Assessing the Layer-by-Layer Assembly of Cellulose Nanofibrils and Polyelectrolytes in Pancreatic Tumor Spheroid Formation.

Three-dimensional (3D) tumor spheroids are regarded as promising models for utilization as preclinical assessments of chemo-sensitivity. However, the creation of these tumor spheroids presents challenges, given that not all tumor cell lines are able to form consistent and regular spheroids. In this context, we have developed a novel layer-by-layer coating of cellulose nanofibril-polyelectrolyte bilayers for the generation of spheroids. This technique builds bilayers of cellulose nanofibrils and polyelectrolytes and is used here to coat two distinct 96-well plate types: nontreated/non-sterilized and Nunclon Delta. In this work, we optimized the protocol aimed at generating and characterizing spheroids on difficult-to-grow pancreatic tumor cell lines. Here, diverse parameters were explored, encompassing the bilayer count (five and ten) and multiple cell-seeding concentrations (10, 100, 200, 500, and 1000 cells per well), using four pancreatic tumor cell lines-KPCT, PANC-1, MiaPaCa-2, and CFPAC-I. The evaluation includes the quantification (number of spheroids, size, and morphology) and proliferation of the produced spheroids, as well as an assessment of their viability. Notably, our findings reveal a significant influence from both the number of bilayers and the plate type used on the successful formation of spheroids. The novel and simple layer-by-layer-based coating method has the potential to offer the large-scale production of spheroids across a spectrum of tumor cell lines.

An idiosyncratic zonated stroma encapsulates desmoplastic liver metastases and originates from injured liver.

A perimetastatic capsule is a strong positive prognostic factor in liver metastases, but its origin remains unclear. Here, we systematically quantify the capsule's extent and cellular composition in 263 patients with colorectal cancer liver metastases to investigate its clinical significance and origin. We show that survival improves proportionally with increasing encapsulation and decreasing tumor-hepatocyte contact. Immunostaining reveals the gradual zonation of the capsule, transitioning from benign-like NGFRhigh stroma at the liver edge to FAPhigh stroma towards the tumor. Encapsulation correlates with decreased tumor viability and preoperative chemotherapy. In mice, chemotherapy and tumor cell ablation induce capsule formation. Our results suggest that encapsulation develops where tumor invasion into the liver plates stalls, representing a reparative process rather than tumor-induced desmoplasia. We propose a model of metastases growth, where the efficient tumor colonization of the liver parenchyma and a reparative liver injury reaction are opposing determinants of metastasis aggressiveness.

FPR2 Shapes an Immune-Excluded Pancreatic Tumor Microenvironment and Drives T-cell Exhaustion in a Sex-Dependent Manner.

Sex-driven immune differences can affect tumor progression and the landscape of the tumor microenvironment. Deeper understanding of these differences in males and females can inform patient selection to improve sex-optimized immunotherapy treatments. In this study, single-cell RNA sequencing and protein analyses uncovered a subpopulation of myeloid cells in pancreatic lesions associated with an immune-excluded tumor phenotype and effector T-cell exhaustion exclusively in females. This myeloid subpopulation was positively correlated with poor survival and genetic signatures of M2-like macrophages and T-cell exhaustion in females. The G-protein coupled receptor formyl peptide receptor 2 (FPR2) mediated these immunosuppressive effects. In vitro, treatment of myeloid cells with a specific FPR2 antagonist prevented exhaustion and enhanced cytotoxicity of effector cells. Proteomic analysis revealed high expression of immunosuppressive secretory proteins PGE2 and galectin-9, enriched integrin pathway, and reduced proinflammatory signals like TNFα and IFNγ in female M2-like macrophages upon FPR2 agonist treatment. In addition, myeloid cells treated with FPR2 agonists induced TIM3 and PD-1 expression only in female T cells. Treatment with anti-TIM3 antibodies reversed T-cell exhaustion and stimulated their ability to infiltrate and kill pancreatic spheroids. In vivo, progression of syngeneic pancreatic tumors was significantly suppressed in FPR2 knockout (KO) female mice compared with wild-type (WT) female mice and to WT and FPR2 KO male mice. In female mice, inoculation of tumors with FPR2 KO macrophages significantly reduced tumor growth compared with WT macrophages. Overall, this study identified an immunosuppressive function of FPR2 in females, highlighting a potential sex-specific precision immunotherapy strategy. SIGNIFICANCE: FPR2 is a sex-dependent mediator of macrophage function in pancreatic cancer and can be targeted to reprogram macrophages and stimulate antitumor immunity in females.

Deletion of Smad7 Ameliorates Intestinal Inflammation and Contributes to Fibrosis.

BACKGROUND: Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of transforming growth factor (TGF)-ß compared with non-IBD controls. SMAD7 negatively regulates TGF-ß signaling. An earlier study aiming to target Smad7 showed a lack of clinical benefit. It remains unknown whether inhibition of SMAD7 is beneficial in specific settings of IBD. We evaluated the effect of Smad7 deficiency on inflammation, fibrogenesis, and wound healing. METHODS: For the initiation of fibrosis in Smad7-/- (Smad7Δex-I) CD-1 mice, the dextran sodium sulfate-induced chronic colitis model and the heterotopic transplantation model of fibrosis were used. Wound closure of fibroblasts from Smad7-/- mice was determined using culture inserts and electric cell-substrate impedance sensing in vitro. RESULTS: In dextran sodium sulfate-induced chronic colitis, Smad7 deficiency was associated with ameliorated inflammation, as evidenced by decreased clinical score, histological score, and myeloperoxidase activity. Absence of SMAD7 decreased T-cell accumulation in colonic tissue and tumor necrosis factor (TNF) mRNA expression levels. Smad7-/- mice showed a significant increase in hydroxyproline and collagen content, as well as ColIVa1 mRNA expression. Wild type mice transplanted with terminal ileum from Smad7-/- mice in the heterotopic animal model for intestinal fibrosis showed a significant increase in collagen content and protein expression of α-smooth muscle actin. CONCLUSIONS: Smad7 deficiency is associated with a decrease in intestinal inflammation and an increase in fibrosis. Targeting SMAD7 constitutes a potential new treatment option for IBD; progression of disease-associated fibrosis should be considered.

We evaluated the effect of Smad7 deficiency on inflammation and fibrogenesis. Smad7 deficiency was associated with ameliorated inflammation and increased collagen deposition. When targeting Smad7 as therapeutic strategy in IBD, potential initiation or aggravation of fibrosis should be considered.

Targeting myeloid suppressive cells revives cytotoxic anti-tumor responses in pancreatic cancer.

Immunotherapy for cancer that aims to promote T cell anti-tumor activity has changed current clinical practice, where some previously lethal cancers have now become treatable. However, clinical trials with low response rates have been disappointing for pancreatic ductal adenocarcinoma (PDAC). One suggested explanation is the accumulation of dominantly immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells in the tumor microenvironment (TME). Using retrospectively collected tumor specimens and transcriptomic data from PDAC, we demonstrate that expression of the scavenger receptor MARCO correlates with poor prognosis and a lymphocyte-excluding tumor phenotype. PDAC cell lines produce IL-10 and induce high expression of MARCO in myeloid cells, and this was further enhanced during hypoxic conditions. These myeloid cells suppressed effector T and natural killer (NK) cells and blocked NK cell tumor infiltration and tumor killing in a PDAC 3D-spheroid model. Anti-human MARCO (anti-hMARCO) antibody targeting triggered the repolarization of tumor-associated macrophages and activated the inflammasome machinery, resulting in IL-18 production. This in turn enhanced T cell and NK cell functions. The targeting of MARCO thus remodels the TME and represents a rational approach to make immunotherapy more efficient in PDAC patients.

Pancreatic Ductal Adenocarcinoma: Preclinical in vitro and ex vivo Models.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most overlooked cancers despite its dismal median survival time of 6 months. The biggest challenges in improving patient survival are late diagnosis due to lack of diagnostic markers, and limited treatment options due to almost complete therapy resistance. The past decades of research identified the dense stroma and the complex interplay/crosstalk between the cancer- and the different stromal cells as the main culprits for the slow progress in improving patient outcome. For better ex vivo simulation of this complex tumor microenvironment the models used in PDAC research likewise need to become more diverse. Depending on the focus of the investigation, several in vitro and in vivo models for PDAC have been established in the past years. Particularly, 3D cell culture such as spheroids and organoids have become more frequently used. This review aims to examine current PDAC in vitro models, their inherent limitations, and their successful implementations in research.

Stabilization of the classical phenotype upon integration of pancreatic cancer cells into the duodenal epithelium.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid tumors. Based on transcriptomic classifiers, basal-like and classical PDAC subtypes have been defined that differ in prognosis. Cells of both subtypes can coexist in individual tumors; however, the contribution of either clonal heterogeneity or microenvironmental cues to subtype heterogeneity is unclear. Here, we report the spatial tumor phenotype dynamics in a cohort of patients in whom PDAC infiltrated the duodenal wall, and identify the duodenal epithelium as a distinct PDAC microniche. MATERIALS AND METHODS: We used serial multiplex quantitative immunohistochemistry (smq-IHC) for 24 proteins to phenotypically chart PDAC tumor cells in patients whose tumors infiltrated the duodenal epithelium. Additionally, we used a genetically engineered mouse model to study the PDAC cell phenotype in the small intestinal epithelium in a controlled genetic background. RESULT: We show that pancreatic cancer cells revert to non-destructive growth upon integration into the duodenal epithelium, where they adopt traits of intestinal cell differentiation, associated with phenotypical stabilization of the classical subtype. The integrated tumor cells replace epithelial cells in an adenoma-like manner, as opposed to invasive growth in the submucosa. Finally, we show that this phenomenon is shared between species, by confirming duodenal integration and phenotypic switching in a genetic PDAC mouse model. DISCUSSION: Our results identify the duodenal epithelium as a distinct PDAC microniche and tightly link microenvironmental cue to cancer transcriptional subtypes. The phenomenon of "intestinal mimicry" provides a unique opportunity for the systematic investigation of microenvironmental influences on pancreatic cancer plasticity.

3D heterospecies spheroids of pancreatic stroma and cancer cells demonstrate key phenotypes of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, partly due to the dense desmoplasia and a lack of suitable model systems to study. In the present work, we developed a 3D heterospecies spheroid model to study the microenvironmental interactions between tumor cells and stellate cells which can also be employed to test therapeutic regimens. We set up monospheroids and heterospheroids made up from murine pancreatic stellate cells (mPSCs) and human PDAC cells (Panc1), which allowed for direct isolation of mRNA from a mixed cell population followed by an in silico separation of the RNA-seq reads. Global transcript level changes for cells in heterospheroids versus monospheroids were calculated, followed by gene set enrichment analysis and molecular subtype analysis. We observed an apparent shift of Panc1 from the classical to the squamous/basal-like phenotype upon co-culture with mPSCs. Moreover, mPSCs acquired a different cancer-associated fibroblast-related phenotype upon co-culture with Panc1. We analyzed the tumor cell-specific chemosensitivities towards gemcitabine, paclitaxel and SN38 and compared these to published pharmacotranscriptomic signatures. In conclusion, our heterospecies spheroid model reflected key aspects of PDAC and facilitated the study of intercellular interactions between tumor and stroma while additionally proving to be a good model for studying therapeutic responses.

Targeting of Smad7 in Mesenchymal Cells Does Not Exacerbate Fibrosis During Experimental Chronic Pancreatitis.

OBJECTIVES: Transforming growth factor-ß (TGF-ß)-mediated accumulation of extracellular matrix proteins such as collagen I is a common feature of fibrosis. Pancreatic stellate cells play an integral role in the pathogenesis of pancreatitis, and their profibrotic ability is mainly mediated by TGF-ß signaling. To specifically address the role of fibrogenic cells in experimental pancreatic fibrosis, we deleted Smad7, the main feedback inhibitor of TGF-ß signaling in this cell type in mice. METHODS: A mouse strain harboring a conditional knockout allele of Smad7 (Smad7fl/fl) with the tamoxifen-inducible inducible Col1a2-CreERT allele was generated and compared with wild-type mice challenged with the cerulein-based model of chronic pancreatitis. RESULTS: Pancreatic stellate cells lacking Smad7 had significantly increased collagen I and fibronectin production and showed a higher activation level in vitro. Surprisingly, the fibrotic index in the pancreata of treated conditional knockout mice was only slightly increased, without statistical significance. Except for fibronectin, the expression of different extracellular matrix proteins and the numbers of fibroblasts and inflammatory cells were similar between Smad7-mutant and control mice. CONCLUSIONS: There was no clear evidence that the lack of Smad7 in pancreatic stellate cells plays a major role in experimental pancreatitis, at least in the mouse model investigated here.

Multimodal Imaging of Pancreatic Ductal Adenocarcinoma Using Multifunctional Nanoparticles as Contrast Agents.

Late diagnosis and refractory behavior toward current treatment protocols make pancreatic ductal adenocarcinoma (PDAC) one of the most difficult cancer forms to treat. The imaging-based approach plays an important role to identify potentially curable PDAC patients in high-risk groups at the early stage. In the present study, we developed a core-shell structured gold nanorod (AuNR) as a contrast agent for multimodal imaging and investigated its application for PDAC diagnosis. The composite nanoparticles composed of a AuNR core inside a layer of mesoporous silica that was then coated with a gadolinium oxide carbonate shell (AuNR-SiO2-Gd) are designed to be used in magnetic resonance imaging (MRI), X-ray computed tomography (CT), and photoacoustic imaging (PAI). A phantom study with the AuNR-SiO2-Gd NPs demonstrated higher MRI contrast compared to Gadovist and higher X-ray attenuation than Visipaque. A strong, stable, and broad wavelength range signal with a peak at 800 nm was observed in PAI. The AuNR-SiO2-Gd NPs showed significant contrast enhancement under PAI/MRI/CT in both the liver and spleen of control mice after intravenous administration. The utility in PDAC was studied in a genetically engineered mouse model carrying Kras and p53 mutations, which develops spontaneous tumors and keeps the desmoplasia and hypovascularity feature of PDAC in patients. The AuNR-SiO2-Gd NPs were highly accumulated in the surrounding soft tissues but were sparsely distributed throughout the tumor due to dense stroma infiltration and poor tumor vascularization. Hence, a negative contrast within the tumor area in CT/PAI and a positive contrast in MRI were observed. In conclusion, AuNR-SiO2-Gd NPs have good potential to be developed as a multimodal contrast agent for PDAC, which might improve early diagnosis and benefit the clinical outcome for PDAC patients.

The vitamin D analogue calcipotriol promotes an anti-tumorigenic phenotype of human pancreatic CAFs but reduces T cell mediated immunity.

The pancreatic tumour stroma is composed of phenotypically heterogenous cancer-associated fibroblasts (CAFs) with both pro- and anti-tumorigenic functions. Here, we studied the impact of calcipotriol, a vitamin D3 analogue, on the activation of human pancreatic CAFs and T cells using 2- and 3-dimensional (2D, 3D) cell culture models. We found that calcipotriol decreased CAF proliferation and migration and reduced the release of the pro-tumorigenic factors prostaglandin E2, IL-6, periostin, and leukemia inhibitory factor. However, calcipotriol promoted PD-L1 upregulation, which could influence T cell mediated tumour immune surveillance. Calcipotriol reduced T cell proliferation and production of IFN-γ, granzyme B and IL-17, but increased IL-10 secretion. These effects were even more profound in the presence of CAFs in 2D cultures and in the presence of CAFs and pancreatic tumour cell line (PANC-1) spheroids in 3D cultures. Functional assays on tumour infiltrating lymphocytes also showed a reduction in T cell activation by calcipotriol. This suggests that calcipotriol reduces the tumour supportive activity of CAFs but at the same time reduces T cell effector functions, which could compromise the patients' tumour immune surveillance. Thus, vitamin D3 analogues appear to have dual functions in the context of pancreatic cancer, which could have important clinical implications.

Immunohistochemical profiling of liver metastases and matched-pair analysis in patients with metastatic pancreatic ductal adenocarcinoma.

BACKGROUND: The purpose of the current study was to investigate the immunohistochemical (IHC) profile of liver metastases (LM) in patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: Expression of 15 IHC markers in liver biopsies from 77 patients with PDAC, who were diagnosed between 2010 and 2014, were evaluated. In a separate subgroup analysis (n = 12), paired samples (LM and primary tumor) from the same patient were investigated for IHC profile differences. RESULTS: LM samples were classified as pancreatobiliary-type (PB-type) in 72 patients (93.5%), intestinal-type (INT-type) in four patients (5.2%), and squamous in one patient (1.3%). There was no significant difference in overall survival (OS) between LM of the PB-type or INT-type (p = 0.097). In a multivariate analysis, age <70 years (p = 0.047), absence of SMAD4 mutation (p = 0.026), absence of CDX2 expression (p = 0.003), and well to moderate differentiation were significant prognostic factors for better OS in patients with LM (p = 0.031). Analysis of paired tissue samples from LM and the primary tumor revealed a difference in CDX2 (50% increase, p = 0.125) and SMAD4 (33% loss of SMAD4, p = 0.375). CONCLUSIONS: CDX2 expression and SMAD4 mutation indicate a poor outcome in patients with LM of PDAC. Matched-pair analysis revealed differences in distinct IHC marker expression.

Tamoxifen affects chronic pancreatitis-related fibrogenesis in an experimental mouse model: an effect beyond Cre recombination.

Tamoxifen is very successfully used for the induction of CreERT -mediated genomic recombination in conditional mouse models. Recent studies, however, indicated that tamoxifen might also affect the fibrotic response in several disease models following administration, both in vitro and in vivo. In order to investigate a possible effect of tamoxifen on pancreatic fibrogenesis and to evaluate an optimal treatment scheme in an experimental pancreatitis mouse model, we administered tamoxifen by oral gavage to both male and female C57BL/6J mice and then waited for different periods of time before inducing chronic pancreatitis by cerulein. We observed a sex-specific and time-dependent effect of tamoxifen on the fibrotic response as measured by collagen deposition and the number of myofibroblasts and macrophages. The findings of in vitro studies, in which cerulein was administrated with or without 4-hydroxytamoxifen to stimulate primary murine female and male pancreatic stellate cells, supported our in vivo observations. Real-time PCR also indicated that this effect may be related to differences in ERα expression between female and male stellate cells. Our data demonstrate that tamoxifen administration has unignorable side effects, which affect the experimental outcome in a cerulein-based model of chronic pancreatitis in mice. We suggest a 2-week waiting period before cerulein administration to reduce side effects to a minimum for the described fibrosis model in female mice.

Role of c-MET Inhibitors in Overcoming Drug Resistance in Spheroid Models of Primary Human Pancreatic Cancer and Stellate Cells.

Pancreatic stellate cells (PSCs) are a key component of tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and contribute to drug resistance. c-MET receptor tyrosine kinase activation plays an important role in tumorigenesis in different cancers including PDAC. In this study, effects of PSC conditioned medium (PCM) on c-MET phosphorylation (by immunocytochemistry enzyme-linked immunosorbent assay (ELISA)) and drug response (by sulforhodamine B assay) were investigated in five primary PDAC cells. In novel 3D-spheroid co-cultures of cyan fluorescence protein (CFP)-firefly luciferase (Fluc)-expressing primary human PDAC cells and green fluorescence protein (GFP)-expressing immortalized PSCs, PDAC cell growth and chemosensitivity were examined by luciferase assay, while spheroids' architecture was evaluated by confocal microscopy. The highest phospho-c-MET expression was detected in PDAC5 and its subclone sorted for "stage specific embryonic antigen-4" (PDAC5 (SSEA4)). PCM of cells pre-incubated with PDAC conditioned medium, containing increased hepatocyte growth factor (HGF) levels, made PDAC cells significantly more resistant to gemcitabine, but not to c-MET inhibitors. Hetero-spheroids containing both PSCs and PDAC5 (SSEA4) cells were more resistant to gemcitabine compared to PDAC5 (SSEA4) homo-spheroids. However, c-MET inhibitors (tivantinib, PHA-665752 and crizotinib) were equally effective in both spheroid models. Experiments with primary human PSCs confirmed the main findings. In conclusion, we developed spheroid models to evaluate PSC-PDAC reciprocal interaction, unraveling c-MET inhibition as an important therapeutic option against drug resistant PDAC.

RCAN1 is a marker of oxidative stress, induced in acute pancreatitis.

BACKGROUND: To date, there still is a lack of specific acute pancreatitis markers and specifically an early marker that can reliably predict disease severity. The inflammatory response in acute pancreatitis is mediated in part through oxidative stress and calcineurin-NFAT (Nuclear Factor of Activated T-cells) signaling, which is inducing its own negative regulator, regulator of calcineurin 1 (RCAN1). Caerulein induction is a commonly used in vivo model of experimental acute pancreatitis. Caerulein induces CN-NFAT signaling, reactive oxygen species and inflammation. METHODS: To screen for potential markers of acute pancreatitis, we used the caerulein model of experimental acute pancreatitis (AP) in C57Bl/6 J mice. Pancreata from treated and control mice were used for expression profiling. Promising gene candidates were validated in cell culture experiments using primary murine acinar cells and rat AR42J cells. These candidates were then further tested for their usefulness as biomarkers in mouse and human plasma. RESULTS: We identified a number of novel genes, including Regulator of calcineurin 1 (Rcan1) and Sestrin 2 (Sesn2) and demonstrated that they are induced by oxidative stress, by stimulation with H2O2 and by inhibiting caerulein stimulated expression with the antioxidant N-acetylcysteine. We found Rcan1 protein to be significantly elevated in AP-induced mouse plasma as well as in plasma from AP patients. CONCLUSION: We demonstrated that Rcan1 is regulated by oxidative stress and identified RCAN1 as a potential diagnostic marker of AP.

Publisher Correction: Experimental orthotopic transplantation of a tissue-engineered oesophagus in rats.

This corrects the article DOI: 10.1038/ncomms4562.

Pdx1-Cre-driven conditional gene depletion suggests PAK4 as dispensable for mouse pancreas development.

Constitutive depletion of p21-activated kinase 4 (PAK4) in the mouse causes embryonic lethality associated with heart and brain defects. Given that conventional gene depletion of PAK1 or PAK3 caused functional deficits in the mouse pancreas, while gene depletion of PAK5 or PAK6 did not, we asked if PAK4 might have a functional role in pancreas development. We therefore introduced conditional, Pdx1-Cre-mediated, pancreatic PAK4 gene depletion in the mouse, verified by loss of PAK4 protein expression in the pancreas. PAK4 knock-out (KO) mice were born at Mendelian ratios in both genders. Further, morphological and immunohistochemical examinations and quantifications indicated that exocrine, endocrine and ductal compartments retained the normal proportions and distributions upon PAK4 gene depletion. In addition, body weight records and a glucose tolerance test revealed no differences between WT and PAK4 KO mice. Together, this suggests that PAK4 is dispensable for mouse pancreas development. This will facilitate future use of our Pdx1-Cre-driven conditional PAK4 KO mouse model for testing in vivo potential functions of PAK4 in pancreatic disease models such as for pancreatitis and different pancreatic cancer forms.

Bioinformatory-assisted analysis of next-generation sequencing data for precision medicine in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a tumor with an extremely poor prognosis, predominantly as a result of chemotherapy resistance and numerous somatic mutations. Consequently, PDAC is a prime candidate for the use of sequencing to identify causative mutations, facilitating subsequent administration of targeted therapy. In a feasibility study, we retrospectively assessed the therapeutic recommendations of a novel, evidence-based software that analyzes next-generation sequencing (NGS) data using a large panel of pharmacogenomic biomarkers for efficacy and toxicity. Tissue from 14 patients with PDAC was sequenced using NGS with a 620 gene panel. FASTQ files were fed into treatmentmap. The results were compared with chemotherapy in the patients, including all side effects. No changes in therapy were made. Known driver mutations for PDAC were confirmed (e.g. KRAS, TP53). Software analysis revealed positive biomarkers for predicted effective and ineffective treatments in all patients. At least one biomarker associated with increased toxicity could be detected in all patients. Patients had been receiving one of the currently approved chemotherapy agents. In two patients, toxicity could have been correctly predicted by the software analysis. The results suggest that NGS, in combination with an evidence-based software, could be conducted within a 2-week period, thus being feasible for clinical routine. Therapy recommendations were principally off-label use. Based on the predominant KRAS mutations, other drugs were predicted to be ineffective. The pharmacogenomic biomarkers indicative of increased toxicity could be retrospectively linked to reported negative side effects in the respective patients. Finally, the occurrence of somatic and germline mutations in cancer syndrome-associated genes is noteworthy, despite a high frequency of these particular variants in the background population. These results suggest software-analysis of NGS data provides evidence-based information on effective, ineffective and toxic drugs, potentially forming the basis for precision cancer medicine in PDAC.

Stroma-regulated HMGA2 is an independent prognostic marker in PDAC and AAC.

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