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Simple Summary Pancreatic ductal adenocarcinoma (PDAC) has still a dismal prognosis. To improve treatment, personalized medicine uses next-generation DNA sequencing to monitor disease and guide treatment decisions. Tumor samples for sequencing are usually obtained by invasive fine-needle biopsy. Recently, the focus has been increasingly shifting to blood-based liquid biopsies, including circulating free (cf)DNA or DNA isolated from extracellular vesicles (evDNA). To evaluate the detection performance of DNA alterations, we directly compared tumor-, cf- and evDNA from patients with advanced PDAC upon panel sequencing. Copy number variations (CNVs), single nucleotide variants (SNVs) and insertions and deletions (indels) were compared for their concordance with tumorDNA. Compared to cfDNA, evDNA contained significantly larger DNA fragments, which improved the concordance of SNVs and indels with tumorDNA. In line with previous observations, CNV detection was mostly uninformative for cf- and evDNA. However, the combination of both liquid biopsy analytes was clearly superior for SNV detection, pointing to potentially improved actionable variant prediction. Abstract Pancreatic ductal adenocarcinomas (PDACs) are tumors with poor prognosis and limited treatment options. Personalized medicine aims at characterizing actionable DNA variants by next-generation sequencing, thereby improving treatment strategies and outcomes. Fine-needle tumor biopsies are currently the gold standard to acquire samples for DNA profiling. However, liquid biopsies have considerable advantages as they are minimally invasive and frequently obtainable and thus may help to monitor tumor evolution over time. However, which liquid analyte works best for this purpose is currently unclear. Our study aims to directly compare tumor-, circulating free (cf-) and extracellular vesicle-derived (ev)DNA by panel sequencing of matching patient material. We evaluated copy number variations (CNVs), single nucleotide variants (SNVs) and insertions and deletions (indels). Our data show that evDNA contains significantly larger DNA fragments up to 5.5 kb, in line with previous observations. Stringent bioinformatic processing revealed a significant advantage of evDNA with respect to cfDNA concerning detection performance for SNVs and a numerical increase for indels. A combination of ev- and cfDNA was clearly superior for SNV detection, as compared to either single analyte, thus potentially improving actionable variant prediction upon further optimization. Finally, calling of CNVs from liquid biopsies still remained challenging and uninformative., publishedVersion

Trauma is the leading cause of death in individuals under 44 years of age. Thorax trauma (TxT) is strongly associated with trauma‐related death, an unbalanced innate immune response, sepsis, acute respiratory distress syndrome, and multiple organ dysfunction. It is shown that different in vivo traumata, such as TxT or an in vitro polytrauma cytokine cocktail trigger secretion of small extracellular nanovesicles (sEVs) from endothelial cells with pro‐inflammatory cargo. These sEVs transfer transcripts for ICAM‐1, VCAM‐1, E‐selectin, and cytokines to systemically activate the endothelium, facilitate neutrophil‐endothelium interactions, and destabilize barrier integrity. Inhibition of sEV‐release after TxT in mice ameliorates local as well as systemic inflammation, neutrophil infiltration, and distant organ damage in kidneys (acute kidney injury, AKI). Vice versa, injection of TxT‐plasma‐sEVs into healthy animals is sufficient to trigger pulmonary and systemic inflammation as well as AKI. Accordingly, increased sEV concentrations and transfer of similar cargos are observed in polytrauma patients, suggesting a fundamental pathophysiological mechanism., It is shown that thorax trauma in mice facilitates enhanced release of small extracellular nanovesicles (sEVs) with proinflammatory features, which originate from endothelial cells. Injection of TxT‐plasma‐sEVs into healthy animals triggers local and systemic inflammation. Mechanistically, endothelial‐derived sEVs transfer transcripts for cytokines and adhesion molecules to propagate the inflammatory response. Similar sEVs are detected in polytrauma patients suggesting a fundamental pathophysiological mechanism.

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Background and Purpose: Post-traumatic cardiac dysfunction often occurs in multiply injured patients (ISS ≥ 16). Next to direct cardiac injury, post-traumatic cardiac dysfunction is mostly induced by the release of inflammatory biomarkers. One of these is the heparin-binding factor Midkine, which is elevated in humans after fracture, burn injury and traumatic spinal cord injury. Midkine is associated with cardiac pathologies but the exact role of Midkine in the development of those diseases is ambiguous. The systemic profile of Midkine after multiple trauma, its effects on cardiomyocytes and the association with post-traumatic cardiac dysfunction, remain unknown. Experimental Approach: Midkine levels were investigated in blood plasma of multiply injured humans and pigs. Furthermore, human cardiomyocytes (iPS) were cultured in presence/absence of Midkine and analyzed regarding viability, apoptosis, calcium handling, metabolic alterations, and oxidative stress. Finally, the Midkine filtration capacity of the therapeutic blood absorption column CytoSorb ®300 was tested with recombinant Midkine or plasma from multiply injured patients. Key Results: Midkine levels were significantly increased in blood plasma of multiply injured humans and pigs. Midkine acts on human cardiomyocytes, altering their mitochondrial respiration and calcium handling in vitro. CytoSorb®300 filtration reduced Midkine concentration ex vivo and in vitro depending on the dosage. Conclusion and Implications: Midkine is elevated in human and porcine plasma after multiple trauma, affecting the functionality and metabolism of human cardiomyocytes in vitro. Further examinations are required to determine whether the application of CytoSorb®300 filtration in patients after multiple trauma is a promising therapeutic approach to prevent post-traumatic cardiac disfunction., Frontiers in Immunology, 10, ISSN:1664-3224

The pancreas ductal adenocarcinoma (PDAC) is one of the most common types of cancer, accounting for a large number of cancer related deaths. Rapid tumour metastasis is a major problem in pancreatic cancer, and little is known on the molecular events governing this process, however, features like changes in cell shape, modulation of cell-to-cell adhesion, enhanced cell motility and matrix degrading potential seem to be important. In the literature the Protein kinase D (PKD) family of serine/threonine kinases, which consists of 3 structurally related isoforms, PKD1/PKCμ, PKD2 and PKD3/PKCnu has been implicated in the regulation of some of these processes, yet the molecular mechanisms involved largely remained unclear. That's why this study, using PDAC cell lines, focused on the function of PKD in the metastatic progression of cancer cells. In this work, PKD was found to be strongly expressed and also active in Panc89 cells. Focusing on this cell line, PKD was shown to colocalise with F-Actin and respective markers, indicating active Actin remodeling. For example, Arp3, a member of the Actin-related Arp2/3 protein complex, which is responsible for de novo Actin nucleation and dendritic branching of Actin filaments, colocalised with PKD. Further, PKD colocalised with a subcellular pool of Vinculin at the edge of membrane protrusions, again indicating active Actin turnover, possibly at nascent focal complexes, however PKD was not localised to Vinculin-positive mature focal adhesions to the substratum. In addition, PKD localised with Cortactin, which is enriched within lamellipodia and membrane ruffles. It is implicated in the stabilisation of F-Actin branch points and therefore exhibits important functions at the cortical F-Actin cytoskeleton, which are directly linked to cell migration. Surprisingly, PKD was also found to directly bind to F-Actin in vitro. The binding domain was mapped to 46 amino acids in the N-terminal region of PKD. An alignment of the respective PKD sequence indicated highly conserved motifs, both amongst PKD isoforms and also between human, mouse, and Drosophila species, pointing to a general feature of PKDs. Apart from the binding to F-Actin, PKD and Cortactin also interacted biochemically and PKD was shown to phosphorylate Cortactin in vitro at S298, as well as at additional unspecified sites. Unfortunately an in vivo phosphorylation of Cortactin could not be demonstrated up to now. Employing stable Panc89 cell lines, PKD1 impaired 3D cell migration, while for PKD1KD expressing cells, migration was enhanced. These effects can either be explained by a model implicating Cortactin phosphorylation by PKD in the regulation of F-Actin turnover and rigidity, or by the phosphorylation of yet unknown PKD substrates at the respective F-Actin-rich structures, negatively regulating cell migration. Since mutation of the potential Cortactin phosphorylation site Ser298 to an alanine residue also increased cell migration in stable Panc89 cells, Cortactin might be a potential target of PKD in the regulation of F-Actin dynamics. PKD has also been implicated in the regulation of Ca2+-dependent cell-to-cell adhesion. PKD2KD and, to lesser extent, PKD1KD, strongly enhanced cell-to-cell adhesion of stable Panc89 cells, whereas E-Cadherin expression in the respective parental cell lines was reduced. At least in the case of PKD2KD cells, aggregation was only partially dependent on E-Cadherin, pointing to the expression of additional Cadherin isoforms. In the PKD2 expressing cell line, aggregation was very weak, in some assays even resembling the vector control. This phenotype could be correlated with processed E-Cadherin fragments in the supernatant of PKD2 expressing cells, which have been implicated in the literature with the inhibition of cell-to-cell adhesion of cancer cell lines, thereby also increasing their invasive potential. Initial results demonstrated that the E-Cadherin fragments from the supernatant of stable PKD2 expressing Panc89 cells were processed by a serine-protease, possibly Plasmin or cationic Trypsin. How the PKD2 isoform is implicated in this process remains to be investigated further. Yet, these findings are in line with data obtained from expression profiling experiments with the respective stable Panc89 cells, indicating that the urokinase-like Plasminogen-activator-receptor (PLAUR) is strongly up-regulated in PKD2 expressing cells, triggering the activation of the uPA-Plasminogen-Plasmin-cascade, which has been demonstrated to be involved in the processing of E-Cadherin. During the course of this work novel aspects concerning the role of PKD in cell migration and cellular adhesion were revealed. The localisation of PKD at the F-Actin cytoskeleton and its in vitro binding to F-Actin implicate possible functions for the PKD protein family in the regulation of F-Actin dynamics, which might influence cell adhesion and motility., Pankreaskarzinome zählen zu häufigsten Tumorarten und sie weisen dabei eine der geringsten 5 Jahre Überlebensraten bei Krebserkrankungen auf. Bis heute ist nicht viel über die molekularen Ereignisse bekannt, die mit der Metastasierung von Krebszellen einhergehen, jedoch spielen Prozesse wie Veränderung der Zellstruktur, Modulation der Zell-Zell-Adhäsion, verstärkte Zellmigration und verändertes invasives Verhalten der Krebszellen eine wichtige Rolle. In der Fachliteratur wurde auch die Proteinkinase D (PKD) Familie mit diesen Prozessen in Verbindung gebracht, die genauen molekularen Mechanismen sind jedoch noch unklar. Auf Grund von Homologien innerhalb ihrer Kinase Domaine wurden die drei Isoenzyme der PKD Familie: PKD1/PKCµ, PKD2, PKD3/PKCnu in eine Subklasse der Calcium/Calmodulin-abhängigen Kinasen (CAMK) eingeordnet. Ziel dieser Arbeit war es, die Rolle der Proteinkinase D bei den oben genannten Prozessen in Pankreasductaladenokarzinom (PDAK) Zelllinien näher zu untersuchen. Dazu wurden zunächst mehrere PDAK Linien charakterisiert. In Panc89 Zellen konnte dabei eine starke Expression kinase-aktiver PKD festgestellt werden, weshalb weitere Untersuchungen sich zunächst auf diese Linie konzentrierten. Durch konfokale Immunfluoreszenzmikroskopie konnte eine Kolokalisation zwischen PKD und F-Aktin, sowie mit Arp3, einem Teil des Arp2/3 Proteinkomplexes, der für die de novo Aktin-Nukleation verantwortlich ist, festgestellt werden. PKD wurde außerdem mit Cortactin, einem Marker, der in Lamellopodien und "membran ruffles" angereichert ist, kolokalisiert. Laut Literatur, ist Cortactin für die Stabilisierung von F-Aktin-Verzweigungen im kortikalen Aktin Zytoskelett verantwortlich und übt somit wichtige Funktionen bei der molekularen Kontrolle der Zellmigration aus. Desweiteren konnte auch eine direkte in vitro Bindung von PKD an F-Aktin nachgewiesen werden. Die Aktin-Bindedomäne wurde dabei auf 46 Aminosäuren im N-terminalen Bereich von PKD eingegrenzt. Ein Sequenzvergleich der entsprechenden Region zeigte hoch konservierte Motive zwischen den einzelnen PKD Isoformen und auch bei unterschiedlichen Spezies, was auf eine mögliche konservierte Funktion innerhalb der PKD Familie hindeutet. Außer dieser Bindung von PKD an F-Aktin konnte auch eine direkte Interaktion von PKD und Cortactin nachgewiesen werden. Im in vitro Kinasetest wurde Cortactin von PKD am Serin-Rest 298 und an weiteren, noch unbekannten Aminosäuren phosphoryliert. Eine Phosphorylierung an Ser298 konnte bisher jedoch in vivo nicht nachgewiesen werden. Mit Hilfe von stabil PKD exprimierenden Zelllinien wurde das Migrationsverhalten in 3D Transwell-Migrationsexperimenten untersucht. PKD1 verringerte die gerichtete Zellmigration, während eine Kinase-inaktive Variante (PKD1KD) die Zellmigration verstärkte. Diese Effekte von PKD lassen sich entweder durch ein Modell erklären, in dem die Phosphorylierung von Cortactin bei der Regulation des Aktinumbaus und der Stabilisierung von Aktin-Verzweigungen eine Rolle spielt, oder aber über die Phosphorylierung bisher noch unbekannter Substrate am Aktin Zytoskelett, welche die Zellmigration negative regulieren. Da jedoch die Mutation von Ser298 in einen Alanin-Rest ebenfalls zu gesteigerter Zellmigration führte, ist es möglich, dass Cortactin ein potentielles physiologisches Substrat von PKD bei der Regulation des Aktinumbaus darstellt. Im Rahmen dieser Arbeit wurde auch der Einfluss der unterschiedlichen PKD Isoformen und Kinase-inaktiven (KD) Mutanten auf die Ca2+-abhängige Zell-Adhäsion untersucht. Die Expression einer Kinase-inaktiven PKD2-, und in geringerem Ausmaß auch einer PKD1KD-Mutante, steigerte deutlich die Zell-Zell-Adhäsion der Panc89 Zellen, bei gleichzeitig reduzierter E-Cadherin Expression. Da ein E-Cadherin spezifischer Antikörper bei der PKD2-Linie nur partiell mit der Ca2+--abhängigen Zell-Aggreagtion interferierte, deutet dies auf die zusätzliche Expression von anderen Cadherin Isoformen in Panc89 Zellen hin, welche die gesteigerte Zell-Zell-Adhäsion bewirkt haben könnten. Die Expression von PKD2 in Panc89 Zellen führte zu einer deutlich reduzierten Aggregation. Dieser Phänotyp konnte mit N-terminalen E-Cadherin Fragmenten im Zellkulturüberstand korreliert werden, die von einer Serin-Protease abgespalten werden. Als potentielle Kandidaten konnten Plasmin und kationisches Trypsin ausgemacht werden. Dieses Ergebnis ist in guter Übereinstimmung mit "Microarray-Genprofiling"-Experimenten, bei denen eine verstärkte Expression des "urokinase-like Plasminogen-activator-receptor" Gens (PLAUR) in PKD2 exprimierenden Panc89 Zellen festgestellt werden konnte, was wiederum zu einer Aktivierung der Plasmin-Kaskade und somit auch zu der beobachteten E-Cadherin Prozessierung beitragen koennte.