DNA-methylation signature accurately differentiates pancreatic cancer from chronic pancreatitis in tissue and plasma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Differentiation from chronic pancreatitis (CP) is currently inaccurate in about one-third of cases. Misdiagnoses in both directions, however, have severe consequences for patients. We set out to identify molecular markers for a clear distinction between PDAC and CP. DESIGN: Genome-wide variations of DNA-methylation, messenger RNA and microRNA level as well as combinations thereof were analysed in 345 tissue samples for marker identification. To improve diagnostic performance, we established a random-forest machine-learning approach. Results were validated on another 48 samples and further corroborated in 16 liquid biopsy samples. RESULTS: Machine-learning succeeded in defining markers to differentiate between patients with PDAC and CP, while low-dimensional embedding and cluster analysis failed to do so. DNA-methylation yielded the best diagnostic accuracy by far, dwarfing the importance of transcript levels. Identified changes were confirmed with data taken from public repositories and validated in independent sample sets. A signature of six DNA-methylation sites in a CpG-island of the protein kinase C beta type gene achieved a validated diagnostic accuracy of 100% in tissue and in circulating free DNA isolated from patient plasma. CONCLUSION: The success of machine-learning to identify an effective marker signature documents the power of this approach. The high diagnostic accuracy of discriminating PDAC from CP could have tremendous consequences for treatment success, once the result from still a limited number of liquid biopsy samples would be confirmed in a larger cohort of patients with suspected pancreatic cancer.

Transcriptional variations in the wider peritumoral tissue environment of pancreatic cancer.

Transcriptional profiling was performed on 452 RNA preparations isolated from various types of pancreatic tissue from tumour patients and healthy donors, with a particular focus on peritumoral samples. Pancreatic ductal adenocarcinomas (PDAC) and cystic tumours were most different in these non-tumorous tissues surrounding them, whereas the actual tumours exhibited rather similar transcript patterns. The environment of cystic tumours was transcriptionally nearly identical to normal pancreas tissue. In contrast, the tissue around PDAC behaved a lot like the tumour, indicating some kind of field defect, while showing far less molecular resemblance to both chronic pancreatitis and healthy tissue. This suggests that the major pathogenic difference between cystic and ductal tumours may be due to their cellular environment rather than the few variations between the tumours. Lack of correlation between DNA methylation and transcript levels makes it unlikely that the observed field defect in the peritumoral tissue of PDAC is controlled to a large extent by such epigenetic regulation. Functionally, a strikingly large number of autophagy-related transcripts was changed in both PDAC and its peritumoral tissue, but not in other pancreatic tumours. A transcription signature of 15 autophagy-related genes was established that permits a prognosis of survival with high accuracy and indicates the role of autophagy in tumour biology.

Expression of DRD2 Is Increased in Human Pancreatic Ductal Adenocarcinoma and Inhibitors Slow Tumor Growth in Mice.

BACKGROUND & AIMS: Incidence of and mortality from pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, are almost equivalent, so better treatments are needed. We studied gene expression profiles of PDACs and the functions of genes with altered expression to identify new therapeutic targets. METHODS: We performed microarray analysis to analyze gene expression profiles of 195 PDAC and 41 non-tumor pancreatic tissue samples. We undertook an extensive analysis of the PDAC transcriptome by superimposing interaction networks of proteins encoded by aberrantly expressed genes over signaling pathways associated with PDAC development to identify factors that might alter regulation of these pathways during tumor progression. We performed tissue microarray analysis to verify changes in expression of candidate protein using an independent set of 152 samples (40 nontumor pancreatic tissues, 63 PDAC sections, and 49 chronic pancreatitis samples). We validated the functional relevance of the candidate molecule using RNA interference or pharmacologic inhibitors in pancreatic cancer cell lines and analyses of xenograft tumors in mice. RESULTS: In an analysis of 38,276 human genes and loci, we identified 1676 genes that were significantly up-regulated and 1166 genes that were significantly down-regulated in PDAC compared with nontumor pancreatic tissues. One gene that was up-regulated and associated with multiple signaling pathways that are dysregulated in PDAC was G protein subunit αi2, which has not been previously associated with PDAC. G protein subunit αi2 mediates the effects of dopamine receptor D2 (DRD2) on cyclic adenosine monophosphate signaling; PDAC tissues had a slight but significant increase in DRD2 messenger RNA. Levels of DRD2 protein were substantially increased in PDACs, compared with non-tumor tissues, in tissue microarray analyses. RNA interference knockdown of DRD2 or inhibition with pharmacologic antagonists (pimozide and haloperidol) reduced proliferation of pancreatic cancer cells, induced endoplasmic reticulum stress and apoptosis, and reduced cell migration. RNA interference knockdown of DRD2 in pancreatic tumor cells reduced growth of xenograft tumors in mice, and administration of the DRD2 inhibitor haloperidol to mice with orthotopic xenograft tumors reduced final tumor size and metastasis. CONCLUSIONS: In gene expression profile analysis of PDAC samples, we found the DRD2 signaling pathway to be activated. Inhibition of DRD2 in pancreatic cancer cells reduced proliferation and migration, and slowed growth of xenograft tumors in mice. DRD2 antagonists routinely used for management of schizophrenia might be tested in patients with pancreatic cancer.

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.

Combined evaluation of a panel of protein and miRNA serum-exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity.

Late diagnosis contributes to pancreatic cancer (PaCa) dismal prognosis, urging for reliable, early detection. Serum-exosome protein and/or miRNA markers might be suitable candidates, which we controlled for patients with PaCa. Protein markers were selected according to expression in exosomes of PaCa cell line culture supernatants, but not healthy donors' serum-exosomes. miRNA was selected according to abundant recovery in microarrays of patients with PaCa, but not healthy donors' serum-exosomes and exosome-depleted serum. According to these preselections, serum-exosomes were tested by flow cytometry for the PaCa-initiating cell (PaCIC) markers CD44v6, Tspan8, EpCAM, MET and CD104. Serum-exosomes and exosome-depleted serum was tested for miR-1246, miR-4644, miR-3976 and miR-4306 recovery by qRT-PCR. The majority (95%) of patients with PaCa (131) and patients with nonPa-malignancies reacted with a panel of anti-CD44v6, -Tspan8, -EpCAM and -CD104. Serum-exosomes of healthy donors' and patients with nonmalignant diseases were not reactive. Recovery was tumor grading and staging independent including early stages. The selected miR-1246, miR-4644, miR-3976 and miR-4306 were significantly upregulated in 83% of PaCa serum-exosomes, but rarely in control groups. These miRNA were also elevated in exosome-depleted serum of patients with PaCa, but at a low level. Concomitant evaluation of PaCIC and miRNA serum-exosome marker panels significantly improved sensitivity (1.00, CI: 0.95-1) with a specificity of 0.80 (CI: 0.67-0.90) for PaCa versus all others groups and of 0.93 (CI: 0.81-0.98) excluding nonPa-malignancies. Thus, the concomitant evaluation of PaCIC and PaCa-related miRNA marker panels awaits retrospective analyses of larger cohorts, as it should allow for a highly sensitive, minimally-invasive PaCa diagnostics.

Complementary induction of immunogenic cell death by oncolytic parvovirus H-1PV and gemcitabine in pancreatic cancer.

UNLABELLED: Novel therapies employing oncolytic viruses have emerged as promising anticancer modalities. The cure of particularly aggressive malignancies requires induction of immunogenic cell death (ICD), coupling oncolysis with immune responses via calreticulin, ATP, and high-mobility group box protein B1 (HMGB1) release from dying tumor cells. The present study shows that in human pancreatic cancer cells (pancreatic ductal adenocarcinoma [PDAC] cells n=4), oncolytic parvovirus H-1 (H-1PV) activated multiple interconnected death pathways but failed to induce calreticulin exposure or ATP release. In contrast, H-1PV elevated extracellular HMGB1 levels by 4.0±0.5 times (58%±9% of total content; up to 100 ng/ml) in all infected cultures, whether nondying, necrotic, or apoptotic. An alternative secretory route allowed H-1PV to overcome the failure of gemcitabine to trigger HMGB1 release, without impeding cytotoxicity or other ICD activities of the standard PDAC medication. Such broad resistance of H-1PV-induced HMGB1 release to apoptotic blockage coincided with but was uncoupled from an autocrine interleukin-1ß (IL-1ß) loop. That and the pattern of viral determinants maintained in gemcitabine-treated cells suggested the activation of an inflammasome/caspase 1 (CASP1) platform alongside DNA detachment and/or nuclear exclusion of HMGB1 during early stages of the viral life cycle. We concluded that H-1PV infection of PDAC cells is signaled through secretion of the alarmin HMGB1 and, besides its own oncolytic effect, might convert drug-induced apoptosis into an ICD process. A transient arrest of cells in the cyclin A1-rich S phase would suffice to support compatibility of proliferation-dependent H-1PV with cytotoxic regimens. These properties warrant incorporation of the oncolytic virus H-1PV, which is not pathogenic in humans, into multimodal anticancer treatments. IMPORTANCE: The current therapeutic concepts targeting aggressive malignancies require an induction of immunogenic cell death characterized by exposure of calreticulin (CRT) as well as release of ATP and HMGB1 from dying cells. In pancreatic tumor cells (PDAC cells) infected with the oncolytic parvovirus H-1PV, only HMGB1 was released by all infected cells, whether nondying, necrotic, or succumbing to one of the programmed death pathways, including contraproductive apoptosis. Our data suggest that active secretion of HMGB1 from PDAC cells is a sentinel reaction emerging during early stages of the viral life cycle, irrespective of cell death, that is compatible with and complements cytotoxic regimens. Consistent induction of HMGB1 secretion raised the possibility that this reaction might be a general "alarming" phenomenon characteristic of H-1PV's interaction with the host cell; release of IL-1ß points to the possible involvement of a danger-sensing inflammasome platform. Both provide a basis for further virus-oriented studies.

Stratification of pancreatic tissue samples for molecular studies: RNA-based cellular annotation procedure.

BACKGROUND/OBJECTIVES: Meaningful profiling of pancreatic cancer samples is particularly challenging due to their complex cellular composition. Beyond tumor cells, surgical biopsies contain desmoplastic stroma with infiltrating inflammatory cells, adjacent normal parenchyma, and "non-pancreatic tissues". The risk of misinterpretation rises when the heterogeneous cancer tissues are sub-divided into smaller fragments for multiple analytic procedures. Pre-analytic histological evaluation is the best option to characterize pancreatic tissue samples. Our aim was to develop a complement or alternative procedure to determine the cellular composition of pancreatic cancerous biopsies, basing on intra-analytic molecular annotation. A standard process for sample stratification at a molecular level does not yet exist. Particularly in the case of retrospective or data depository-based studies, when hematoxylin-eosin stained sections are not available, it supports the correct interpretation of expression profiles. METHODS: A five-gene transcriptional signature (RNACellStrat) was defined that allows cell type-specific stratification of pancreatic tissues. Testing biopsy material from biobanks with this procedure demonstrated high correspondence of molecular (qRT-PCR and microarray) and histologic (hematoxylin-eosin stain) evaluations. RESULTS: Notably, about a quarter of randomly selected samples (tissue fragments) were exposed as inappropriate for subsequent clinico-pathological interpretation. CONCLUSIONS: Via immediate intra-analytical procedure, our RNA-based stratification RNACellStrat increases the accuracy and reliability of the conclusions drawn from diagnostic and prognostic molecular information.

Multiscale and multimodal imaging for three-dimensional vascular and histomorphological organ structure analysis of the pancreas.

Exocrine and endocrine pancreas are interconnected anatomically and functionally, with vasculature facilitating bidirectional communication. Our understanding of this network remains limited, largely due to two-dimensional histology and missing combination with three-dimensional imaging. In this study, a multiscale 3D-imaging process was used to analyze a porcine pancreas. Clinical computed tomography, digital volume tomography, micro-computed tomography and Synchrotron-based propagation-based imaging were applied consecutively. Fields of view correlated inversely with attainable resolution from a whole organism level down to capillary structures with a voxel edge length of 2.0 µm. Segmented vascular networks from 3D-imaging data were correlated with tissue sections stained by immunohistochemistry and revealed highly vascularized regions to be intra-islet capillaries of islets of Langerhans. Generated 3D-datasets allowed for three-dimensional qualitative and quantitative organ and vessel structure analysis. Beyond this study, the method shows potential for application across a wide range of patho-morphology analyses and might possibly provide microstructural blueprints for biotissue engineering.

The impact of MFG-E8 in chronic pancreatitis: potential for future immunotherapy?

BACKGROUND: The glycoprotein MFG-E8 mediates phagocytic clearance of apoptotic cells and influences the pathogenesis and progression of inflammatory diseases. MFG-E8 was shown to attenuate the progression of inflammation and to improve survival in septic rats. Accumulating evidence suggests an immunomodulatory link between MFG-E8 and the pro-inflammatory chemokine fractalkine, which may determine the severity of pain, fibrosis, and inflammation in chronic pancreatitis (CP). METHODS: The expression and localization of MFG-E8 was investigated in CP (n=62), and normal pancreas (NP; n=34) by QRT-PCR, Western-blot and immunohistochemistry analyses. Results were correlated with mRNA expression of fractalkine, CX3CR1, and with the presence and degree of pain and fibrosis. Human pancreatic stellate cells (hPSCs) were isolated from CP tissues and evaluated for MFG-E8 mRNA expression after fractalkine stimulation. RESULTS: MFG-E8-mRNA was significantly overexpressed in CP and isolated hPSCs when compared to NP. Western-blot and immunohistochemistry analysis confirmed accumulation of MFG-E8 in CP, with noticeably increased MFG-E8 immunoreactivity in tubular complexes. MFG-E8 expression correlated significantly with fractalkine expression, severe fibrosis, and the presence of pain in CP patients. Stimulation of hPSCs with fractalkine led to a significant increase in MFG-E8 expression. CONCLUSIONS: In the present study, we demonstrated for the first time that MFG-E8 is significantly up-regulated in CP patients and together with fractalkine correlated noticeably with severe fibrosis and the presence of pain. hPSCs overexpress MFG-E8 upon fractalkine stimulation in vitro, which underlines the suggested immunmodulatory link in CP and may be a key mechanism in CP fibrogenesis and pain generation. Taken together, these novel findings suggest that MFG-E8 blockade may be a promising tool for future immunotherapy in CP to attenuate both fibrosis and pain sensation.

Seromic profiling of ovarian and pancreatic cancer.

Autoantibodies, a hallmark of both autoimmunity and cancer, represent an easily accessible surrogate for measuring adaptive immune responses to cancer. Sera can now be assayed for reactivity against thousands of proteins using microarrays, but there is no agreed-upon standard to analyze results. We developed a set of tailored quality control and normalization procedures based on ELISA validation to allow patient comparisons and determination of individual cutoffs for specificity and sensitivity. Sera from 60 patients with pancreatic cancer, 51 patients with ovarian cancer, and 53 age-matched healthy donors were used to assess the binding of IgG antibodies against a panel of >8000 human antigens using protein microarrays and fluorescence detection. The resulting data interpretation led to the definition and ranking of proteins with preferred recognition by the sera from cancer patients in comparison with healthy donors, both by frequency and strength of signal. We found that 202 proteins were preferentially immunogenic in ovarian cancer sera compared to 29 in pancreatic cancer, with few overlaps. Correlates of autoantibody signatures with known tumor expression of corresponding antigens, functional pathways, clinical stage, and outcome were examined. Serological analysis of arrays displaying the complete human proteome (seromics) represents a new era in cancer immunology, opening the way to defining the repertoire of the humoral immune response to cancer.

Blood-Based Diagnosis and Risk Stratification of Patients with Pancreatic Intraductal Papillary Mucinous Neoplasm (IPMN).

PURPOSE: Intraductal papillary mucinous neoplasm (IPMN) is a precursor of pancreatic ductal adenocarcinoma. Low-grade dysplasia has a relatively good prognosis, whereas high-grade dysplasia and IPMN invasive carcinoma require surgical intervention. However, diagnostic distinction is difficult. We aimed to identify biomarkers in peripheral blood for accurate discrimination. EXPERIMENTAL DESIGN: Sera were obtained from 302 patients with IPMNs and 88 healthy donors. For protein biomarkers, serum samples were analyzed on microarrays made of 2,977 antibodies. A support vector machine (SVM) algorithm was applied to define classifiers, which were validated on a separate sample set. For microRNA biomarkers, a PCR-based screen was performed for discovery. Biomarker candidates confirmed by quantitative PCR were used to train SVM classifiers, followed by validation in a different sample set. Finally, a combined SVM classifier was established entirely independent of the earlier analyses, again using different samples for training and validation. RESULTS: Panels of 26 proteins or seven microRNAs could distinguish high- and low-risk IPMN with an AUC value of 95% and 94%, respectively. Upon combination, a panel of five proteins and three miRNAs yielded an AUC of 97%. These values were much better than those obtained in the same patient cohort by using the guideline criteria for discrimination. In addition, accurate discrimination was achieved between other patient subgroups. CONCLUSIONS: Protein and microRNA biomarkers in blood allow precise diagnosis and risk stratification of IPMN cases, which should improve patient management and thus the prognosis of IPMN patients. See related commentary by Löhr and Pantel, p. 1387.

Neuronal plasticity in chronic pancreatitis is mediated via the neurturin/GFRα2 axis.

The glial cell line-derived neurotrophic factor (GDNF) family member neurturin (NRTN) and its receptor GFRα2 play a deciding role in the normal development of pancreatic parasympathetic innervation. In this study, we aimed at investigating the role of NRTN/GFRα2 axis in pancreatic neuropathy in human chronic pancreatitis (CP). Expression of NRTN/GFRα2 was compared between normal human pancreas (NP) and CP tissues via immunohistochemistry, immunoblotting, and quantitative RT-PCR and correlated to abdominal pain sensation. To elucidate the impact of NRTN in pancreatic neuroplasticity, neuronal phenotype and glial density were quantified via an in vitro neuroplasticity assay in dissociated newborn rat dorsal root ganglia (DRG) cultured 1) in CP tissue extracts depleted from NRTN, 2) in NP, 3) in untreated CP tissue extracts, and 4) CP extracts in which nerve growth factor, glial cell derived-neurotrophic factor, or TGF-ß(1) was depleted. NRTN and GFRα2 were highly upregulated in CP, especially in intrapancreatic nerves and the extracellular matrix. CP tissue demonstrated increased amounts of mature multimeric NRTN and elevated levels of GFRα2. The noticeable neurotrophic effect of CP tissue extracts on DRG neurons was diminished upon blockade of NRTN from these extracts. However, blockade of NRTN from CP extracts did not influence the density of DRG glia cells. In conclusion, the NRTN/GFRα2 axis is activated during the course of CP and represents a major key player in the reactive neural alterations in CP. This is the first study to provide functional evidence for the contribution of neurotrophic factors to neuroplasticity in CP.

Real-imaging cDNA-AFLP transcript profiling of pancreatic cancer patients: Egr-1 as a potential key regulator of muscle cachexia.

BACKGROUND: Cancer cachexia is a progressive wasting syndrome and the most prevalent characteristic of cancer in patients with advanced pancreatic adenocarcinoma. We hypothesize that genes expressed in wasted skeletal muscle of pancreatic cancer patients may determine the initiation and severity of cachexia syndrome. EXPERIMENTAL DESIGN: We studied gene expression in skeletal muscle biopsies from pancreatic cancer patients with and without cachexia utilizing Real-Imaging cDNA-AFLP-based transcript profiling for genome-wide expression analysis. RESULTS: Our approach yielded 183 cachexia-associated genes. Ontology analysis revealed characteristic changes for a number of genes involved in muscle contraction, actin cytoskeleton rearrangement, protein degradation, tissue hypoxia, immediate early response and acute-phase response. CONCLUSIONS: We demonstrate that Real-Imaging cDNA-AFLP analysis is a robust method for high-throughput gene expression studies of cancer cachexia syndrome in patients with pancreatic cancer. According to quantitative RT-PCR validation, the expression levels of genes encoding the acute-phase proteins α-antitrypsin and fibrinogen α and the immediate early response genes Egr-1 and IER-5 were significantly elevated in the skeletal muscle of wasted patients. By immunohistochemical and Western immunoblotting analysis it was shown, that Egr-1 expression is significantly increased in patients with cachexia and cancer. This provides new evidence that chronic activation of systemic inflammatory response might be a common and unifying factor of muscle cachexia.

Toward 3D-bioprinting of an endocrine pancreas: A building-block concept for bioartificial insulin-secreting tissue.

Three-dimensional bioprinting of an endocrine pancreas is a promising future curative treatment for patients with insulin secretion deficiency. In this study, we present an end-to-end concept from the molecular to the macroscopic level. Building-blocks for a hybrid scaffold device of hydrogel and functionalized polycaprolactone were manufactured by 3D-(bio)printing. Pseudoislet formation from INS-1 cells after bioprinting resulted in a viable and proliferative experimental model. Transcriptomics showed an upregulation of proliferative and ß-cell-specific signaling cascades, downregulation of apoptotic pathways, overexpression of extracellular matrix proteins, and VEGF induced by pseudoislet formation and 3D-culture. Co-culture with endothelial cells created a natural cellular niche with enhanced insulin secretion after glucose stimulation. Survival and function of pseudoislets after explantation and extensive scaffold vascularization of both hydrogel and heparinized polycaprolactone were demonstrated in vivo. Computer simulations of oxygen, glucose and insulin flows were used to evaluate scaffold architectures and Langerhans islets at a future perivascular transplantation site.

The HIF-prolyl hydroxylases have distinct and nonredundant roles in colitis-associated cancer.

Colitis-associated colorectal cancer (CAC) is a severe complication of inflammatory bowel disease (IBD). HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) control cellular adaptation to hypoxia and are considered promising therapeutic targets in IBD. However, their relevance in the pathogenesis of CAC remains elusive. We induced CAC in Phd1-/-, Phd2+/-, Phd3-/-, and WT mice with azoxymethane (AOM) and dextran sodium sulfate (DSS). Phd1-/- mice were protected against chronic colitis and displayed diminished CAC growth compared with WT mice. In Phd3-/- mice, colitis activity and CAC growth remained unaltered. In Phd2+/- mice, colitis activity was unaffected, but CAC growth was aggravated. Mechanistically, Phd2 deficiency (i) increased the number of tumor-associated macrophages in AOM/DSS-induced tumors, (ii) promoted the expression of EGFR ligand epiregulin in macrophages, and (iii) augmented the signal transducer and activator of transcription 3 and extracellular signal-regulated kinase 1/2 signaling, which at least in part contributed to aggravated tumor cell proliferation in colitis-associated tumors. Consistently, Phd2 deficiency in hematopoietic (Vav:Cre-Phd2fl/fl) but not in intestinal epithelial cells (Villin:Cre-Phd2fl/fl) increased CAC growth. In conclusion, the 3 different PHD isoenzymes have distinct and nonredundant effects, promoting (PHD1), diminishing (PHD2), or neutral (PHD3), on CAC growth.

Patient-specific modeling of stroma-mediated chemoresistance of pancreatic cancer using a three-dimensional organoid-fibroblast co-culture system.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are considered to play a fundamental role in pancreatic ductal adenocarcinoma (PDAC) progression and chemoresistance. Patient-derived organoids have demonstrated great potential as tumor avatars for drug response prediction in PDAC, yet they disregard the influence of stromal components on chemosensitivity. METHODS: We established direct three-dimensional (3D) co-cultures of primary PDAC organoids and patient-matched CAFs to investigate the effect of the fibroblastic compartment on sensitivity to gemcitabine, 5-fluorouracil and paclitaxel treatments using an image-based drug assay. Single-cell RNA sequencing was performed for three organoid/CAF pairs in mono- and co-culture to uncover transcriptional changes induced by tumor-stroma interaction. RESULTS: Upon co-culture with CAFs, we observed increased proliferation and reduced chemotherapy-induced cell death of PDAC organoids. Single-cell RNA sequencing data evidenced induction of a pro-inflammatory phenotype in CAFs in co-cultures. Organoids showed increased expression of genes associated with epithelial-to-mesenchymal transition (EMT) in co-cultures and several potential receptor-ligand interactions related to EMT were identified, supporting a key role of CAF-driven induction of EMT in PDAC chemoresistance. CONCLUSIONS: Our results demonstrate the potential of personalized PDAC co-cultures models not only for drug response profiling but also for unraveling the molecular mechanisms involved in the chemoresistance-supporting role of the tumor stroma.

Radiotherapy orchestrates natural killer cell dependent antitumor immune responses through CXCL8.

Radiotherapy is a mainstay cancer therapy whose antitumor effects partially depend on T cell responses. However, the role of Natural Killer (NK) cells in radiotherapy remains unclear. Here, using a reverse translational approach, we show a central role of NK cells in the radiation-induced immune response involving a CXCL8/IL-8-dependent mechanism. In a randomized controlled pancreatic cancer trial, CXCL8 increased under radiotherapy, and NK cell positively correlated with prolonged overall survival. Accordingly, NK cells preferentially infiltrated irradiated pancreatic tumors and exhibited CD56dim-like cytotoxic transcriptomic states. In experimental models, NF-κB and mTOR orchestrated radiation-induced CXCL8 secretion from tumor cells with senescence features causing directional migration of CD56dim NK cells, thus linking senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined high-dose radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice, suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy.

Reduced α-dystroglycan expression correlates with shortened patient survival in pancreatic cancer.

BACKGROUND: Deregulation of cell-matrix interactions is considered an important factor in malignant transformation. Dystroglycan forms the interface between the basement membrane and the cell membrane in epithelia by its two subunits, α- and ß-dystroglycan. Aberrant expression of dystroglycan has been observed in various human cancers. Here we assessed the expression of dystroglycan in pancreatic ductal adenocarcinoma (PDAC) and analyzed its association with clinical parameters. METHODS: mRNA levels of dystroglycan were determined by real-time quantitative PCR in tissue samples from 60 patients with PDAC, from 48 patients with CP, and from 18 healthy donors. Furthermore, pancreatic cancer specimens of 53 patients were analyzed by immunohistochemistry using specific α- and ß-dystroglycan antibodies. The staining was semiquantitatively evaluated and correlated to patient survival using the Kaplan-Meier method. RESULTS: On the mRNA level, dystroglycan was down-regulated in PDAC compared with the normal pancreas. In normal pancreatic tissues, α- and ß-dystroglycans were mainly expressed on the basolateral cell membrane of acinar and ductal cells, while islet cells showed a cytoplasmatic staining. In contrast, in PDAC tissues, this membrane staining pattern was lost and replaced by a cytoplasmatic staining, suggesting impairments in the membrane translocation of both dystroglycans. Semiquantitative analysis revealed a significant inverse correlation of α- (but not ß-) dystroglycan staining and postoperative survival (P=0.039). CONCLUSION: Reduced expression and altered localization of dystroglycan is common in PDAC, potentially contributing to the aggressive behavior of this disease.

Human Retrotransposons and the Global Shutdown of Homeostatic Innate Immunity by Oncolytic Parvovirus H-1PV in Pancreatic Cancer.

Although the oncolytic parvovirus H-1PV has entered clinical trials, predicting therapeutic success remains challenging. We investigated whether the antiviral state in tumor cells determines the parvoviral oncolytic efficacy. The interferon/interferon-stimulated genes (IFN/ISG)-circuit and its major configurator, human endogenous retroviruses (HERVs), were evaluated using qRT-PCR, ELISA, Western blot, and RNA-Seq techniques. In pancreatic cancer cell lines, H-1PV caused a late global shutdown of innate immunity, whereby the concomitant inhibition of HERVs and IFN/ISGs was co-regulatory rather than causative. The growth-inhibitory IC50 doses correlated with the power of suppression but not with absolute ISG levels. Moreover, H-1PV was not sensitive to exogenous IFN despite upregulated antiviral ISGs. Such resistance questioned the biological necessity of the oncotropic ISG-shutdown, which instead might represent a surrogate marker for personalized oncolytic efficacy. The disabled antiviral homeostasis may modify the activity of other viruses, as demonstrated by the reemergence of endogenous AluY-retrotransposons. This way of suppression may compromise the interferogenicity of drugs having gemcitabine-like mechanisms of action. This shortcoming in immunogenic cell death induction is however amendable by immune cells which release IFN in response to H-1PV.

Promoter Hypermethylation Promotes the Binding of Transcription Factor NFATc1, Triggering Oncogenic Gene Activation in Pancreatic Cancer.

Studies have indicated that some genes involved in carcinogenesis are highly methylated in their promoter regions but nevertheless strongly transcribed. It has been proposed that transcription factors could bind specifically to methylated promoters and trigger transcription. We looked at this rather comprehensively for pancreatic ductal adenocarcinoma (PDAC) and studied some cases in more detail. Some 2% of regulated genes in PDAC exhibited higher transcription coupled to promoter hypermethylation in comparison to healthy tissue. Screening 661 transcription factors, several were found to bind specifically to methylated promoters, in particular molecules of the NFAT family. One of them-NFATc1-was substantially more strongly expressed in PDAC than control tissue and exhibited a strong oncogenic role. Functional studies combined with computational analyses allowed determining affected genes. A prominent one was gene ALDH1A3, which accelerates PDAC metastasis and correlates with a bad prognosis. Further studies confirmed the direct up-regulation of ALDH1A3 transcription by NFATc1 promoter binding in a methylation-dependent process, providing insights into the oncogenic role of transcription activation in PDAC that is promoted by DNA methylation.