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

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

International Journal of Cancer / Neuroblastoma cells undergo transcriptomic alterations upon dissemination into the bone marrow and subsequent tumor progression

Neuroblastoma is the most common extracranial solid tumor in childhood. The vast majority of metastatic (M) stage patients present with disseminated tumor cells (DTCs) in the bone marrow (BM) at diagnosis and relapse. Although these cells represent a major obstacle in the treatment of neuroblastoma patients, insights into their expression profile remained elusive. The present RNASeq study of stage 4/M primary tumors, enriched BMderived diagnostic and relapse DTCs, as well as the corresponding BMderived mononuclear cells (MNCs) from 53 patients revealed 322 differentially expressed genes in DTCs as compared to the tumors (q2). Particularly, the levels of transcripts encoded by mitochondrial DNA were elevated in DTCs, whereas, for example, genes involved in angiogenesis were downregulated. Furthermore, 224 genes were highly expressed in DTCs and only slightly, if at all, in MNCs (q6). Interestingly, we found the transcriptome of relapse DTCs largely resembling those of diagnostic DTCs with only 113 differentially expressed genes under relaxed cutoffs (q0.5). Notably, relapse DTCs showed a positional enrichment of 31 downregulated genes on chromosome 19, including five tumor suppressor genes: SIRT6, BBC3/PUMA, STK11, CADM4 and GLTSCR2. This first RNASeq analysis of neuroblastoma DTCs revealed their unique expression profile in comparison to the tumors and MNCs, and less pronounced differences between diagnostic and relapse DTCs. The latter preferentially affected downregulation of genes encoded by chromosome 19. As these alterations might be associated with treatment failure and disease relapse, further functional studies on DTCs should be considered.(VLID)481812

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

Effects of freezing, storage and thawing of samples on NB gene expression.

<p>In (a-b) we analyzed the effect of freezing, storage at -80°C and thawing by qPCR array on the expression of 64 genes of NB cells spiked into PB. In (a) we show the effect when after thawing the samples were only enriched by magnetic bead-based separation, whereas in (b) we introduced an additional density gradient separation prior to magnetic bead-based separation. Red dots represent genes that are significantly changed (p<0.05, |log₂FC|>1) at given conditions compared to the baseline (LAN-1 cells enriched and homogenized in TRIzol immediately after spiking). Genes with |log₂FC|>1 and p>0.05 are represented by black dots (not significant), as their expression was not coherently changed between the three biological replicates. The log₂ fold change is indicated on the y-axis and the mean Ct values in the x-axis. In (c-d) we analyzed the same effects on NB cells spiked into BM by microarrays. The mean log₂ expression is shown in the x-axis, and the log₂ fold change on the y-axis.</p

Altered gene expression of NB cells kept at 4°C and at RT up to 72h.

<p>70 genes were analyzed by qPCR array. The altered gene expression of LAN-1 cells kept for 24 hours in PB on 4°C (a) and at room temperature (b) is shown. In (c) and (d) the same is shown for 48h, and in (e) and (f) for 72h. Red dots represent genes that are significantly changed (p<0.05, |log₂FC|>1) at given time points compared to the baseline (time point 0h). Genes with |log₂FC|>1 and labeled by black dots, did not show significant changes (p>0.05) between the three biological replicates. The log₂ fold change for a given conditions is indicated on the y-axis, whereas the mean Ct value is shown on the x-axis.</p

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

Experimental design.

<p><b>(a)</b> We spiked LAN-1 NB cells into fresh PB and kept the samples for 0, 24, 48 and 72h at room temperature and, for the same time periods, at 4°C prior to density gradient separation. The LAN-1 cells were enriched from the MNC fraction with magnetic beads to a 99% purity of the tumor cell fractions prior to homogenization in TRIzol. RNA was isolated from all seven samples simultaneously and used for the qPCR array. (<b>b)</b> LAN-1 cells were spiked into PB and tumor-free BM, and density gradient separation was immediately performed. The MNCs were frozen in 20% DMSO for seven days at -80°C. After thawing, the LAN-1 cells were either directly enriched by magnetic bead-based separation, or an additional density gradient separation (*) was performed prior to magnetic bead-based separation. The samples were homogenized in TRIzol and the isolated RNA was used for qPCR (in case of PB) and microarrays (in case of BM). <b>(c)</b> LAN-1 cells were spiked into PB and density gradient separation of MNCs was performed without delay, following two enrichment steps in a row. The >99% LAN-1 cell fractions were homogenized in TRIzol and RNA was isolated from all samples simultaneously. qPCR arrays were performed in order to analyze the effect of enrichment on selected genes.</p