Refined high-content imaging-based phenotypic drug screening in zebrafish xenografts

Zebrafish xenotransplantation models are increasingly applied for phenotypic drug screening to identify small compounds for precision oncology. Larval zebrafish xenografts offer the opportunity to perform drug screens at high-throughput in a complex in vivo environment. However, the full potential of the larval zebrafish xenograft model has not yet been realized and several steps of the drug screening workflow still await automation to increase throughput. Here, we present a robust workflow for drug screening in zebrafish xenografts using high-content imaging. We established embedding methods for high-content imaging of xenografts in 96-well format over consecutive days. In addition, we provide strategies for automated imaging and analysis of zebrafish xenografts including automated tumor cell detection and tumor size analysis over time. We also compared commonly used injection sites and cell labeling dyes and show specific site requirements for tumor cells from different entities. We demonstrate that our setup allows us to investigate proliferation and response to small compounds in several zebrafish xenografts ranging from pediatric sarcomas and neuroblastoma to glioblastoma and leukemia. This fast and cost-efficient assay enables the quantification of anti-tumor efficacy of small compounds in large cohorts of a vertebrate model system in vivo. Our assay may aid in prioritizing compounds or compound combinations for further preclinical and clinical investigations

The transcriptomic landscape of bone marrow derived disseminated neuroblastoma cells

Das Neuroblastom, der häufigste Tumor bei Säuglingen, ist durch ein extrem vielfältiges und häufig dramatisches Krankheitsbild gekennzeichnet. Das vielgestaltige klinische Verhalten ist primär durch die hohe genetische Diversität dieses Tumors erklärbar. Während eine Reihe von Tumoren, die meist mit numerischen Chromosomenveränderungen assoziiert sind, eine spontane Regression oder Ausreifung aufweisen, zeigen andere hoch maligne Eigenschaften und sind durch Chromosomenumbauten und/oder Genamplifikationen charakterisiert. Etwa die Hälfte aller Neuroblastom Patienten ist durch Dissemination der Tumorzellen in das Knochenmark (KM) gekennzeichnet, was in weiterer Folge zum metastatischen Rezidiv führen kann. Die Anzahl der disseminierten Tumorzellen (disseminated tumor cells, DTCs) im KM ist jedoch oftmals sehr gering, was ihre Charakterisierung bisher erschwert hat. In der vorliegenden Dissertation wurde daher der dringenden Notwendigkeit nachgegangen die DTCs auf Transkriptionsebene zu charakterisieren, um Einblicke in die Biologie dieser Zellen zu erlangen. Um dies zu ermöglichen haben wir eine immuno-magnetische Anreicherungsmethode etabliert, getestet und angewandt, um DTCs von den restlichen mononukleären Zellen (mononuclear cells MNCs) zu trennen und anzureichern, ohne dabei die Transkriptionssignatur der Tumorzellen zu verändern. Anschließend wurde die RNA von Primärtumoren, DTCs und MNCs von Hoch-Risiko Neuroblastom Patienten sequenziert um folgende Fragen zu beantworten: (i) wie unterscheidet sich die Transkriptionssignatur primärer Tumoren von jener diagnostischer DTCs? (ii) Wie unterscheidet sich das Expressionsverhalten von DTCs von den korrespondierenden MNCs? Und, (iii) wie verändert sich das Transkriptionsprofil von DTCs im Verlauf der Erkrankung (Diagnose Rezidiv)? Unsere RNA-seq Daten haben gezeigt, dass, obwohl die Transkriptionssignatur der DTCs jener der primären Tumoren großteils ähnelte, 322 Gene signifikant differenziell transkribiert waren. Am auffälligsten waren die in den DTCs hoch-regulierten Gene welche in der mitochondrialen DNA kodiert sind. Eine substantielle Anzahl von Genen war jedoch in den DTCs herab-reguliert, unter anderem Gene, die für die Angiogenese notwendig sind. Zudem haben wir 224 Gene gefunden, die in den DTCs hoch-transkribiert waren, deren Transkription jedoch in den MNCs nur sehr gering bis gar nicht nachweisbar war. Diese 224 differentiell exprimierten Gene können behilflich sein, die DTCs an Hand ihres spezifischen Expressionsverhaltens zu detektieren. Wir konnten auch zeigen, dass sich interessanterweise das Transkriptom der DTCs im Krankheitsverlauf kaum ändert. Lediglich 113 Gene wiesen zwischen Diagnose und Rezidiv ein differentielles Transkriptionsverhalten auf. Auffällig war jedoch die Häufung der differentiell exprimierten Gene am Chromosom 19. Von den insgesamt 113 differentiell exprimierten Genen waren 31 am Chromosom 19 kodiert und interessanterweise herab-reguliert. Unter den 31 Genen befinden sich auch fünf Tumorsuppressorgene (SIRT6, PUMA, STK11, CADM4 und GLTSCR2) die im Zusammenhang mit dem Neuroblastom kaum beschrieben sind, die jedoch in einer Reihe von anderen Tumoren herab-reguliert sind. Von den insgesamt 31 deregulierten Genen befanden sich 11 auf dem langen (q) Arm und 20 auf dem kurzen (p) Arm von Chromosom 19. Obwohl Deletionen am q-Arm viel häufiger waren als am p-Arm, entsprach dies nicht dem Verteilungsmuster der differentiell exprimierten Gene. Durch die erste RNA Sequenzierung von DTCs konnten wir wesentliche Erkenntnisse über deren Biologie erlangen. Neben der Beschreibung von DTC spezifischen Markern, ist besonders die Herabregulation von Tumorsupressorgenen in DTCs beim Rezidiv von potentieller Tragweite, da diese für das Scheitern der heutigen Therapiestrategien verantwortlich sein könnte. Somit sind diese Erkenntnisse ein wichtiger Ausgangspunkt für zukünftige funktionelle Studien an DTCs.Neuroblastoma is the most common tumor diagnosed in the first year of life. It exhibits diverse and often dramatic clinical behaviors, a property which is mainly attributed to its genetic diversity. While tumors which present only with numerical chromosomal aberrations are often characterized by spontaneous regression or maturation, others are characterized by structural chromosomal aberrations and/or gene amplifications, thus being highly malignant and often causing death. The malignant tumors are defined by a frequent dissemination of tumor cells into the bone marrow (BM), a feature that is assumed to cause metastatic relapse in a substantial number of patients. However, disseminated tumor cells (DTCs) frequently represent only a minor cell population in the BM of neuroblastoma patients, which is why their characterization was hampered in the past. In the present PhD thesis, the urgent necessity for analyzing the transcriptomic landscape of DTCs was pursued in order to improve our knowledge about the emergence of relapse metastatic disease. By establishing, testing and applying an immunomagnetic enrichment method, we were able to separate and enrich DTCs from the remaining mononuclear cells (MNCs) of the BM without altering their gene expression profile. Thus, RNA-Seq of tumors, DTCs, and MNCs of high-risk neuroblastoma patients was performed in order to analyze: (i) whether and how the expression profile of diagnostic DTCs differs from the primary tumors, (ii) how the expression of DTCs varies from the corresponding BM-derived MNCs, and (iii) whether the transcription profile of DTCs changes during the course of disease (diagnosis relapse). As a result, we found that DTCs largely retained the gene expression signature of primary tumors, including concordant transcription levels of the neuroblastoma hallmark genes MYCN and PHOX2B. Nevertheless, we identified 322 genes that were differentially expressed between DTCs and the primary tumors. Particularly genes encoded by the mitochondrial DNA were highly up-regulated in DTCs, whereas genes involved in angiogenesis and initiation of metastasis were down-regulated in DTCs. Furthermore, we identified 224 genes that were highly expressed in DTCs and only slightly, if at all, in MNCs. These genes may be considered as markers for the detection of minimal residual disease (MRD) in the BM during or after treatment. Finally, we found that diagnostic DTCs resembled those at relapse with only 113 differentially expressed genes under relaxed cut-offs. Notably, relapse DTCs showed a positional enrichment of 31 down-regulated genes encoded by chromosome 19, with twenty genes (n = 20) encoded by the p-arm and eleven genes (n = 11) encoded by the q-arm of chromosome 19. SNP array data indicated frequent deletions on chromosome 19 in DTCs of relapse patients. However, these genomic deletions were mainly affecting genes that are encoded by the q-arm of chromosome 19, while genes encoded by the p-arm were rarely deleted. Interestingly, we found five tumor suppressor genes, namely SIRT6, BBC3 (PUMA), STK11, CADM4 and GLTSCR2, among the 31 down-regulated genes encoded by chromosome 19 in relapse DTCs. Not much is known about these five tumor suppressor genes in association with neuroblastoma, but their down-regulation is highly associated with an increase of the oncogenic potential in various other cancers. With the first RNA-Seq data of BM-derived disseminated neuroblastoma cells, we were able to obtain substantial insights into the biology of these cells. We identified DTC-specific markers and transcriptional alterations occurring in DTCs, particularly the down-regulation of tumor suppressor genes encoded by chromosome 19 in relapse patients. As these alterations might be associated with treatment failure and disease relapse in high-risk neuroblastoma patients, they should be considered for further functional studies.submitted by Mag.rer.nat. Fikret RifatbegovicZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität, Dissertation, 2017OeBB(VLID)191468

Multivisceral Resection with Performing a Double Roux-en-Y Reconstruction for Advanced Gastric Cancer

Background. The role of multivisceral resection, in the setting of locally advanced gastric cancer, is still debated. Previous studies have reported a higher risk for perioperative morbidity and mortality, with limited objective benefit in terms of survival. Patient. A male patient, 55 years old, was admitted to the clinic of surgery for surgical treatment of bleeding gastric ulceration. Preoperative diagnostic evaluation was performed, and patient had undergone a surgical treatment which revealed a large mass in head of the pancreas, infiltrating the hepatoduodenal ligament and transverse mesocolon. Total gastrectomy, duodenopancreatectomy, and right hemicolectomy were performed. The digestive tube continuity was reestablished by deriving the double Roux limbs. Conclusion. The aim of this case presentation is to demonstrate a method of digestive tube reconstruction by performing the double Roux-en-Y reconstruction in advanced gastric cancer when the multivisceral resection is performed

Vascular Endothelial Growth Factor Receptor-1 Expression in Breast Cancer and Its Correlation to Vascular Endothelial Growth Factor A

VEGF-A is the most potent angiogenic factor in tumour angiogenesis. Its effects are mediated via two receptors VEGFR-1 and VEGFR-2. Primary aim of our study was to examine the expression of VEGFR-1 in breast cancer and its correlation to VEGF expression, lymph node status, tumour size, histological grade, and hormone receptor status. To examine the VEGFR-1 and VEGF expressions in tumour and surrounding tissue of 51 breast cancer patients, and in healthy breast tissue of 30 benign breast diseases patients, we used three-step immunohistochemical staining. VEGFR-1 and VEGF expressions were significantly increased in breast cancer tumour in relation to surrounding tissue (P<0.01), and the VEGF expression was significantly increased in lymph node positive breast cancer patients (P<0.01). VEGFR-1 and VEGF expressions were significantly higher in breast cancer tumour compared with healthy breast tissue (P<0.01). Significant correlation between VEGF and VEGFR-1 expressions was found (P<0.05). No significant correlations between VEGF and VEGFR-1 expressions and tumour size, histological grade, and hormone receptor status were found. Increased expression of VEGFR-1 and VEGF in breast cancer tumour and significant correlation between these proteins suggest the possible role of VEGF/VEGFR-1 signalization in breast cancer development, although VEGFR-1 potential prognostic value was not confirmed

Enriched Bone Marrow Derived Disseminated Neuroblastoma Cells Can Be a Reliable Source for Gene Expression Studies-A Validation Study.

Metastases in the bone marrow (BM) in form of disseminated tumor cells (DTCs) are frequent events at diagnosis and also at relapse in high-risk neuroblastoma patients. The frequently highly diluted occurrence of DTCs requires adequate enrichment strategies to enable their detailed characterization. However, to avoid methodical artifacts we tested whether pre-analytical processing steps-including transport duration, temperature and, importantly, tumor cell enrichment techniques-are confounding factors for gene expression analysis in DTCs.LAN-1 neuroblastoma cells were spiked into tumor free BM and/or peripheral blood and: i) kept at room temperature or at 4°C for 24, 48 and 72 hours; ii) frozen down at -80°C and thawed; iii) enriched via magnetic beads. The effect on the gene expression signature of LAN-1 cells was analyzed by qPCR arrays and gene expression microarrays.Neither storage at -80°C in DMSO and subsequent thawing nor enrichment of spiked-in neuroblastoma cells changed the expression of the analyzed genes significantly. Whereas storage at 4°C altered the expression of analyzed genes (14.3%) only at the 72h-timepoint in comparison to the 0h-timepoint, storage at room temperature had a much more profound effect on gene expression by affecting 20% at 24h, 26% at 48h and 43% at 72h of the analyzed genes.Using neuroblastoma as a model, we show that tumor cell enrichment by magnetic bead separation has virtually no effect on gene expression in DTCs. However, transport time and temperature can influence the expression profile remarkably. Thus, the expression profile of routinely collected BM samples can be analyzed without concern as long as the transport conditions are monitored

Effects of magnetic bead-based enrichment of NB cells on their gene expression.

<p>qPCR arrays were used to analyze the effects of magnetic bead-based enrichment on the expression of 71 genes in NB cells. In (a) the altered gene expression is shown for cells that have been enriched only once after density gradient separation, whereas in (b) the effect of two following magnetic bead-based enrichment steps is shown. Red dots represent genes that are significantly changed (p<0.05, |log₂FC|>1) at given conditions compared to the baseline (LAN-1 cells before spiking into PB). The expression of genes with |log₂FC|>1 but p>0.05 are not considered as significant, as their expression was not coherently changed in the different biological replicates. The log₂ fold change is indicated on the y-axis and the mean Ct values in the x-axis.</p

Unsupervised clustering of NB samples after freezing, storage and thawing procedure.

<p>Microarray analysis of three biological replicates (A-C) and the three different pretreatment conditions: fresh (no freezing/thawing), frozen 1 (thawing > magnetic bead-based separation of LAN-1 cells) and frozen 2 (thawing > density gradient separation > magnetic bead-based separation of LAN-1 cells). In the unsupervised clustering of the expression of the analyzed genes, the fresh and the two differently frozen samples (1, 2) did not cluster. The correlation coefficient (R) is illustrated by the color key: white (0) = no correlation and red (1) = high correlation.</p