A multicenter phase 4 geriatric assessment directed trial to evaluate gemcitabine +/- nab-paclitaxel in elderly pancreatic cancer patients (GrantPax).

BACKGROUND: In the group of elderly patients (≥70 years) with metastatic pancreatic ductal adenocarcinoma (mPDAC), it is not known who benefits from intensive 1st line nab-paclitaxel/gemcitabine (nab-p/gem) combination chemotherapy or who would rather suffer from increased toxicity. We aim to determine whether treatment individualization by comprehensive geriatric assessments (CGAs) improves functional outcome of the patients. METHODS/DESIGN: GrantPax is a multicenter, open label phase 4 interventional trial. We use a CGA to stratify elderly patients into three parallel treatment groups (n = 45 per arm): 1) GOGO (nab-p/gem), 2) SLOWGO (gem mono) or 3) FRAIL (best supportive care). After the 1st cycle of chemotherapy (or 4 weeks in FRAIL group) another CGA and safety assessment is performed. CGA-stratified patients may not decline in their CGA performance in response to the first cycle of chemotherapy (primary objective), measured as a loss of 5 points or less in Barthels activities of daily living. Based on the second CGA, patients are re-assigned to their definite treatment arm and undergo further CGAs to monitor the course of treatment. Secondary endpoints include CGA scores during the course of therapy (CGA1-4), response rates, safety and survival rates. DISCUSSION: GrantPax is the first trial implementing a CGA-driven treatment to personalize therapy for elderly patients with pancreatic cancer. This may lead to standardization of therapy decisions for elderly patients and may optimize standard of care for this increasing group of patients. TRIAL REGISTRATION: NCT02812992 , registered 24.06.2016.

Diagnosing autoimmune pancreatitis with the Unifying-Autoimmune-Pancreatitis-Criteria.

BACKGROUND/OBJECTIVES: We had developed the Unifying-Autoimmune-Pancreatitis-Criteria (U-AIP) to diagnose autoimmune pancreatitis (AiP) within the M-ANNHEIM classification of chronic pancreatitis. In 2011, International-Consensus-Diagnostic-Criteria (ICDC) to diagnose AiP have been published. We had applied the U-AIP long before the ICDC were available. The aims of the study were, first, to describe patients with AiP diagnosed by the U-AIP; second, to compare diagnostic accuracies of the U-AIP and other diagnostic systems; third, to evaluate the clinical applicability of the U-AIP. METHODS: From 1998 until 2008, we identified patients with AiP using U-AIP, Japanese-, Korean-, Asian-, Mayo-HISORt-, Revised-Mayo-HISORt- and Italian-criteria. We retrospectively verified the diagnosis by ICDC and Revised-Japanese-2011-criteria, compared diagnostic accuracies of all systems and evaluated all criteria in consecutive patients with pancreatitis (2009 until 2010, Pancreas-Outpatient-Clinic-Cohort, n = 84). We retrospectively validated our diagnostic approach in consecutive patients with a pancreatic lesion requiring surgery (Surgical-Cohort, n = 98). RESULTS: Overall, we identified 21 patients with AiP. Unifying-Autoimmune-Pancreatitis-Criteria and ICDC presented the highest diagnostic accuracies (each 98.8%), highest Youden indices (each 0.95238), and highest proportions of diagnosed patients (each n = 20/21, U-AIP/ICDC vs. other diagnostic systems, p < 0.05, McNemar test). In the Pancreas-Outpatient-Clinic-Cohort, seven patients were diagnosed with AiP (n = 6 by U-AIP, n = 1 by Asian-criteria). International-Consensus-Diagnostic-Criteria confirmed the diagnosis in these individuals. Based on partial fulfillment of U-AIP, AiP was initially suspected in 13% (n = 10/77) of remaining patients from the Pancreas-Outpatient-Clinic-Cohort. In the Surgical-cohort, we identified one patient with AiP by U-AIP and ICDC. CONCLUSIONS: Unifying-Autoimmune-Pancreatitis-Criteria revealed a satisfactory clinical applicability and offered an additional approach to diagnose AiP.

Biodegradable biliary stents have a different effect than covered metal stents on the expression of proteins associated with tissue healing in benign biliary strictures.

BACKGROUND: Benign biliary strictures (BBS) are primarily treated endoscopically with covered self-expandable metal stents (CSEMS). Biodegradable biliary stents (BDBS) may be the future of endoscopic therapy of BBS. The aim was to assess the expression of proteins related to tissue healing in BBS compared with the intact bile duct (BD), and to study the protein expression after therapy with CSEMS or BDBS. METHODS: Pigs with ischemic BBS were endoscopically treated either with BDBS or CSEMS. Samples were harvested from pigs with intact BD (n = 5), untreated BBS (n = 5), and after six months of therapy with BDBS (n = 4) or CSEMS (n = 5) with subsequent histologic analysis. Two-dimensional electrophoresis with protein identification was performed to evaluate protein expression patterns. RESULTS: In BBS, the expression of galectin-2 and annexin-A4 decreased, compared to intact BD. Treatment with biodegradable stents normalized galectin-2 level; with CSEMS therapy it remained low. Transgelin expression of intact BD and BBS remained low after BDBS treatment but increased after CSEMS therapy. Histologic analysis did not show unwanted foreign body reaction or hyperplasia in the BD in either group. CONCLUSIONS: The expression of proteins related to tissue healing in BBS is different after treatment with biodegradable stents and CSEMS. Treatment with biodegradable stents may bring protein expression towards what is seen in intact BD. BDBS seem to have a good biocompatibility.

Isolation and culture of primary human pancreatic stellate cells that reflect the context of their tissue of origin.

BACKGROUND: Pancreatic stellate cells (PSCs) play a critical role in pancreatic ductal adenocarcinoma (PDAC). Activated PSCs are the main source of fibrosis in chronic pancreatitis and of desmoplasia in PDAC. The majority of studies on PSC are based on in vitro experiments relying on immortalized cell lines derived from diseased human pancreas or from animal models. These PSCs are usually activated and may not represent the biological context of their tissue of origin. PURPOSE: (1) To isolate and culture primary human PSC from different disease contexts with minimal impact on their state of activation. (2) To perform a comparative analysis of phenotypes of PSC derived from different contexts. METHODS: PSCs were isolated from normal pancreas, chronic pancreatitis, and PDAC using a hybrid method of digestion and outgrowth. To minimize activation by serum compounds, cells were cultured in a low-serum environment (2.5 % fetal bovine serum (FBS)). Expression patterns of commonly used markers for PSC phenotype and activity were compared between primary PSC lines derived from different contexts and correlated to expression in their original tissues. RESULTS: Isolation was successful from 14 of 17 tissues (82 %). Isolated PSC displayed stable viability and phenotype in low-serum environment. Expression profiles of isolated PSC and matched original tissues were closely correlated. PDAC-derived PSC tended to have a higher status of activation if compared to PSC derived from non-cancerous tissues. CONCLUSIONS: Primary human PSCs isolated from different contexts and cultured in a low-serum environment maintain a phenotype that reflects the stromal activity present in their tissue of origin.

Transforming growth factor-α activates pancreatic stellate cells and may be involved in matrix metalloproteinase-1 upregulation.

The role that transforming growth factor-α (TGF-α) has in chronic pancreatitis and pancreatic cancer has not been fully elucidated. We evaluated the effects of TGF-α on the human pancreatic stellate cell (PSC) line RLT-PSC and primary human PSCs, and the expression levels of TGF-α and metalloproteinase-1 (MMP-1) in human chronic pancreatitis and pancreatic cancer tissues. TGF-α stimulated the proliferation and migration of PSCs. Although the mRNA expression levels of tissue inhibitor of metalloproteinase-1 and α1(I) collagen were unchanged, the mRNA expression levels of MMP-1 increased concomitant with increases in MMP-1 protein levels and collagenase activity. TGF-α-stimulated migration of RLT-PSC cells was partially blocked by tissue inhibitor of metalloproteinase-1 protein and MMP-1 small interfering RNA. MMP-1 was also observed to stimulate the migration of PSCs. TGF-α-induced MMP-1 expression was completely blocked by gefitinib in PSCs. The Ras-ERK and PI3/Akt pathways appear to be involved in the activation of MMP-1 in PSCs. Immunohistochemical analyses showed that MMP-1 expression was significantly increased in the pancreatic interstitial tissues in case of chronic pancreatitis or pancreatic cancer compared with those in case of normal pancreas. In conclusion, TGF-α increased proliferation and migration of PSCs. TGF-α-induced migration of cells may be partly due to upregulation of MMP-1. TGF-α and MMP-1 upregulation may contribute to the pathogenesis of chronic pancreatitis and pancreatic cancer.

Treatment of experimental pancreatic cancer by doxorubicin-, mitoxantrone-, and irinotecan-drug eluting beads.

BACKGROUND AND AIMS: Peritoneal carcinomatosis is a common cause of death in pancreatic cancer patients. In this metastatic stage of the disease, few patients show a sustained response to therapy. In the palliative situation, targeted and compartment restricted delivery of drugs offers the opportunity to focus drugs directly to the tumor site, which is a prerequisite for avoiding toxic side effects. Here, we demonstrate the therapeutic efficiency of biocompatible polyvinyl-alcohol hydrogel drug eluting beads (DEBs) containing doxorubicin, mitoxantrone and irinotecan in vitro and in vivo in a syngenic model of experimental pancreatic cancer. METHODS: Panc02 murine pancreatic carcinoma cells were exposed to doxorubicin, mitoxantrone and irinotecan DEBs and free compounds. The effect on cell proliferation and apoptosis induction was compared. Using this cell line, peritoneal carcinomatosis was induced in C57 black6 mice. Mortality, tumor load and therapy-associated weight loss were compared after treatment of tumor-bearing mice with DEBs or free compounds. RESULTS: In vitro treatment with DEBs decreases tumor cell proliferation and induces apoptosis. The effect is less pronounced than with corresponding doses of the free drug. Repeated applications of the free drugs in vivo, however, induce significantly higher lethality and weight loss than corresponding doses of DEBs. No relevant differences in antitumoral activity were observed. Using computer tomography and HE-histology after subcutaneous and intraperitoneal injection of radiopaque beads no systemic spread of the beads could be found. CONCLUSION: DEBs show the advantage of delivering potent cytotoxic activity to the intraperitoneal tumor manifestation while maintaining a low systemic toxicity.

Neglected geriatric assessment and overtreatment of older patients with pancreatic cancer - Results from a prospective phase IV clinical trial.

BACKGROUND: Older patients with metastatic pancreatic cancer may suffer increased toxicity from intensive chemotherapy. Treatment individualization by geriatric assessment (GA) might improve functional outcome. METHODS: We performed a multicenter, phase IV, open label trial in patients ≥70 years with metastatic pancreatic adenocarcinoma. Patients underwent GA and were assigned to one of three categories based on their scores: Go-Go, Slow-Go, or Frail. These categories were intended to guide physician's treatment decisions when choosing to treat patients with nab-paclitaxel/gemcitabine (arm A), gemcitabine (arm B), or best supportive care (arm C). Primary objective was a stable (loss of five points or less) Barthel's Activities of Daily Living (ADL) score during chemotherapy; secondary endpoints included GA scores during therapy, safety, quality of life, response and survival rates. RESULTS: Thirty-two patients were enrolled in the trial in six centers in Germany (out of 135 planned), resulting in termination due to low recruitment. Fifteen patients were allocated to nab-paclitaxel/gemcitabine, fifteen to gemcitabine, and two to best supportive care by their physicians, although according to their GA scores 29 patients (91%) were categorized as Slow-Go and three (9%) as Go-Go. Thus, fifteen of 32 (47%) patients were misclassified and given a course of treatment inconsistent with their GA scores. Median progression-free survival (PFS) were 3.3 months and 9.1 months and median time to quality-of-life deterioration 13 days and 29 days in the nab-paclitaxel/gemcitabine and gemcitabine monotherapy arms, respectively. Serious adverse events were reported in 11 (78.6%) patients in the nab-paclitaxel/gemcitabine and 8 (53.3%) patients in the gemcitabine arm. CONCLUSIONS: Clinical evaluations by investigators differed markedly from geriatric assessments, leading to potential overtreatment. In our modest sample size study, those patients undergoing more intensive therapy had a less favorable course.

Sequential Geriatric Assessment in Older Patients with Colorectal Cancer during Chemotherapy: Subgroup Analysis of a Prospective, Multicenter Study EpiReal 75.

INTRODUCTION: Colorectal cancer (CRC) is a disease of older patients, but evidence-based guidelines for chemotherapy in older patients are scarce. Geriatric assessment (GA) evaluates a patient's functional status (FS) and helps in decision-making when choosing chemotherapy for older patients. However, the change of FS during chemotherapy is rarely studied as GA is mostly performed once instead of sequentially. METHODS: We performed a subgroup analysis of a prospective, multicenter study EpiReal 75. Patients aged ≥75 years with gastrointestinal malignancy prior to initiation of chemotherapy or receiving palliative chemotherapy were screened. We defined geriatric core assessments including the Eastern Cooperative Oncology Group score, Barthel's activities of daily living (ADL) scale, Lawton's instrumental activities of daily living (IADL) scale, and G-8 questionnaire, which were performed at baseline and repeated every 3 months. Quality of life (QoL) assessed by QLQ-C30 questionnaire was also re-evaluated every 3 months. We defined any deterioration in any of the geriatric parameters as unstable in the corresponding function. RESULTS: 28 patients with CRC were enrolled between April 2014 and December 2018. 20 patients were evaluable for statistical analysis with a mean age of 78.5 years (range, 75-88). Most patients received chemotherapy in palliative setting. During 3 months of chemotherapy, 25% of patients became more dependent as measured by ADL or IADL. During a median follow-up of 15 months, patients with unstable ADL or IADL had a significantly shorter overall survival (OS) than those with stable ADL or IADL (plogrank = 0.0055 and 0.0253, respectively), without a significant difference in progression-free survival (PFS). Also, unstable IADL correlated with a deterioration in aspects of QoL such as role functioning and emotional functioning (p = 0.0189 and 0.0239, respectively). 20% of patients experienced treatment-related grade 3 adverse events (AEs), no grade 4-5 AEs occurred. CONCLUSION: Sequential GA revealed changes in FS in older patients with CRC receiving chemotherapy. A deterioration of FS during chemotherapy did not influence PFS but had a negative impact on OS and QoL. It is therefore important to maintain FS in older patients with cancer, and regular performance of geriatric core assessments should be encouraged in the clinical practice.

The drug-induced phenotypic landscape of colorectal cancer organoids.

Patient-derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases. They have heterogeneous morphologies with unclear biological causes and relationship to treatment response. Here, we use high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with >500 small molecules. Integration of data using multi-omics modeling identifies axes of morphological variation across organoids: Organoid size is linked to IGF1 receptor signaling, and cystic vs. solid organoid architecture is associated with LGR5 + stemness. Treatment-induced organoid morphology reflects organoid viability, drug mechanism of action, and is biologically interpretable. Inhibition of MEK leads to cystic reorganization of organoids and increases expression of LGR5, while inhibition of mTOR induces IGF1 receptor signaling. In conclusion, we identify shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them.

Autoantibodies against the exocrine pancreas in autoimmune pancreatitis: gene and protein expression profiling and immunoassays identify pancreatic enzymes as a major target of the inflammatory process.

OBJECTIVES: Autoimmune pancreatitis (AIP) is thought to be an immune-mediated inflammatory process, directed against the epithelial components of the pancreas. The objective was to identify novel markers of disease and to unravel the pathogenesis of AIP. METHODS: To explore key targets of the inflammatory process, we analyzed the expression of proteins at the RNA and protein level using genomics and proteomics, immunohistochemistry, western blot, and immunoassay. An animal model of AIP with LP-BM5 murine leukemia virus-infected mice was studied in parallel. RNA microarrays of pancreatic tissue from 12 patients with AIP were compared with those of 8 patients with non-AIP chronic pancreatitis. RESULTS: Expression profiling showed 272 upregulated genes, including those encoding for immunoglobulins, chemokines and their receptors, and 86 downregulated genes, including those for pancreatic proteases such as three trypsinogen isoforms. Protein profiling showed that the expression of trypsinogens and other pancreatic enzymes was greatly reduced. Immunohistochemistry showed a near-loss of trypsin-positive acinar cells, which was also confirmed by western blotting. The serum of AIP patients contained high titers of autoantibodies against the trypsinogens PRSS1 and PRSS2 but not against PRSS3. In addition, there were autoantibodies against the trypsin inhibitor PSTI (the product of the SPINK1 gene). In the pancreas of AIP animals, we found similar protein patterns and a reduction in trypsinogen. CONCLUSIONS: These data indicate that the immune-mediated process characterizing AIP involves pancreatic acinar cells and their secretory enzymes such as trypsin isoforms. Demonstration of trypsinogen autoantibodies may be helpful for the diagnosis of AIP.

Detection of mutational patterns in cell-free DNA of colorectal cancer by custom amplicon sequencing.

Monitoring the mutational patterns of solid tumors during cancer therapy is a major challenge in oncology. Analysis of mutations in cell-free (cf) DNA offers a noninvasive approach to detect mutations that may be prognostic for disease survival or predictive for primary or secondary drug resistance. A main challenge for the application of cfDNA as a diagnostic tool is the diverse mutational landscape of cancer. Here, we developed a flexible end-to-end experimental and bioinformatic workflow to analyze mutations in cfDNA using custom amplicon sequencing. Our approach relies on open-software tools to select primers suitable for multiplex PCR using minimal cfDNA as input. In addition, we developed a robust linear model to identify specific genetic alterations from sequencing data of cfDNA. We used our workflow to design a custom amplicon panel suitable for detection of hotspot mutations relevant for colorectal cancer and analyzed mutations in serial cfDNA samples from a pilot cohort of 34 patients with advanced colorectal cancer. Using our method, we could detect recurrent and patient-specific mutational patterns in the majority of patients. Furthermore, we show that dynamic changes of mutant allele frequencies in cfDNA correlate well with disease progression. Finally, we demonstrate that sequencing of cfDNA can reveal mechanisms of resistance to anti-Epidermal Growth Factor Receptor(EGFR) antibody treatment. Thus, our approach offers a simple and highly customizable method to explore genetic alterations in cfDNA.

Changes in the microarchitecture of the pancreatic cancer stroma are linked to neutrophil-dependent reprogramming of stellate cells and reflected by diffusion-weighted magnetic resonance imaging.

In pancreatic cancer (PDAC) intratumor infiltration of polymorphonuclear neutrophils (PMN) is associated with histologically apparent alterations of the tumor growth pattern. The aim of this study was to examine possible associations between PMN infiltration, tumor microarchitecture, and water diffusivity in diffusion-weighted magnetic resonance imaging (DW-MRI), and to further asses the underlying mechanisms. Methods: DW-MRI was performed in 33 PDAC patients prior to surgery. In parallel, tissue specimen were examined histologically for growth pattern, azurocidin-positive PMN infiltrates, and the presence of alpha-smooth muscle actin (α-SMA) and metalloproteinase 9 (MMP9)-positive myofibroblastic cells. For confirmation of the histological findings, a tissue microarray of a second cohort of patients (n=109) was prepared and examined similarly. For in vitro studies, the pancreatic stellate cell line RLT was co-cultivated either with isolated PMN, PMN-lysates, or recombinant azurocidin and characterized by Western blot, flow cytometry, and proteome profiler arrays. Results: Tumors with high PMN density showed restricted water diffusion in DW-MRI and histologic apparent alterations of the tumor microarchitecture (microglandular, micropapillary, or overall poorly differentiated growth pattern) as opposed to tumors with scattered PMN. Areas with altered growth pattern lacked α-SMA-positive myofibroblastic cells. Tissue microarrays confirmed a close association of high PMN density with alterations of the tumor microarchitecture and revealed a significant association of high PMN density with poor histologic grade of differentiation (G3). In vitro experiments provided evidence for direct effects of PMN on stellate cells, where a change to a spindle shaped cell morphology in response to PMN and to PMN-derived azurocidin was seen. Azurocidin incorporated into stellate cells, where it associated with F-actin. Down-regulation of α-SMA was seen within hours, as was activation of the p38-cofilin axis, up-regulation of MMP9, and acquisition of intracellular lipid droplets, which together indicate a phenotype switch of the stellate cells. Conclusion: In PDAC, PMN infiltrates are associated with alterations of the tumor microarchitecture. As a causal relationship, we propose a reprogramming of stellate cells by PMN-derived azurocidin towards a phenotype, which affects the microarchitecture of the tumor.

Enhanced iNOS gene expression in the steatotic rat liver after normothermic ischemia.

BACKGROUND: Impaired hepatic microcirculation in the steatotic liver has been identified as a considerable factor for increased vulnerability after ischemia/reperfusion (I/R). Changes in regulation and synthesis of vasoactive mediators, such as nitric oxide (NO) and endothelin (ET-1), may result in functional impairment of postischemic sinusoidal perfusion. The aim of the current study was to assess the impact of I/R injury on postischemic gene expression of NO and ET-1 in steatotic livers. MATERIALS AND METHODS: Male Sprague-Dawley rats with or without hepatic steatosis (induced by carbon tetrachloride treatment) were subjected to normothermic I/R injury. Steady-state mRNA levels were assessed using RT-PCR to study the expression of genes encoding ET-1, NO synthase (endothelial cell NO synthase and inducible NO synthase, iNOS). Immunohistochemistry was performed for detection of iNOS. RESULTS: I/R injury was followed by increased iNOS gene expression (RT-PCR/immunohistochemistry) in animals with hepatic steatosis, predominately in hepatocytes with fatty degeneration. A mild increase in mRNA levels for ET-1 was found in steatotic rat livers. I/R induced a further increase in ET-1 gene expression in some but not all reperfused steatotic livers. CONCLUSIONS: We show an enhanced gene expression of iNOS in postischemic steatotic rat livers. Hepatocytes with fatty degeneration appear to be the major source for NO generation. Furthermore, I/R may also induce ET-1 gene expression. Dysregulation of sinusoidal perfusion by NO and ET-1 is therefore likely to contribute to I/R injury of the steatotic liver.

Interleukin-15 stimulates natural killer cell-mediated killing of both human pancreatic cancer and stellate cells.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death in Western countries with a 5-year survival rate below 5%. One of the hallmarks of this cancer is the strong desmoplastic reaction within the tumor microenvironment (TME), orchestrated by activated pancreatic stellate cells (PSC). This results in a functional and mechanical shield which causes resistance to conventional therapies. Aiming to overcome this resistance by tackling the stromal shield, we assessed for the first time the capacity of IL-15 stimulated natural killer (NK) cells to kill PSC and pancreatic cancer cells (PCC). The potency of IL-15 to promote NK cell-mediated killing was evaluated phenotypically and functionally. In addition, NK cell and immune checkpoint ligands on PSC were charted. We demonstrate that IL-15 activated NK cells kill both PCC and PSC lines (range 9-35% and 20-50%, respectively) in a contact-dependent manner and significantly higher as compared to resting NK cells. Improved killing of these pancreatic cell lines is, at least partly, dependent on IL-15 induced upregulation of TIM-3 and NKG2D. Furthermore, we confirm significant killing of primary PSC by IL-15 activated NK cells in an ex vivo autologous system. Screening for potential targets for immunotherapeutic strategies, we demonstrate surface expression of both inhibitory (PD-L1, PD-L2) and activating (MICA/B, ULBPs and Galectin-9) ligands on primary PSC. These data underscore the therapeutic potential of IL-15 to promote NK cell-mediated cytotoxicity as a treatment of pancreatic cancer and provide promising future targets to tackle remaining PSC.

Chronic hyperglycemia induces trans-differentiation of human pancreatic stellate cells and enhances the malignant molecular communication with human pancreatic cancer cells.

BACKGROUND: Diabetes mellitus is linked to pancreatic cancer. We hypothesized a role for pancreatic stellate cells (PSC) in the hyperglycemia induced deterioration of pancreatic cancer and therefore studied two human cell lines (RLT-PSC, T3M4) in hyperglycemic environment. METHODOLOGY/PRINCIPAL FINDINGS: The effect of chronic hyperglycemia (CHG) on PSCs was studied using mRNA expression array with real-time PCR validation and bioinformatic pathway analysis, and confirmatory protein studies. The stress fiber formation (IC: αSMA) indicated that PSCs tend to transdifferentiate to a myofibroblast-like state after exposure to CHG. The phosphorylation of p38 and ERK1/2 was increased with a consecutive upregulation of CDC25, SP1, cFOS and p21, and with downregulation of PPARγ after PSCs were exposed to chronic hyperglycemia. CXCL12 levels increased significantly in PSC supernatant after CHG exposure independently from TGF-ß1 treatment (3.09-fold with a 2.73-fold without TGF-ß1, p<0.05). The upregualtion of the SP1 transcription factor in PSCs after CHG exposure may be implicated in the increased CXCL12 and IGFBP2 production. In cancer cells, hyperglycemia induced an increased expression of CXCR4, a CXCL12 receptor that was also induced by PSC's conditioned medium. The receptor-ligand interaction increased the phosphorylation of ERK1/2 and p38 resulting in activation of MAP kinase pathway, one of the most powerful stimuli for cell proliferation. Certainly, conditioned medium of PSC increased pancreatic cancer cell proliferation and this effect could be partially inhibited by a CXCR4 inhibitor. As the PSC conditioned medium (normal glucose concentration) increased the ERK1/2 and p38 phosphorylation, we concluded that PSCs produce other factor(s) that influence(s) pancreatic cancer behaviour. CONCLUSIONS: Hyperglycemia induces increased CXCL12 production by the PSCs, and its receptor, CXCR4 on cancer cells. The ligand-receptor interaction activates MAP kinase signaling that causes increased cancer cell proliferation and migration.

Gene expression profiles of microdissected pancreatic ductal adenocarcinoma.

In a search for new molecular markers of pancreatic ductal adenocarcinoma (PDAC), we compared the gene expression profiles of seven pancreatic carcinomas and one carcinoma of the papilla Vateri with those of duct cells from three non-neoplastic pancreatic tissues. In addition, the human pancreatic duct cell line and five PDAC cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, HPAF) were examined. RNA was extracted from microdissected tissue or cultured cell lines and analysed using a custom-made Affymetrix Chip containing 3023 genes, of which 1000 were known to be tumour associated. Hierarchical clustering revealed 81 differentially expressed genes. Of all the genes, 26 were downregulated in PDAC and 14 were upregulated in PDAC. In PDAC cell lines versus normal pancreatic duct cells, 21 genes were downregulated and 20 were upregulated. Of these 81 differentially expressed genes, 15 represented human genes previously implicated in the tumourigenesis of PDAC. From the genes that were so far not known to be associated with PDAC tumorigenesis, we selected ADAM9 for further validation because of its distinct overexpression in tumour tissue. Using immunohistochemistry, the over-expressed gene, ADAM9, was present in 70% of the PDACs analysed. In conclusion, using microarray technology we were able to identify a set of genes whose aberrant expression was associated with PDAC and may be used to target the disease.

Fluorescent resonance energy transfer imaging of VEGFR dimerization.

Activation of vascular endothelial growth factor receptor 1 (VEGFR1/FLT1) and 2 (VEGFR2/KDR) involves receptor dimerization. Formation of VEGFR dimer has so far not been visualized in single intact cells. In the present study we describe different optical assays which can be used to observe dimerization of VEGFR1 and VEGFR2. Bimolecular fluorescence complementation (BIFC) assays confirmed homo,- and heterodimerization of transfected receptors. Fluorescence resonance energy transfer (FRET) techniques in living and fixed CHO-K1 cells allowed observation of VEGFR1 homodimer,- and VEGFR1 and VEGFR2 heterodimer formation after ligand stimulation. After inhibition of ligand binding by the VEGFA JH121 antibody VEGFR1 homodimerization was completely abolished. Under the same conditions, cells transfected by VEGFR1 and VEGFR2 maintained relevant receptor heterodimerization. These techniques to monitor VEGFR1 and VEGFR2 homo- and heterodimerization in living and fixed cells may help in the search for new angiogenesis-directed inhibitors of VEGFR dimerization.

Desmoplasia and chemoresistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) occurs mainly in people older than 50 years of age. Although great strides have been taken in treating PDAC over the past decades its incidence nearly equals its mortality rate and it was quoted as the 4th leading cause of cancer deaths in the U.S. in 2012. This review aims to focus on research models and scientific developments that help to explain the extraordinary resistance of PDAC towards current therapeutic regimens. Furthermore, it highlights the main features of drug resistance including mechanisms promoted by cancer cells or cancer stem cells (CSCs), as well as stromal cells, and the acellular components surrounding the tumor cells-known as peritumoral desmoplasia-that affects intra-tumoral drug delivery. Finally, therapeutic concepts and avenues for future research are suggested, based on the topics discussed.

Antitumor effects of proteasome inhibition in anaplastic thyroid carcinoma.

UNLABELLED: The ubiquitin-proteasome pathway has been identified as a potential molecular target for cancer therapy. In this study, we investigated the effect of the proteasome inhibitor bortezomib on anaplastic thyroid carcinoma (ATC) characterized by complete refractoriness to multimodal therapeutic approaches. METHODS: The ATC cell lines C643 and SW1736 were treated with bortezomib (1 nM to 1 µM) for 12-72 h. Thereafter, growth inhibition was analyzed by thymidine uptake experiments and determination of the viable cell number. Apoptosis was measured and a cell cycle analysis was done. Using gene chip analysis and the real-time quantitative PCR system, we measured transcriptional changes. The activity of the nuclear factor (NF)-κB and p53 signal transduction pathways was monitored using the reporter constructs pNF-κB-TA-Luc and pp53-TA-Luc in the luciferase activity assay. Uptake measurements using (3)H-FDG, (14)C-aminoisobutyric acid, and Na(125)iodide were performed to investigate metabolic changes and iodide symporter activity in vitro. Moreover, the (18)F-FDG uptake was evaluated in ATC tumor-bearing nude mice 1 or 2 d after treatment with bortezomib. RESULTS: Bortezomib induced growth inhibition, apoptosis, and G(2)-M cell cycle arrest associated with upregulation of p21(CIP1/WAF1) expression in SW1736 and C643 cells. Moreover, the glucose metabolism and aminoisobutyric acid uptake significantly decreased in vitro in both of the ATC cell lines in vivo only in SW1736 tumors at 2 d after the bortezomib treatment. The transcriptional profile in bortezomib-treated SW1736 and C643 cells revealed increased expression of genes involved in stress response, apoptosis, regulation of the cell cycle, and differentiation. Using real-time quantitative PCR for the quantification of gene expression, we additionally noticed upregulation of the tumor necrosis factor-related apoptosis-inducing ligand and the thyroid-specific transcription factors Pax8 and TTF-1, leading to expression of the thyroid-specific target genes thyroglobulin, sodium iodide symporter, thyroperoxidase, and thyroid-stimulating hormone receptor and to a moderate accumulation of iodide in ATC cells. CONCLUSION: On the basis of our data, bortezomib represents a promising antineoplastic agent for the treatment of ATC. To improve the clinical outcome, further investigation into the potential of bortezomib therapy of thyroid cancer is clearly warranted.