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Objective: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models., Design: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D ; Trp53 LSL-R172H ; Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D ; Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice ( Loxl2 Exon2 fl/fl ) or conditional Loxl2 overexpressing mice (R26 Loxl2 KI/KI ) to generate KPCL2 KO or KCL2 KO and KPCL2 KI or KCL2 KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation., Results: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment., Conclusion: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The literature on liver cysts is highly conflicting, mostly owing to definitional variations. Two hundred and fifty-eight ≥1 cm cysts evaluated pathologically using updated criteria were classifiable as: I. Ductal plate malformation related (63%); that is, cystic bile duct hamartoma or not otherwise specified-type benign biliary cyst (35 with polycystic liver disease). These were female predominant (F/M=2.4), large (10 cm), often multifocal with degenerative/inflammatory changes and frequently misclassified as "hepatobiliary cystadenoma." II. Neoplastic (13%); 27 (10.5%) had ovarian-type stroma (OTS) and qualified as mucinous cystic neoplasm (MCN) per World Health Organization (WHO). These were female, solitary, mean age 52, mean size 11 cm, and 2 were associated with carcinoma (1 in situ and 1 microinvasive). There were 3 intraductal papillary neoplasms, 1 intraductal oncocytic papillary neoplasm, 1 cystic cholangiocarcinoma, and 2 cystic metastasis. III. Infectious/inflammatory (12%). These included 23 hydatid cysts (including 2 Echinococcus alveolaris both misdiagnosed preoperatively as cancer), nonspecific inflammatory cysts (abscesses, inflammatory cysts: 3.4%). IV. Congenital (7%). Mostly small (<3 cm); choledochal cyst (5%), foregut cyst (2%). V. Miscellaneous (4%). In conclusion, hepatic cysts occur predominantly in women (3/1), are mostly (90%) non-neoplastic, and seldom (<2%) malignant. Cystic bile duct hamartomas and their relative not otherwise specified-type benign biliary cysts are frequently multifocal and often misdiagnosed as "cystadenoma/carcinoma." Defined by OTS, MCNs (the true "hepatobiliary cystadenoma/carcinoma") are solitary, constitute only 10.5% of hepatic cysts, and have a significantly different profile than the impression in the literature in that essentially all are perimenopausal females, and rarely associated with carcinoma (7%). Since MCNs can only be diagnosed by demonstration of OTS through complete microscopic examination, it is advisable to avoid the term "cystadenoma/cystadenocarcinoma" solely based on radiologic examination, and the following simplified terminology would be preferable in preoperative evaluation to avoid conflicts with the final pathologic diagnosis: (1) noncomplex (favor benign), (2) complex (in 3 subsets, as favor benign, cannot rule out malignancy, or favor malignancy), (3) malignant features., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Pancreatic stellate cells (PSCs) constitute important cells of the pancreatic microenvironment and their close interaction with cancer cells is important in pancreatic cancer. It is currently not known whether PSCs accumulate genetic alterations that contribute to tumor biology. Our aim was to analyze genetic alterations in cancer associated PSCs. PSC DNA was matched to DNA isolated from pancreatic cancer patients' blood (n = 5) and analyzed by Next-Generation Sequencing (NGS). Bioinformatic analysis was performed using the GATK software and pathogenicity prediction scores. Sanger sequencing was carried out to verify specific genetic alterations in a larger panel of PSCs (n = 50). NGS and GATK analysis identified on average 26 single nucleotide variants in PSC DNA as compared to the matched blood DNA that could be visualized with the Integrative Genomics Viewer. The absence of PDAC driver mutations (KRAS, p53, p16/INK4a, SMAD4) confirmed that PSC isolations were not contaminated with cancer cells. After filtering the variants, using different pathogenicity scores, ten genes were identified (SERPINB2, CNTNAP4, DENND4B, DPP4, FGFBP2, MIGA2, POLE, SNRNP40, TOP2B, and ZDHHC18) in single samples and confirmed by Sanger sequencing. As a proof of concept, functional analysis using control and SERPINB2 knock-out fibroblasts revealed functional effects on growth, migration, and collagen contraction. In conclusion, PSC DNA exhibit a substantial amount of single nucleotide variants that might have functional effects potentially contributing to tumor aggressiveness., (© 2022. The Author(s).)

The advancing edge profile is a powerful determinant of tumor behavior in many organs. In this study, a grading system assessing the tumor-host interface was developed and tested in 181 pancreatic neuroendocrine tumors (PanNETs), 63 of which were <=2 cm. Three tumor slides representative of the spectrum (least, medium, and most) of invasiveness at the advancing edge of the tumor were selected, and then each slide was scored as follows. Well-demarcated/encapsulated, 1 point; Mildly irregular borders and/or minimal infiltration into adjacent tissue, 2 points; Infiltrative edges with several clusters beyond the main tumor but still relatively close, and/or satellite demarcated nodules, 3 points; No demarcation, several cellular clusters away from the tumor, 4 points; Exuberantly infiltrative pattern, scirrhous growth, dissecting the normal parenchymal elements, 5 points. The sum of the rankings on the three slides was obtained. Cases with scores of 3-6 were defined as "non/minimally infiltrative" (NI; n = 77), 7-9 as "moderately infiltrative" (MI; n = 68), and 10-15 as "highly infiltrative" (HI; n = 36). In addition to showing a statistically significant correlation with all the established signs of aggressiveness (grade, size, T-stage), this grading system was found to be the most significant predictor of adverse outcomes (metastasis, progression, and death) on multivariate analysis, more strongly than T-stage, while Ki-67 index did not stand the multivariate test. As importantly, cases <=2 cm were also stratified by this grading system rendering it applicable also to this group that is currently placed in "watchful waiting" protocols. In conclusion, the proposed grading system has a strong, independent prognostic value and therefore should be considered for integration into routine pathology practice after being evaluated in validation studies with larger series., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Patient-derived in vivo models of human cancer have become a reality, yet their turnaround time is inadequate for clinical applications. Therefore, tailored ex vivo models that faithfully recapitulate in vivo tumour biology are urgently needed. These may especially benefit the management of pancreatic ductal adenocarcinoma (PDAC), where therapy failure has been ascribed to its high cancer stem cell (CSC) content and high density of stromal cells and extracellular matrix (ECM). To date, these features are only partially reproduced ex vivo using organoid and sphere cultures. We have now developed a more comprehensive and highly tuneable ex vivo model of PDAC based on the 3D co-assembly of peptide amphiphiles (PAs) with custom ECM components (PA-ECM). These cultures maintain patient-specific transcriptional profiles and exhibit CSC functionality, including strong in vivo tumourigenicity. User-defined modification of the system enables control over niche-dependent phenotypes such as epithelial-to-mesenchymal transition and matrix deposition. Indeed, proteomic analysis of these cultures reveals improved matrisome recapitulation compared to organoids. Most importantly, patient-specific in vivo drug responses are better reproduced in self-assembled cultures than in other models. These findings support the use of tuneable self-assembling platforms in cancer research and pave the way for future precision medicine approaches., (© 2021. The Author(s).)

Cancer-associated fibroblasts (CAF) are major contributors to pancreatic ductal adenocarcinoma (PDAC) progression through protumor signaling and the generation of fibrosis, the latter of which creates a physical barrier to drugs. CAF inhibition is thus an ideal component of any therapeutic approach for PDAC. SLC7A11 is a cystine transporter that has been identified as a potential therapeutic target in PDAC cells. However, no prior study has evaluated the role of SLC7A11 in PDAC tumor stroma and its prognostic significance. Here we show that high expression of SLC7A11 in human PDAC tumor stroma, but not tumor cells, is independently prognostic of poorer overall survival. Orthogonal approaches showed that PDAC-derived CAFs are highly dependent on SLC7A11 for cystine uptake and glutathione synthesis and that SLC7A11 inhibition significantly decreases CAF proliferation, reduces their resistance to oxidative stress, and inhibits their ability to remodel collagen and support PDAC cell growth. Importantly, specific ablation of SLC7A11 from the tumor compartment of transgenic mouse PDAC tumors did not affect tumor growth, suggesting the stroma can substantially influence PDAC tumor response to SLC7A11 inhibition. In a mouse orthotopic PDAC model utilizing human PDAC cells and CAFs, stable knockdown of SLC7A11 was required in both cell types to reduce tumor growth, metastatic spread, and intratumoral fibrosis, demonstrating the importance of targeting SLC7A11 in both compartments. Finally, treatment with a nanoparticle gene-silencing drug against SLC7A11, developed by our laboratory, reduced PDAC tumor growth, incidence of metastases, CAF activation, and fibrosis in orthotopic PDAC tumors. Overall, these findings identify an important role of SLC7A11 in PDAC-derived CAFs in supporting tumor growth. SIGNIFICANCE: This study demonstrates that SLC7A11 in PDAC stromal cells is important for the tumor-promoting activity of CAFs and validates a clinically translatable nanomedicine for therapeutic SLC7A11 inhibition in PDAC., (©2021 American Association for Cancer Research.)

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: The extent of the COVID-19 pandemic and the resulting response has varied globally. The European and African Hepato-Pancreato-Biliary Association (E-AHPBA), the premier representative body for practicing HPB surgeons in Europe and Africa, conducted this survey to assess the impact of COVID-19 on HPB surgery., Methods: An online survey was disseminated to all E-AHPBA members to assess the effects of the pandemic on unit capacity, management of HPB cancers, use of COVID-19 screening and other aspects of service delivery., Results: Overall, 145 (25%) members responded. Most units, particularly in COVID-high countries (>100,000 cases) reported insufficient critical care capacity and reduced HPB operating sessions compared to COVID-low countries. Delayed access to cancer surgery necessitated alternatives including increased neoadjuvant chemotherapy for pancreatic cancer and colorectal liver metastases, and locoregional treatments for hepatocellular carcinoma. Other aspects of service delivery including COVID-19 screening and personal protective equipment varied between units and countries., Conclusion: This study demonstrates that the COVID-19 pandemic has had a profound adverse impact on the delivery of HPB cancer care across the continents of Europe and Africa. The findings illustrate the need for safe resumption of cancer surgery in a "new" normal world with screening of patients and staff for COVID-19., (Copyright © 2020 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Cancer-associated fibroblasts (CAF) are orchestrators of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Stromal heterogeneity may explain differential pathophysiological roles of the stroma (pro- versus anti-tumoural) in PDAC. We hypothesised that multiple CAF functional subtypes exist in PDAC, that contribute to stromal heterogeneity through interactions with cancer cells. Using molecular and functional analysis of patient-derived CAF primary cultures, we demonstrated that human PDAC-derived CAFs display a high level of inter- and intra-tumour heterogeneity. We identified at least four subtypes of CAFs based on transcriptomic analysis, and propose a classification for human PDAC-derived CAFs (pCAFassigner). Multiple CAF subtypes co-existed in individual patient samples. The presence of these CAF subtypes in bulk tumours was confirmed using publicly available gene expression profiles, and immunostainings of CAF subtype markers. Each subtype displayed specific phenotypic features (matrix- and immune-related signatures, vimentin and α-smooth muscle actin expression, proliferation rate), and was associated with an assessable prognostic impact. A prolonged exposure of non-tumoural pancreatic stellate cells to conditioned media from cancer cell lines (cancer education experiment) induced a CAF-like phenotype, including loss of capacity to revert to quiescence and an increase in the expression of genes related to CAF subtypes B and C. This classification demonstrates molecular and functional inter- and intra-tumoural heterogeneity of CAFs in human PDAC. Our subtypes overlap with those identified from single-cell analyses in other cancers, and pave the way for the development of therapies targeting specific CAF subpopulations in PDAC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

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., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with an overall 5-year survival rate of less than 7%. After many years of basic and clinical research efforts, pancreatic cancer patients presenting with locally advanced, unresectable tumors remain a therapeutic challenge. Despite the lack of high quality randomized controlled trials, perioperative/neoadjuvant treatment strategies seem to be beneficial in these patients. At present the FOLFIRINOX regimen, which was established in the palliative setting, is increasingly recognized as the backbone of neoadjuvant therapy for locally advanced PDAC. Surgical resection follows the same principles and guidelines as upfront surgery specifically regarding the extent of resection including lymphadenectomy, vascular resections and multivisceral resections. Because of the limited diagnostic accuracy of restaging after neoadjuvant treatment, an adjusted intraoperative strategy is necessary to minimize the risk of debulking procedures and maximize the chance of a potential curative resection. Locally advanced PDAC requires a multidisciplinary and individualized treatment approach, and further research efforts for novel and innovative therapies. This article provides an updated overview on strategies to improve the outcome in locally advanced PDAC.

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) metastasizes to liver at early stages, making this disease highly lethal. Tissue inhibitor of metalloproteinases-1 (TIMP1) creates a metastasis-susceptible environment in the liver. We investigated the role of TIMP1 and its receptor CD63 in metastasis of early-stage pancreatic tumors using mice and human cell lines and tissue samples., Methods: We obtained liver and plasma samples from patients in Germany with chronic pancreatitis, pancreatic intra-epithelial neoplasia, or PDAC, as well as hepatic stellate cells (HSCs). We performed studies with Ptf1a + /Cre;Kras + /LSL-G12D;Trp53loxP/loxP (CPK) mice, Pdx-1 + /Cre;Kras + /LSL-G12D;Trp53 + /LSL-R172H (KPC) mice, and their respective healthy littermates as control, and Cd63 -/- mice with their wild-type littermates. KPC mice were bred with Timp1 -/- mice to produce KPCxTimp1 -/- mice. TIMP1 was overexpressed and CD63 was knocked down in mice using adenoviral vectors AdTIMP1 or AdshCD63, respectively. Hepatic susceptibility to metastases was determined after intravenous inoculation of syngeneic 9801L pancreas carcinoma cells. Pancreata and liver tissues were collected and analyzed by histology, immunohistochemical, immunoblot, enzyme-linked immunosorbent assay, and quantitative polymerase chain reaction analyses. We analyzed the effects of TIMP1 overexpression or knockdown and CD63 knockdown in transduced human primary HSCs and HSC cell lines., Results: Chronic pancreatitis, pancreatic intra-epithelial neoplasia, and PDAC tissues from patients expressed higher levels of TIMP1 protein than normal pancreas. The premalignant pancreatic lesions that developed in KPC and CPK mice expressed TIMP1 and secreted it into the circulation. In vitro and in vivo, TIMP1 activated human or mouse HSCs, which required interaction between TIMP1 and CD63 and signaling via phosphatidylinositol 3-kinase, but not TIMP1 protease inhibitor activity. This signaling pathway induced expression of endogenous TIMP1. TIMP1 knockdown in HSCs reduced their activation. Cultured TIMP1-activated human and mouse HSCs began to express stromal-derived factor-1, which induced neutrophil migration, a marker of the premetastatic niche. Mice with pancreatic intra-epithelial neoplasia-derived systemic increases in TIMP1 developed more liver metastases after injections of pancreatic cancer cells than mice without increased levels of TIMP1. This increase in formation of liver metastases from injected pancreatic cancer cells was not observed in TIMP1 or CD63 knockout mice., Conclusions: Expression of TIMP1 is increased in chronic pancreatitis, pancreatic intra-epithelial neoplasia, and PDAC tissues from patients. TIMP1 signaling via CD63 leads to activation of HSCs, which create an environment in the liver that increases its susceptibility to pancreatic tumor cells. Strategies to block TIMP1 signaling via CD63 might be developed to prevent PDAC metastasis to the liver., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a transmembrane glycoprotein that is involved in tumor progression and metastasis. In the present study, the expression and functional role of ALCAM in pancreatic cancer cells and pancreatic stellate cells (PSCs) was investigated. Tissue specimens were obtained from patients with pancreatic ductal adenocarcinoma (n=56) or chronic pancreatitis (CP; n=10), who underwent pancreatic resection, and from normal pancreatic tissue samples (n=10). Immunohistochemistry was used to analyze the localization and expression of ALCAM in pancreatic tissues. Subsequently, reverse transcription‑quantitative polymerase chain reaction and immunoblotting were applied to assess the expression of ALCAM in pancreatic cancer Panc‑1 and T3M4 cells, as well as in PSCs. An enzyme‑linked immunosorbent assay was used to measure ALCAM levels in cell culture medium stimulated by hypoxia, tumor necrosis factor (TNF)‑α and transforming growth factor‑β. Silencing of ALCAM was performed using ALCAM small interfering (si)RNA and immunocytochemistry was used to analyze the inhibition efficiency. An invasion assay and a cell interaction assay were performed to assess the invasive ability and co‑cultured adhesive potential of Panc‑1 and T3M4 cells, as well as PSCs. Histologically, ALCAM expression was generally weak or absent in pancreatic cancer cells, but was markedly upregulated in PSCs in pancreatic cancer tissues. ALCAM was highly expressed in PSCs from CP tissues and PSCs surrounding pancreatic intraepithelial neoplasias, as well as in pancreatic cancer cells. ALCAM mRNA was highly expressed in PSCs, with a low to moderate expression in T3M4 and Panc‑1 cells. Similar to the mRNA expression, immunoblotting demonstrated that ALCAM protein levels were high in PSCs and T3M4 cells, but low in Panc‑1 cells. The expression of TNF‑α increased, while hypoxia decreased the secretion of ALCAM in pancreatic cancer Panc‑1 and T3M4 cells, and also in PSCs. Silencing of ALCAM by siRNA revealed no significant alteration in the invasion of pancreatic cancer cells, however, it inhibited the invasive ability of PSCs, and decreased the interaction between Panc‑1 cells and PSCs. In conclusion, ALCAM is upregulated in PSCs of pancreatic cancer tissues, suggesting a potential role of ALCAM in regulating pancreatic cancer cell‑PSC interactions.

Objective: Oncogenic Kras-activated robust Mek/Erk signals phosphorylate to the tuberous sclerosis complex (Tsc) and deactivates mammalian target of rapamycin (mTOR) suppression in pancreatic ductal adenocarcinoma (PDAC); however, Mek and mTOR inhibitors alone have demonstrated minimal clinical antitumor activity., Design: We generated transgenic mouse models in which mTOR was hyperactivated either through the Kras/Mek/Erk cascade, by loss of Pten or through Tsc1 haploinsufficiency. Primary cancer cells were isolated from mouse tumours. Oncogenic signalling was assessed in vitro and in vivo, with and without single or multiple targeted molecule inhibition. Transcriptional profiling was used to identify biomarkers predictive of the underlying pathway alterations and of therapeutic response. Results from the preclinical models were confirmed on human material., Results: Reduction of Tsc1 function facilitated activation of Kras/Mek/Erk-mediated mTOR signalling, which promoted the development of metastatic PDACs. Single inhibition of mTOR or Mek elicited strong feedback activation of Erk or Akt, respectively. Only dual inhibition of Mek and PI3K reduced mTOR activity and effectively induced cancer cell apoptosis. Analysis of downstream targets demonstrated that oncogenic activity of the Mek/Erk/Tsc/mTOR axis relied on Aldh1a3 function. Moreover, in clinical PDAC samples, ALDH1A3 specifically labelled an aggressive subtype., Conclusions: 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., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

One of the key factors that correlates with poor survival of patients with pancreatic cancer is the extent of hypoxic areas within the tumor tissue. The adaptation of pancreatic cancer cells to limited oxygen delivery promotes the induction of an invasive and treatment-resistant phenotype, triggering metastases at an early stage of tumor development, which resist in most cases adjuvant therapies following tumor resection. In this article, the authors summarize the evidence demonstrating the significance of hypoxia in pancreatic cancer pathogenesis and discuss the possible hypoxia-induced mechanisms underlying its aggressive nature. We then conclude with promising strategies that target hypoxia-adapted pancreatic cancer cells.

Background: Pancreatic acinar cell carcinoma (ACC) is a rare tumor entity with an unfavorable prognosis. Recent whole-exome sequencing identified p53 mutations in a subset of human ACC. Activation of the mammalian target of rapamycin (mTOR) pathway is associated with various pancreatic neoplasms. We thus aimed at analyzing whether activation of mTOR with a concomitant loss of p53 may initiate ACC., Methods: We generated transgenic mouse models in which mTOR was hyperactivated through pancreas-specific, homozygous tuberous sclerosis 1 (Tsc1) deficiency, with or without deletion of p53 (Tsc1 (-/-) and Tsc1 (-/-) ; p53 (-/-) ). Activity of mTOR signaling was investigated using mouse tissues and isolated murine cell lines. Human ACC specimens were used to corroborate the findings from the transgenic mouse models., Results: Hyperactive mTOR signaling in Tsc1 (-/-) mice was not oncogenic but rather induced a near-complete loss of the pancreatic acinar compartment. Acinar cells were lost as a result of apoptosis which was associated with p53 activation. Concomitantly, ductal cells were enriched. Ablation of p53 in Tsc1-deficient mice prevented acinar cell death but promoted formation of acinar cells with severe nuclear abnormalities. One out of seven Tsc1 (-/-) ; p53 (-/-) animals developed pancreatic tumors showing a distinctive tumor morphology, reminiscent of human ACC. Hyperactive mTOR signaling was also detected in a subset of human ACC., Conclusion: Hyperactive mTOR signaling combined with loss of p53 in mice induces tumors similar to human ACC.

Objectives: The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance., Design: Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology., Results: We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-β1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R., Conclusions: Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background: FOLFIRINOX is an active but relatively toxic chemotherapeutic regimen for patients with metastatic pancreatic ductal adenocarcinoma (PDAC). The increased frequency of responding tumors shift interest to neoadjuvant approaches. We report our institutional experience with FOLFIRINOX for therapy-naïve patients with locally advanced and initially unresectable PDAC., Methods: All patients with unresectable locally advanced PDAC who underwent treatment with FOLFIRINOX at a single center between 2011 and 2014 were identified and evaluated retrospectively regarding chemotherapy response, toxicity, conversion to resectability, and survival. Resectability, response to chemotherapy, and postoperative complications were reported according to NCCN-guidelines, RECIST-criteria, and Clavien-Dindo-classification, respectively., Results: Overall, 14 patients received FOLFIRINOX as first-line therapy for locally advanced and unresectable PDAC. Fifty-seven percent of the patients had severe tumor-related comorbidities at the time of diagnosis, and in 86 %, dose reduction due to toxicity was necessary during a median of seven cycles. Nevertheless, only one patient had progressive disease during FOLFIRINOX, whereas the others experienced stable disease (n = 6) or partial remission (n = 6; no restaging in one patient). Oncological tumor resection was possible in 4 patients (29 % of all patients) with no postoperative mortality and only one grade 2 surgical complication. After a median follow-up of 10 months, 4 of the 14 patients were still in remission, 5 were alive with stable disease under ongoing systemic chemotherapy, and 5 died tumor-related., Conclusions: FOLFIRINOX is a powerful first-line regimen that leads to resectability in a substantial portion of patients with initially unresectable pancreatic cancer.

Stromal fibrosis and tissue necrosis are major histological sequelae of hypoxia. The hypoxia-to-fibrosis sequence is well-documented in pancreatic ductal adenocarcinoma (PDAC). However, hypoxic and necrotic PDAC phenotypes are insufficiently characterized. Recently, reduction of tuberous sclerosis expression in mice together with oncogenic Kras demonstrated a rapidly metastasizing phenotype with histologically eccentric necrosis, transitional hypoxia and devascularisation. We established cell lines from these tumors and transplanted them orthotopically into wild-type mice to test their abilities to recapitulate the histological features of the primary lesions. Notably, the necrotic phenotype was reproduced by only a subset of cell lines while others gave rise to dedifferentiated tumors with significantly reduced necrosis. In vitro analysis of the necrotic tumor-inducing cell lines revealed that these cells released a significant amount of vascular endothelial growth factor A (Vegfa). However, its release was not further increased under hypoxic conditions. Defective hypoxia-induced Vegfa secretion was not due to impaired Vegfa transcription or hypoxia-inducible factor 1-alpha activation, but rather a result of hypoxia-induced endoplasmic reticulum (ER) stress. We thus identified hypoxia-induced ER stress as an important pathway in PDACs with tissue necrosis and rapid metastasis.

Background: Pancreatic ductal adenocarcinoma (PDAC) patients frequently present with borderline resectable disease, which can be due to invasion of the portal/superior mesenteric vein (PV/SMV). Here, we analyzed this group of patients, with emphasis on short and long-term outcomes., Methods: 156 patients who underwent a resection for PDAC were included in the analysis and sub-stratified into a cohort of patients with PV/SMV resection (n = 54) versus those with standard surgeries (n = 102)., Results: While venous resections could be performed safely, there was a trend towards shorter median survival in the PV/SMV resection group (22.7 vs. 15.8 months, p = 0.157). These tumors were significantly larger (3.5 vs. 4.3 cm; p = 0.026) and margin-positivity was more frequent (30.4% vs. 44.4%, p = 0.046)., Conclusion: Venous resection was associated with a higher rate of margin positivity and a trend towards shorter survival. However, compared to non-surgical treatment, resection offers the best chance for long term survival.

Background: The Translational Genome Research Network in Pancreatic Cancer performed a meta-analysis of publicly available various high-throughput gene analysis panels to identify drugable targets. There, the most differentially expressed gene between normal and cancerous pancreas was Kif20a. The aim of the study was to verify this expression pattern and further characterize Kif20a in pancreatic cancer., Materials and Methods: Detailed expression analyses were carried out in pancreatic tissues and in a wide panel of pancreatic cells including ductal adenocarcinoma (PDAC) and neuroendocrine-cancer cell lines as well as immortalized human pancreatic ductal epithelial and primary stellate cells using quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and immunoblot analyses. Effects on proliferation, apoptosis, and cell cycle were assessed by MTT assays, caspase-cleavage assays, and fluorescence-activated cell sorting analysis after Kif20a silencing. Cell motility was assessed by migration and invasion assays as well as time-lapse microscopy., Results: Mean Kif20a messenger RNA expression was 18.4-fold upregulated in PDAC tissues compared with that in the normal pancreas. In line, neuroendocrine-cancer cell lines display a 1.6-fold increase and ductal adenocarcinoma cell lines a 11-fold increase of Kif20a messenger RNA (P = 0.009) in comparison with primary stellate cells. A 7.3-fold overexpression was also found in immortalized pancreatic ductal epithelial cells. Kif20a silencing with small interfering RNA molecules resulted in an inhibition of proliferation, motility, and invasion of pancreatic cancer cell lines., Conclusions: Targeting Kif20a reduces proliferation, migration, and invasion of pancreatic cancer cells. Together with its significant overexpression in PDAC, this makes it a potential target for diagnostic and interventional purposes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a cancer of the exocrine pancreas with unmet medical need and is strongly promoted by tumor-associated macrophages (TAM). The presence of TAMs is associated with poor clinical outcome, and their overall role, therefore, appears to be protumorigenic. The "don't eat me" signal CD47 on cancer cells communicates to the signal regulatory protein-α on macrophages and prevents their phagocytosis. Thus, inhibition of CD47 may offer a new opportunity to turn TAMs against PDAC cells, including cancer stem cells (CSC), as the exclusively tumorigenic population., Experimental Design: We studied in vitro and in vivo the effects of CD47 inhibition on CSCs using a large set of primary pancreatic cancer (stem) cells as well as xenografts of primary human PDAC tissue., Results: CD47 was highly expressed on CSCs, but not on other nonmalignant cells in the pancreas. Targeting CD47 efficiently enhanced phagocytosis of a representative set of primary human pancreatic cancer (stem) cells and, even more intriguingly, also directly induced their apoptosis in the absence of macrophages during long-term inhibition of CD47. In patient-derived xenograft models, CD47 targeting alone did not result in relevant slowing of tumor growth, but the addition of gemcitabine or Abraxane resulted in sustained tumor regression and prevention of disease relapse long after discontinuation of treatment., Conclusions: These data are consistent with efficient in vivo targeting of CSCs, and strongly suggest that CD47 inhibition could be a novel adjuvant treatment strategy for PDAC independent of underlying and highly variable driver mutations., (©2015 American Association for Cancer Research.)

Pancreatic cancer is a leading cause of cancer-related deaths in Western societies. This poor prognosis is due to chemotherapeutic drug resistance and metastatic spread. Evidence suggests that microtubule proteins namely, β-tubulins are dysregulated in tumor cells and are involved in regulating chemosensitivity. However, the role of β-tubulins in pancreatic cancer are unknown. We measured the expression of different β-tubulin isotypes in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Next, we used RNAi to silence βIII-tubulin expression in pancreatic cancer cells, and measured cell growth in the absence and presence of chemotherapeutic drugs. Finally, we assessed the role of βIII-tubulin in regulating tumor growth and metastases using an orthotopic pancreatic cancer mouse model. We found that βIII-tubulin is highly expressed in pancreatic adenocarcinoma tissue and pancreatic cancer cells. Further, we demonstrated that silencing βIII-tubulin expression reduced pancreatic cancer cell growth and tumorigenic potential in the absence and presence of chemotherapeutic drugs. Finally, we demonstrated that suppression of βIII-tubulin reduced tumor growth and metastases in vivo. Our novel data demonstrate that βIII-tubulin is a key player in promoting pancreatic cancer growth and survival, and silencing its expression may be a potential therapeutic strategy to increase the long-term survival of pancreatic cancer patients.

HNF1 homeobox A (HNF1A)-mediated gene expression constitutes an essential component of the secretory pathway in the exocrine pancreas. Melanoma inhibitory activity 2 (MIA2), a protein facilitating protein secretion, is an HNF1A target. Protein secretion is precisely coordinated by the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) system. Here, we demonstrate that HNFA and MIA2 are expressed in a subset of human PDAC tissues and that HNF1A induced MIA2 in vitro. We identified a common germline variant of MIA2 (c.A617G: p.I141M) associated with a secretory defect of the MIA2 protein in PDAC cells. Patients carrying MIA2(I141M) survived longer after tumor resection but the survival benefit was restricted to those patients who received adjuvant chemotherapy. The MIA2(I141M) variant was associated with high expression of ER stress/UPR genes--in particular those of the ERN1/XBP arm--in human PDAC samples. Accordingly, PDAC cell lines expressing the MIA2(I141M) variant expressed high levels of ERN1 and were more sensitive to gemcitabine. These findings define an interaction between the common MIA2(I141M) variant and the ER stress/UPR system and specify a subgroup of PDAC patients who are more likely to benefit from adjuvant chemotherapy.

Excessive matrix production by pancreatic stellate cells promotes local growth and metastasis of pancreatic ductal adenocarcinoma and provides a barrier for drug delivery. Collagen type V is a fibrillar, regulatory collagen up-regulated in the stroma of different malignant tumors. Here we show that collagen type V is expressed by pancreatic stellate cells in the stroma of pancreatic ductal adenocarcinoma and affects the malignant phenotype of various pancreatic cancer cell lines by promoting adhesion, migration and viability, also after treatment with chemotherapeutic drugs. Pharmacological and antibody-mediated inhibition of β1-integrin signaling abolishes collagen type V-induced effects on pancreatic cancer cells. Ablation of collagen type V secretion of pancreatic stellate cells by siRNA reduces invasion and proliferation of pancreatic cancer cells and tube formation of endothelial cells. Moreover, stable knock-down of collagen type V in pancreatic stellate cells reduces metastasis formation and angiogenesis in an orthotopic mouse model of ductal adenocarcinoma. In conclusion, paracrine loops involving cancer and stromal elements and mediated by collagen type V promote the malignant phenotype of pancreatic ductal adenocarcinoma and underline the relevance of epithelial-stromal interactions in the progression of this aggressive neoplasm., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Pancreatic ductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells in vitro and corresponding patient-derived pancreatic cancer tissue xenografts in vivo. Intriguingly, we found that in vitro treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished in vivo tumorigenicity and invasiveness in a large panel of pancreatic cancers. In vivo treatment in combination with gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer., (©2014 American Association for Cancer Research.)

Gemcitabine is an anticancer drug used in the treatment of different cancer types, including pancreatic ductal adenocarcinoma. The maximum tolerated dose in humans is restricted by its side effects on healty cells. Furthermore, the fibrotic stroma produced by the pancreatic stellate cells prevents effective delivery of chemotherapeutic agents providing a safe-haven for the cancer cells. This becomes more of a problem considering the short half-life of this drug. Magnetic nanoparticle-based targeted drug delivery systems are a promising alternative to overcome the limitations of classical chemotherapies. The aim of this study is to obtain an effective targeted delivery system for gemcitabine using magnetic nanoparticles (MNPs) and all-trans retinoic acid (ATRA). This dual approach targets the tumor cells and its infrastructure - stellate cells - simultaneously. Gemcitabine and ATRA were loaded onto the PAMAM dendrimer-coated magnetic nanoparticles (DcMNPs), which were synthesized and characterized previously. Drug loading and release characteristics, and stability of the nanoparticles were investigated. Gemcitabine and ATRA loaded MNPs are efficiently taken up by pancreatic cancer and stellate cells successfully targeting and eliminating both cells. Results of this study can provide new insights on pancreatic cancer therapy where tumor is seen as a system with its stroma insead of epithelial cells alone., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with an extremely poor prognosis. Inflammatory processes have emerged as key mediators of pancreatic cancer development and progression. In genetically engineered mouse models, induction of pancreatitis accelerates PDAC development, and patients with chronic pancreatitis are known to have a higher risk of developing pancreatic cancer. In recent years, much effort has been given to identify the underlying mechanisms that contribute to inflammation-induced tumorigenesis. Many inflammatory pathways have been identified and inhibitors have been developed in order to prevent cancer development and progression. In this chapter, we discuss the role of inflammatory pathways in the initiation and progression of pancreatic cancer as well as the role of inhibitors used in treatment and prevention of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a notoriously therapy-resistant desmoplastic tumour. Antifibrotic therapy is shown to increase drug delivery in the preclinical setting. However, this approach can be a double-edged sword: first, PDAC is not uniform and some types of (dormant) stroma may in fact be protective; second, conventional chemotherapeutics are not powerful enough to eradicate all cancer cells in the tumour, therefore breaking down the stromal wall non-selectively may also lead to the increased dissemination of cancer cells. Recently, Kadaba et al have analysed the impact of the stromal cells in pancreatic, oesophageal and skin cancers, in bio-engineered, physiomimetic organotypic cultures. These authors show that the maximal effect on increasing cancer cell proliferation and invasion, as well as decreasing cancer cell apoptosis, occurs when pancreatic stellate cells constitute the majority of the cellular population in a three-dimensional (3D) model. This work may be instrumental for better understanding the types of stoma in PDAC before eliminating it non-selectively., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Since conventional and targeted therapies aiming at cancer cells have largely failed to prolong survival in pancreatic cancer, targeting the infrastructure of the tumor, hence its stroma is a novel strategy. It is believed that fibrotic and hypovascular stroma forms a barrier around cancer cells, hindering effective delivery of chemotherapy. Theoretically, antifibrotic therapy should reduce the compactness of the stroma and reduce the interstitial pressure, allowing better delivery of chemotherapy. This approach has worked successfully in a genetically engineered mouse model but failed in humans, paradoxically increasing mortality in the treatment arm. Normally, stromal cells deposit extracellular matrix as an innate defensive reaction to form a barrier between what is harmful and the rest of the body. Despite the significant amount of in vitro data suggesting the pro-tumorigenic roles of activated stellate cells, there is no reason to believe that stellate cells around genetically mutated cells are from the beginning there to support carcinogenesis. Such a stromal activation is also observed around PanIN lesions (which harbor genetically mutated cells) in chronic pancreatitis, where no cancer develops. In pancreatic cancer, the selection pressure created by the fibrotic and hypoxic stroma eventually leads to the evolution of more aggressive clones, indirectly contributing to the aggressiveness of the tumor. Here, the main problem is the late diagnosis of pancreatic cancer, which gives cancer cells enough time for malignant evolution. Therefore, applying antifibrotic therapy at a late stage can be counterproductive. It may increase delivery of chemotherapy, but also lead to the escape of cancer cells., (Copyright © 2013 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is one of the five most lethal malignancies worldwide and survival has not improved substantially in the past 30 years. Desmoplasia (abundant fibrotic stroma) is a typical feature of PDAC in humans, and stromal activation commonly starts around precancerous lesions. It is becoming clear that this stromal tissue is not a bystander in disease progression. Cancer-stroma interactions effect tumorigenesis, angiogenesis, therapy resistance and possibly the metastatic spread of tumour cells. Therefore, targeting the tumour stroma, in combination with chemotherapy, is a promising new option for the treatment of PDAC. In this Review, we focus on four issues. First, how can stromal activity be used to detect early steps of pancreatic carcinogenesis? Second, what is the effect of perpetual pancreatic stellate cell activity on angiogenesis and tissue perfusion? Third, what are the (experimental) antifibrotic therapy options in PDAC? Fourth, what lessons can be learned from Langton's Ant (a simple mathematical model) regarding the unpredictability of genetically engineered mouse models?

Background: We have identified syndecan-2 as a protein potentially involved in perineural invasion of pancreatic adenocarcinoma (PDAC) cells., Methods: Syndecan-2 (SDC-2) expression was analyzed in human normal pancreas, chronic pancreatitis and PDAC tissues. Functional in vitro assays were carried out to determine its role in invasion, migration and signaling., Results: SDC-2 was expressed in the majority of the tested pancreatic cancer cell lines while it was upregulated in nerve-invasive PDAC cell clones. There were 2 distinct expression patterns of SDC-2 in PDAC tissue samples: SDC-2 positivity in the cancer cell cytoplasm and a peritumoral expression. Though SDC-2 silencing (using specific siRNA oligonucleotides) did not affect anchorage-dependent growth, it significantly reduced cell motility and invasiveness in the pancreatic cancer cell lines T3M4 and Su8686. On the transcriptional level, migration-and invasion-associated genes were down-regulated following SDC-2 RNAi. Furthermore, SDC-2 silencing reduced K-ras activity, phosphorylation of Src and--further downstream--phosphorylation of ERK2 while levels of the putative SDC-2 signal transducer p120GAP remained unaltered., Conclusion: SDC-2 is a novel (perineural) invasion-associated gene in PDAC which cooperates with K-ras to induce a more invasive phenotype.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer entity with a high proliferative potential. Uncontrolled cell proliferation is mediated by a number of core signaling pathways. Recently, novel data of PDAC biology suggest that these core signal pathways affect cell proliferation and metabolism simultaneously., Methods: Here, we reviewed the literature on core metabolic signaling pathways in pancreatic carcinogenesis., Results: Results obtained from mouse genetics and in vitro experiments have demonstrated the significance of the Kras, p53, c-Myc, and Lkb1 networks in the proliferation of pancreatic epithelial and cancer cells. At the same time, these major pathways also affect energy metabolism by influencing glucose and glutamine utilization. In particular, Kras-mediated metabolic changes seem to be directly involved in carcinogenesis. However, there is a lack of solid evidence on how metabolism and proliferation are connected in pancreatic carcinogenesis., Conclusion: Understanding early and subtle changes in cellular metabolism of pancreatic epithelial-and specifically of acinar-cells, which accompany or directly influence malignant transformation and uncontrolled proliferation, will be paramount to define novel imaging and other modalities for earlier detection of PDAC.

Glutamate has been implicated in tumorigenesis through activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR). However, the function of a glutamate-to-AMPAR signal in pancreatic ductal adenocarcinoma (PDAC) has remained elusive. We now show that glutamate-mediated AMPA receptor activation increases invasion and migration of pancreatic cancer cells via activation of the classical MAPK pathway. Glutamate levels were increased in pancreatic cancer accompanied by downregulation of GluR subunits 1, 2, and 4. In pancreatic cancer precursor lesions, pancreatic intraepithelial neoplasia (PanIN), GluR1 subunit levels were strikingly and step-wise increased but its expression was rare in PDAC. Pharmacological inhibition or RNAi-mediated suppression of GluR1 or GluR2 did not affect cancer cell growth but significantly decreased invasion. In a K-ras wildtype cell line, AMPA receptor activation enhanced K-ras activity and--further downstream--phosphorylation of p38 and of p44/42. Preemptive blockade of AMPA receptors in a mouse model of pancreatic cancer inhibited tumor cell settling. AMPA receptor activation thus not only activates MAPK signalling but also directly increases activity of K-ras. Glutamate might serve as a molecular switch that decreases the threshold of K-ras-induced oncogenic signalling and increases the chance of malignant transformation of pancreatic cancer precursor lesions., (Copyright © 2011 UICC.)

Objectives: Pigment epithelium-derived factor (PEDF) is a noninhibitory member of the serine protease inhibitor gene family with neuroprotective, neuroproliferative, and anti-angiogenic functions. Its role in pancreatic fibrosis and neuropathy is unknown., Methods: The expression and localization of PEDF were assessed by quantitative real-time (RT)-PCR, immunohistochemistry, and quantitative image analysis and correlated with neural and microvessel densities (MVDs) in the normal pancreas (n=20) and pancreatic cancer (n=55). Primary human pancreatic stellate cells (PSCs), mouse neuroblastoma, and human Schwann cells were used for functional experiments. The effect of hypoxia on PEDF production in cancer cell lines and immortalized pancreatic ductal epithelial cells was assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay. The effect of recombinant PEDF on PSCs was assessed by immunoblot analysis., Results: PEDF expression was homogeneous in epithelial cells of the normal pancreas where some acinar cells consistently displayed stronger staining. A higher expression was found in tubular complexes, PanIN lesions, and inflammatory cells in pancreatic cancer. Cancer cells expressed various levels of PEDF. In cancer cell lines and in human immortalized pancreatic ductal epithelial cells, hypoxia increased PEDF mRNA up to 132-fold. Higher expression of PEDF in cancer cells was significantly correlated with better patient survival (median survival 21.5 months vs. 17.5 months, P=0.043), increased neuropathy (P=0.0251), increased PSC activity, and extracellular matrix protein production., Conclusions: PEDF increases PSC activity, thereby contributing to the desmoplasia of pancreatic cancer. PSC overactivation likely leads to periacinar fibrosis and degeneration of fine acinar innervation. Increased focal PEDF expression in cancer cells correlates with neuropathic changes and better patient survival.

Background: Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells., Methods: Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition., Results: Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt., Conclusion: TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.

Aims: The cyclic adenosine monophosphate-inducible basic helix-loop-helix (bHLH) domain containing class-B2 transcriptional factor BHLHB2 is differentially expressed in a number of human malignancies. In the present study, the expression, regulation, functions and prognostic impact of BHLHB2 in pancreatic cancer were investigated., Methods: Expression analyses were carried out in tissues of the normal pancreas (n=10) and pancreatic ductal adenocarcinoma (n=77) as well as in eight pancreatic cancer cell lines using quantitative RT-PCR, semiquantitative immunohistochemistry, and immunoblot analyses. In vitro functional experiments were conducted using siRNA transfection, hypoxia, serum starvation, apoptosis induction with gemcitabine and actinomycin-D, and invasion assays. Survival analysis was performed using the Kaplan-Meier method. Prognostic factors were determined in a multivariable analysis using a Cox proportional hazards model., Results: BHLHB2 mRNA and protein expressions were strongly induced by hypoxia and by serum starvation in pancreatic cancer cell lines. BHLHB2 silencing with RNAi had no significant effects on growth and invasion but increased apoptosis resistance against gemcitabine by reducing caspace-3 cleavage. In BHLHB2 silenced cells the ED50 of gemcitabine increased from 13.95 ± 1.353 to 38.70 ± 5.262 nM (p<0.05). Ex vivo, the weak/absent nuclear staining in normal pancreatic ducts and acinar cells was replaced by moderate to strong nuclear/cytoplasmic staining in PanIN lesions and pancreatic cancer cells. Patients with weak/absent nuclear BHLHB2 staining had significantly worse median survival compared to those with strong staining (13 months vs. 27 months, p=0.03). In a multivariable analysis, BHLHB2 staining was an independent prognostic factor (Hazard-Ratio=2.348, 95% CI=1.250-4.411, p=0.008)., Conclusions: Hypoxia-inducible BHLHB2 expression is a novel independent prognostic marker in pancreatic cancer patients and indicates increased chemosensitivity towards gemcitabine., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: To further characterize the neurotrophic attributes of pancreatic cancer (PCa)., Summary Background Data: PCa is characterized by neuropathic alterations which are resulting in pancreatic pain. To further characterize pancreatic neuropathy, we aimed: to analyze whether neuropathic alterations in PCa are only limited to the tumor-core or whether they are similarly encountered in neural structures in the noncancerous pancreas, to demonstrate whether PCa features neurotrophic attributes and finally to identify responsible neurotrophic molecules., Methods: Nerve density and area were quantified in normal pancreas (NP, n=45), histologically "normal" pancreas next to pancreatic cancer (NNPCa, n=61) and PCa (n=97). Growth-associated protein-43, nerve growth factor (NGF), and Artemin expressions were assessed by Immunohistochemistry, Western-Blot, and quantitative real time polymerase chain reaction-analyses. Isolated myenteric plexus of newborn rats were exposed to NP, NNPCa, and PCa tissue extracts and supernatants of Panc1 and T3M4 cancer cells with or without Artemin and NGF depletion, followed by neurite density analysis., Results: Dense neural networks and enlarged nerves were not only detected in PCa but were also present in NNPCa. Growth-associated protein-43, NGF, and Artemin expressions were absent/weak in NP, but increased in both NNPCa and PCa and were closely associated with intrapancreatic neuropathy. PCa and NNPCa tissue extracts and Panc1/T3M4 supernatants noticeably increased neurite density in myenteric plexus-cultures, which were attenuated by depletion of NGF and Artemin., Conclusions: The neurotrophic effects of PCa extend into the peritumoral "normal" pancreatic areas without neuro-cancer interactions. The neurotrophic characteristics of PCa can be mimicked by in vitro analyses and reveal NGF and Artemin as potential key players in the generation of pancreatic neuropathy in PCa.

Background: Tissue fibrosis is an integral component of chronic inflammatory (liver and pancreas) diseases and pancreatic cancer. Activated pancreatic- (PSC) and hepatic- (HSC) stellate cells play a key role in fibrogenesis. To identify organ- and disease-specific stellate cell transcriptional fingerprints, we employed genome-wide transcriptional analysis of primary human PSC and HSC isolated from patients with chronic inflammation or cancer., Methods: Stellate cells were isolated from patients with pancreatic ductal adenocarcinoma (n = 5), chronic pancreatitis (n = 6), liver cirrhosis (n = 5) and liver metastasis of pancreatic ductal adenocarcinoma (n = 6). Genome-wide transcriptional profiles of stellate cells were generated using our 51K human cDNA microarray platform. The identified organ- and disease specific genes were validated by quantitative RT-PCR, immunoblot, ELISA, immunocytochemistry and immunohistochemistry., Results: Expression profiling identified 160 organ- and 89 disease- specific stellate cell transcripts. Collagen type 11a1 (COL11A1) was discovered as a novel PSC specific marker with up to 65-fold higher expression levels in PSC compared to HSC (p < 0.0001). Likewise, the expression of the cytokine CCL2 and the cell adhesion molecule VCAM1 were confined to HSC. PBX1 expression levels tend to be increased in inflammatory- vs. tumor- stellate cells. Intriguingly, tyrosine kinase JAK2 and a member of cell contact-mediated communication CELSR3 were found to be selectively up-regulated in tumor stellate cells., Conclusions: We identified and validated HSC and PSC specific markers. Moreover, novel target genes of tumor- and inflammation associated stellate cells were discovered. Our data may be instrumental in developing new tailored organ- or disease-specific targeted therapies and stellate cell biomarkers.

As a molecular chaperone, GRP94 is the most abundant glycoprotein in the endoplasmic reticulum, playing an important role in maintaining cellular homeostasis. Here, we investigated the expression and the role of GRP94 in regulating cell growth and apoptosis in pancreatic cancer cells. GRP94 mRNA levels were analyzed by QRT-PCR. Immunohistochemistry was performed to localize GRP94 in tissues of the normal pancreas (n=20), chronic pancreatitis (n=20) and pancreatic ductal adenocarcinoma (n=44). Silencing of GRP94 expression was carried out by transfection with specific siRNA oligonucleotides. Apoptosis was induced by treatment with actinomycin D. Compared to normal pancreatic tissues, median mRNA levels of GRP94 were 1.5- and 3.7-fold (p<0.05) lower in chronic pancreatitis and pancreatic cancer tissues, respectively. GRP94 protein was strongly expressed in normal acinar cells and moderately expressed in normal ductal cells. GRP94 expression was lost in 48% of the cancer cases. Moderate or strong staining in cancer cells was observed in 32 and 20% of pancreatic cancer tissues, respectively. Silencing GRP94 by siRNA increased apoptosis of pancreatic cancer cells in vitro. Patients with higher than the median expression have a tendency for a worsened survival. When the small number of patients with the highest expression (n=3) were compared with the rest of the group (n=41), the survival difference was significantly worse (5 vs. 18 months, respectively, p=0.006). Down-regulation of GRP94 decreases apoptosis resistance in pancreatic cancer cells. Clinically, patients with high GRP94 expression show a tendency for a worsened survival.

Background and Aims: Although both cancer and stellate cells (PSCs) secrete proangiogenic factors, pancreatic cancer is a scirrhous and hypoxic tumor. The impact of cancer-PSCs interactions on angiogenesis was analyzed., Methods: Expression of periostin, CD31, and alpha-smooth muscle actin was assessed by immunohistochemistry. Human PSCs and cancer cells were cultivated under normoxia and hypoxia alone, or in coculture, to analyze the changes in their angiogenic and fibrogenic attributes, using enzyme-linked immunosorbent assay, immunoblot, and quantitative polymerase chain reaction analyses and growth of cultured endothelial cells in vitro., Results: On the invasive front of the activated stroma, PSCs deposited a periostin-rich matrix around the capillaries in the periacinar spaces. Compared with the normal pancreas, there was a significant reduction in the microvessel density in chronic pancreatitis (five-fold, P < .001) and pancreatic cancer (four-fold, P < .01) tissues. In vitro, hypoxia increased PSCs' activity and doubled the secretion of periostin, type I collagen, fibronectin, and vascular endothelial growth factor (VEGF). Cancer cells induced VEGF secretion of PSCs (390 +/- 60%, P < .001), whereas PSCs increased the endostatin production of cancer cells (210 +/- 14%, P < .001) by matrix metalloproteinase-dependent cleavage. In vitro, PSCs increased the endothelial cell growth, whereas cancer cells alone, or their coculture with PSCs, suppressed it., Conclusions: Although PSCs are the dominant producers of VEGF and increase endothelial cell growth in vitro, in the peritumoral stroma, they contribute to the fibrotic/hypoxic milieu through abnormal extracellular matrix deposition and by amplifying endostatin production of cancer cells.

Aims: Endocrine gland-derived vascular endothelial growth factor (EG-VEGF)/prokineticins have been identified as tissue-specific angiogenic factors. This study investigates the expression and localization of EG-VEGF and its receptors in pancreatic tissues and pancreatic stellate cells (PSCs)., Methods: mRNA levels of EG-VEGF/prokineticin 1 (PK1), prokineticin 2 (PK2) and their receptors 1 (PKR1) and 2 (PKR2) were measured in pancreatic tissues, pancreatic cancer cell lines and PSCs by quantitative reverse-transcriptase polymerase chain reaction (QRT-PCR). Protein expression of PK1, PKR1 and PKR2 was assessed in pancreatic tissues by immunohistochemistry. Growth factor-induced secretion of EG-VEGF was measured by ELISA., Results: QRT-PCR analysis in bulk tissues of normal pancreas, chronic pancreatitis and pancreatic ductal adenocarcinoma showed no significant difference of PK1 mRNA levels, whereas PK2 mRNA was barely detectable. High PK1 mRNA levels were observed only in cultured PSCs and microdissected islet cells, but not in cancer cells, and PK1 protein was localized mainly in islets and cancer-associated stromal cells. PKR1 and PKR2 proteins were present in endothelial cells of small blood vessels. TGF-beta(1) and PDGF-BB specifically stimulated PK1 secretion in PSCs., Conclusions: Islet and/or PSC-derived PK1 might act through its receptors on endothelial cells to increase angiogenesis in pancreatic diseases., (Copyright 2008 S. Karger AG, Basel and IAP.)

The fact that pancreatic ductal adenocarcinoma (PDAC) is still an exceptionally lethal disease with an annual mortality almost equivalent to its annual incidence has stimulated intense research efforts directed at understanding the underlying molecular mechanisms. By enabling simultaneous expression analysis of thousands of genes, microarray technology has significantly contributed to illuminating the pathophysiology of PDAC. Gene expression profiling studies have been performed for molecular classification of clinically relevant tumor subtypes and have shed light on various signaling pathways associated with tumor progression. Altered expression levels of several genes have been identified as correlating with functional in vitro data as well as patient survival, indicating the potential clinical value of transcriptional profiling. However, broad clinical use of array techniques for patient characterization has been hampered by their cost intensity and by limited inter-study comparability. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR), as the most sensitive technique for mRNA detection and quantification, will complement arrays for the confirmation of individual transcripts in larger sample cohorts. This review highlights recent studies that addressed gene expression analysis with both methodologies and that identified components of the TGF-beta signaling pathway, BNIP3, or periostin to be differentially expressed in PDAC. These studies demonstrated that the combination of microarray and RT-PCR technologies is a highly efficient and reliable approach for the identification of clinically important diagnostic and prognostic biomarkers, as well as for the discovery of novel therapeutic target candidates., (Copyright 2009 S. Karger AG, Basel.)

Investigation of gene expression significantly contributes to our knowledge of the regulation and function of genes in many areas of biology. In this protocol, we describe how northern blot analysis is used to identify gene expression patterns at the RNA level in human cancer cells as well as in cancerous and normal tissues. RNA molecules are separated by gel electrophoresis and are subsequently transferred to a porous membrane by capillary action. Specific sequences in the RNA are detected on the membrane by molecular hybridization with radiolabeled nucleic acid probes. Despite the development of newer methods, such as real-time PCR, nuclease protection assays and microarrays, northern blot analysis is still a standard technique used in the detection and quantification of mRNA levels because it allows a direct comparison of the mRNA abundance between samples on a single membrane. This entire northern blotting protocol takes approximately 4 d to complete.

Background: Pim-1 is a proto-oncogene involved in cell survival, differentiation and proliferation in several hematologic and epithelial malignancies. Clinically, absence of Pim-1 expression correlates with poor prognosis in prostate cancer. In the present study, the expression of Pim-1 is analyzed in pancreatic cancer and correlated to clinicopathological parameters., Results: Compared to benign, inflammatory and pre-malignant conditions (i.e., the normal pancreas, chronic pancreatitis and benign intraductal papillary mucinous neoplasm), expression of Pim-1 mRNA and protein increased significantly in pancreatic malignancies. Absence of Pim-1 immunopositivity in cancer cells strongly correlated with a poor prognosis (median survival 13.8 vs. 23.4 months, p = 0.0016). In vitro, rapidly dividing (high versus low serum concentrations) and hypoxic cells displayed higher Pim-1 mRNA and protein levels., Methods: Pim-1 mRNA and protein was evaluated with quantitative real-time RT-PCR, immunofluorescence and immunocytochemistry analyses. Ex vivo expression analysis using semi-quantitative immunohistochemistry was performed using human pancreatic tissues of the normal pancreas (n = 10), chronic pancreatitis (n = 30), pancreatic ductal adenocarcinoma (n = 59) and other pancreatic tumors (n = 42). In consecutive sections HIF1-alpha was used as a marker of hypoxia. Survival of patients (n = 35) was compared using the Kaplan-Meier method and a log-rank test. In vitro analyses were performed using cultured pancreatic cancer cell lines (n = 8) and primary human pancreatic stellate cells., Conclusion: Hypoxia is a novel inducer of Pim-1 expression. Compared to non-malignant tissues Pim-1 significantly increases in pancreatic cancer. However, the presence of Pim-1 in cancer cells has a positive prognostic impact.

Cannabinoids exert antiproliferative properties in a variety of malignant tumors, including pancreatic ductal adenocarcinoma (PDAC). In our study, we quantitatively evaluated the immunoreactivity for cannabinoid-1 (CB1) and cannabinoid-2 (CB2) receptors as well as for the endocannabinoid metabolizing enzymes fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MGLL). Furthermore, quantitative real-time RT-PCR for CB1, CB2, FAAH and MGLL in normal pancreas and pancreatic cancer tissues was performed. Levels of endocannabinoids were determined by liquid chromatography/mass spectrometry. Immunoreactivity scores and QRT-PCR expression levels were correlated with the clinico-pathological (TNM, survival, pain) status of the patients. Evaluation of endocannabinoid levels revealed that these remained unchanged in PDAC compared to the normal pancreas. Patients with high CB1 receptor levels in enlarged nerves in PDAC had a lower combined pain score (intensity, frequency, duration; p = 0.012). There was a significant relationship between low CB1 receptor immunoreactivity or mRNA expression levels (p = 0.0011 and p = 0.026, respectively), or high FAAH and MGLL cancer cell immunoreactivity (p = 0.036 and p = 0.017, respectively) and longer survival of PDAC patients. These results are underlined by a significant correlation of high pain scores and increased survival (p = 0.0343). CB2 receptor immunoreactivity, CB2 receptor, FAAH and MGLL mRNA expression levels did not correlate with survival. Therefore, changes in the levels of endocannabinoid metabolizing enzymes and cannabinoid receptors on pancreatic cancer cells may affect prognosis and pain status of PDAC patients., ((c) 2007 Wiley-Liss, Inc.)

Resection of liver metastases for locally resectable pancreatic cancer has rarely been performed. Recently, promising results regarding morbidity and mortality as well as long-term survival have been shown. Thus, we conducted a systematic review of the literature on pancreatic cancer resection with associated liver metastasis resection. There are 3 case reports and 18 studies including less than 10 patients. Only three studies are larger series with 10 or more patients in whom pancreatic resections and hepatic metastasectomies were performed. Here, morbidity and mortality ranged from 24.1 to 26% and from 0 to 4.3%, respectively. Median survival was reported to be between 5.8 and 11.4 months. In total, all identified studies included 103 patients in whom a metastasis resection was performed. Liver metastasis resection for locally resectable pancreatic cancer can be performed in selected cases with low morbidity and mortality. Overall survival in cases with one or few liver metastases which are concomitantly resected seems to be comparable to cases without evidence of metastasis. Therefore, randomized controlled clinical trials will have to be initiated to determine the value of such resections and to identify factors which will allow for selection of patients in whom the extension of the resectability criteria might confer a survival benefit., ((c) 2009 S. Karger AG, Basel.)

Background: The value of re-exploration for pancreatic ductal adenocarcinoma after the initial diagnosis of unresectability is unclear., Methods: In this study, we analyzed 33 patients who were re-explored after an initial diagnosis of unresectability., Results: At the time of reoperation, a resectable tumor was found in 18 patients: therefore, 15 pancreaticoduodenectomies, two total pancreatectomies and one left resection were performed with three vascular resections. Morbidity and mortality rates for the cohort were 6/33 and 1/33, without significant differences between resectable and nonresectable patients. Length of stay, duration of operation, and blood loss were significantly increased in the resection group. Kaplan-Meier survival analysis demonstrated increased median survival for resected patients (1078 days after the initial operation versus 547 days in the group of unresectable patients; p = 0.018). Analysis of the reasons against initial resection showed that, if the patients had been sent to a tertiary referral center for pancreatic surgery, a different decision in favor of resection would probably have been made in 14 out of 33 patients. A review of 10 published reports on reoperation for pancreatic cancer revealed results comparable to our study in terms of low morbidity and mortality as well as a survival benefit., Conclusions: Reoperation for pancreatic ductal adenocarcinoma that is initially deemed unresectable can be safely performed in a selected group of patients by experienced surgeons, supporting the concept of patient centralization in pancreatic surgery. Resection at the second operation may confer a survival benefit even when the initial findings preclude a potentially curative approach.

Background & Aims: Pancreatic cancer creates desmoplasia by stimulating stellate cells (PSCs), thereby influencing tumor aggressiveness. The aim of this study was to analyze the impact of the PSC-specific matrix protein periostin on tumor responses to radiochemotherapy., Methods: PSCs and cancer cells in primary and metastatic lesions of patients treated with or without neoadjuvant radiochemotherapy were evaluated by immunohistochemistry. Periostin messenger-RNA levels determined by quantitative reverse-transcription polymerase chain reaction were correlated to patient survival. Interactions between PSCs and cancer cells and the effects of periostin in modulating cellular responses under conditions of hypoxia, starvation, and radiochemotherapy were assessed by immunoblotting and by growth, clonogenicity, and invasion assays., Results: Periostin messenger-RNA levels were elevated 42-fold in cancer, and patients with increased expression had a tendency toward shorter survival (19 vs 12 months; P = .14). Stromal cells were the only source of periostin in the pancreas and in metastatic sites. Cancer cell supernatants stimulated periostin secretion from PSCs. Recombinant periostin increased alpha-smooth muscle actin, periostin, collagen-1, fibronectin, and transforming growth factor-beta1 expression while decreasing PSC invasiveness. These effects were reversed by silencing periostin expression and secretion by small interfering RNA transfection. In cancer cells, periostin stimulated growth and conferred resistance to starvation and hypoxia. In addition, the periostin downstream target collagen-1 significantly increased chemoresistance., Conclusions: Once stimulated by cancer cells, PSCs remain active via an autocrine periostin loop even under radiotherapy and produce excessive extracellular matrix proteins, creating a tumor-supportive microenvironment. Increased periostin expression may therefore reflect a more aggressive tumor phenotype.