rarb/Runx3/Col6a1 is pharmaceutically accessible to a widely used antidiabetic substance, metformin, and Rar modulator. Finally, PDAC tissues from patients with diabetes show an increased expression of COL6A1.ConclusionsGlycemic variability promotes both local invasion and metastatic colonization of PDAC. A pro-metastatic signal axis Rarb/Runx3/Col6a1 whose activity is controlled by glycemic variability is identified. The therapeutic relevance of this pathway needs to be explored in PDAC patients, especially in those with diabetes.

Measurement of cellular tracer uptake is widely applied to learn the physiologic status of cells and their interactions with imaging agents and pharmaceuticals. In-culture measurements have the advantage of less stress to cells. However, the tracer solution still needs to be loaded, unloaded, and purged from the cell culture during the measurements. Here, we propose a continuously infused microfluidic radioassay (CIMR) system for continuous in-culture measurement of cellular uptake. The system was tested to investigate the influence of the glucose concentration in cell culture media on 18 F-FDG uptake kinetics. Methods: The CIMR system consists of a microfluidic chip integrated with a flow-control unit and a positron camera. Medium diluted with radioactive tracer flows through a cell chamber continuously at low speed. Positrons emitted from the cells and from tracer in the medium are measured with the positron camera. The human cell lines SkBr3 and Capan-1 were incubated with media of 3 different glucose concentrations and then measured with 18 F-FDG on the CIMR system. In addition, a conventional uptake experiment was performed. The relative uptake ratios between different medium conditions were compared. A cellular 2-compartment model was applied to estimate the cellular pharmacokinetics on CIMR data. The estimated pharmacokinetic parameters were compared with expressions of glucose transporter-1 (GLUT1) and hexokinase-2 measured by quantitative real-time polymerase chain reaction. Results: The relative uptake ratios obtained from CIMR measurements correlated significantly with those from the conventional uptake experiments. The relative SDs of the relative uptake ratios obtained from the CIMR uptake experiments were significantly lower than those from the conventional uptake experiments. The fit of the cellular 2-compartment model to the 18 F-FDG CIMR measurements was of high quality. For SkBr3, the estimated pharmacokinetic parameters k 1 and k 3 were consistent with the messenger RNA expression of GLUT1 and hexokinase-2: culturing with low glucose concentrations led to higher GLUT1 and hexokinase-2 expression as well as higher estimated k 1 and k 3 . For Capan-1, the estimated k 1 and k 3 increased as the glucose concentration in the culture medium decreased, and this finding did not match the corresponding messenger RNA expression. Conclusion: The CIMR system captures dynamic uptake within the cell culture and enables estimation of the cellular pharmacokinetics. Thei nvestigation of molecular uptake inside cells is valuable for the understanding of physiologic status (1,2), for the interpretation of molecular imaging results (3,4), and for the development of imaging agents and pharmaceuticals (5). The cellular uptake of a molecule is usually assessed by measuring labeling signals such as radioactivity (2,6), optical signal (7), or magnetic signal (8). Radiometric measurement of cellular uptake is a typical experiment in nuclear medicine (6) and provides high sensitivity for a very small mass of tracer. The...

Background Pancreatic adenocarcinoma (PDAC) is one of the most fatal malignancies. Surgical resection supplemented by chemotherapy remains the major therapeutic regimen, but with unavoidable resistance and systemic toxic reaction. Curcumin is a known safe natural compound that can effectively eliminate pancreatic adenocarcinoma cells in vitro, making it a promising candidate for substitution in subsequent chemotherapy. However, due to its extremely low bioavailability caused by its insolubility and circular elimination, curcumin had an unexpectedly modest therapeutic effect in clinical trials. Results Here, we electrospun curcumin/gelatin-blended nanofibrous mat to largely improve curcumin’s bioavailability by local controlled-release. With characterization by scanning electron microscopy, fluorescence microscopy, Fourier transform infrared spectroscopy, X-ray diffraction and high-performance liquid chromatography, it was revealed that curcumin was uniformly dispersed in the fiber of the mats with nanoscopic dimensions and could be continuously released into the surrounding medium for days. The cancer inhibitory effects of nano-curcumin and underlying mechanisms were further explored by assays using pancreatic adenocarcinoma cell and experiments using xenograft model. The results showed the released nano-curcumin could effectively inhibit pancreatic adenocarcinoma cell proliferation not only in vitro, but more importantly in vivo. This cytotoxic effect of nano-curcumin against pancreatic adenocarcinoma was achieved through provoking the production of intracellular reactive oxygen species and activating endoplasmic reticulum stress, which leads to enhanced cell apoptosis via decreased phosphorylation of signal transducer and activator of transcription 3. Conclusions Clinically, curcumin/gelatin-blended nanofibrous mat could be a promising, secure, efficient and affordable substitutional agent for the elimination of residual cancer cells after tumor resection. Moreover, our strategy to obtain curcumin released from nanofibrous mat may provide a universally applicable approach for the study of the therapeutic effects and molecular mechanisms of other potential medicines with low bioavailability.

Hypoxia-inducible factor 1α (Hif1α) is a key regulator of cellular adaptation and survival under hypoxic conditions. In pancreatic ductal adenocarcinoma (PDAC), it has been recently shown that genetic ablation of Hif1α accelerates tumour development by promoting tumour-supportive inflammation in mice, questioning its role as the key downstream target of many oncogenic signals of PDAC. Likely, Hif1α has a context-dependent role in pancreatic tumorigenesis. To further analyse this, murine PDAC cell lines with reduced Hif1α expression were generated using shRNA transfection. Cells were transplanted into wild-type mice through orthotopic or portal vein injection in order to test the in vivo function of Hif1α in two major tumour-associated biological scenarios: primary tumour growth and remote colonization/metastasis. Although Hif1α protects PDAC cells from stress-induced cell deaths in both scenarios—in line with the general function Hif1α—its depletion leads to different oncogenic consequences. Hif1α depletion results in rapid tumour growth with marked hypoxia-induced cell death, which potentially leads to a persistent tumour-sustaining inflammatory response. However, it simultaneously reduces tumour colonization and hepatic metastases by increasing the susceptibility to anoikis induced by anchorage-independent conditions. Taken together, the role of Hif1α in pancreatic tumorigenesis is context-dependent. Clinical trials of Hif1α inhibitors need to take this into account, targeting the appropriate scenario, for example palliative vs adjuvant therapy.

Pancreatitis is a significant risk factor for pancreatic ductal adenocarcinoma (PDAC). Previous studies in mice have demonstrated that pancreatitis contributes to oncogenic Kras‐driven carcinogenesis, probably initiated in acinar cells; however, oncogenic Kras alone or in combination with caerulein‐induced pancreatitis is not sufficient in initiating PDAC from the ductal compartment. We thus introduced ductal obstruction – which induces a more severe form of pancreatitis – by pancreatic ductal ligation in mice harbouring oncogenic Kras. This induced a particular phenotype with highly proliferative nonmucinous cells with nuclear atypia. Around these lesions, there was a significant proliferation of activated fibroblasts and infiltration of immune cells, corroborating the pathological features of preneoplastic lesions. Lineage‐tracing experiments revealed that these preneoplastic cells derived from two distinctive cellular sources: acinar and ductal cells. Phenotypic characterisation revealed that the duct‐derived preneoplastic lesions show a high proliferative potential with persistent activation of tumour‐promoting inflammatory pathways while the acinar‐derived ones were less proliferative with persistent p53 activation. Furthermore, the duct‐derived preneoplastic cells have a particularly high nuclear‐to‐cytoplasmic ratio. These data demonstrate that ductal obstruction promotes preneoplastic lesion formation from the pancreatic ductal compartment.

Background Transpathology highlights the interpretation of the underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between trans-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation. Methods The proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on an athymic nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, tumour models of the human colorectal adenocarcinoma cell line HT-29 were imaged. The tissue within the window chamber was sectioned, fixed and haematoxylin–eosin (HE) stained for comparison with multimodal in vivo imaging. Results The final MIMI system had a maximum field of view (FOV) of 18 mm × 18 mm. Using the fiducial markers and the tubing phantom, the co-registration errors are 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, vasculatures and metabolism of the in vivo tumour microenvironment, which was consistent with ex vivo pathology. Conclusions The established multimodal intravital imaging system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.