Biology subjects

Eising, S.

Publications and source records attributed to Eising, S..

11 recordsLinked to original sources

bioRxiv2026

A microscopy-based readout to assess tumour-specific viability in neuroblastoma co-cultures and short-term cultured patient samples

High-risk neuroblastoma patients face poor survival despite intensive treatment. Drug testing using patient-derived models can support therapy prioritization for precision medicine and drug development. Models incorporating tumour microenvironmental components, such as co-cultures and short-term cultured patient samples containing substantial non-malignant cell fractions, could better recapitulate microenvironment-dependent drug responses. However, conventional viability assays measure the combined signal from all viable cells in a well and therefore cannot determine tumour-specific drug responses. Here, we establish a microscopy-based readout to quantify cell-type-specific viability in two complementary settings: neuroblastoma-PBMC co-cultures and freshly dissociated patient tumour samples. In the co-cultures, PBMCs were pre-labelled with a cell-tracking dye, and Calcein staining was used to independently quantify the viability of tumour cells and PBMCs in the same well. The Calcein-based viability readout correlated strongly with conventional CellTiter-Glo measurements and was compatible with automated high-throughput drug screening. The imaging workflow enabled identification of compounds with differential efficacy in co-culture versus monoculture and distinguished tumour-specific effects from PBMC toxicity. The microscopy-based viability readout was further adapted to short-term cultured patient samples. Neuroblastoma tumour cells were distinguished from the non-malignant cells using a combination of tumour-specific surface markers NCAM, L1CAM and B7H3. This enabled determination of tumour fractions and measurement of tumour-specific drug responses. Tumour fractions varied substantially between patient samples, highlighting the importance of tumour-specific viability measurements. Together, the microscopy-based viability readout for co-cultures and patient samples enables scalable assessment of tumour-specific drug responses.

Cancer Biology↗

bioRxiv2026

Sensitivity profiling reveals consistent drug responses across preclinical neuroblastoma models

Despite intensive treatment, overall survival for high-risk and relapse neuroblastoma patients remains below 50%. Even though comprehensive molecular profiling enables treatment stratification, druggable alterations have been identified for only a subset of patients. In vitro drug screening offers a complementary approach. Here, we compare the translational potential of three preclinical drug screening methods: ex vivo short-term, in vitro patient-derived organoid and in vivo patient-derived xenograft (PDX) drug testing. In total, 55 screens were performed from 38 neuroblastoma samples and five pediatric non-malignant samples, testing 77-224 drugs per screen. Ex vivo short-term drug screens achieved higher success rates than organoid screens (65% versus 23%) and shorter turnaround times (14 days versus 3-12 months). Matched samples showed consistent drug sensitivities across sample origin (patient versus PDX-derived; mean r = 0.84) and method (ex vivo short-term versus organoids; mean r = 0.87), demonstrating that ex vivo short-term screens recapitulate drug sensitivities found in long-term organoid models. In parallel, as part of the ITCC-P4 consortium, ten compounds were tested in vivo in eight PDX models, with samples matching the ex vivo screens. For seven out of ten clinically available compounds, ex vivo drug responses were comparable with in vivo responses in matched PDX models. These results demonstrate that, while organoids and PDX models remain essential for drug discovery, ex vivo short-term drug screening provides a rapid alternative for functional precision oncology in neuroblastoma.

cell biology↗

bioRxiv2026

Lipid Droplet Targeting Drives Substrate Access during Very Long Chain Fatty Acid Activation by Fat1

Fatty acids, the most abundant building blocks for lipid synthesis, require conjugation with Coenzyme A (CoA) for their incorporation into membrane lipids. Of those, very long chain fatty acids (VLCFAs), the precursors of yeast ceramides, are particularly hydrophobic. Yet it remains unresolved where and how the sole VLCFA-CoA synthetase Fat1 activates them. Here we show that lipid droplets (LDs) are both the site of free VLCFAs storage, as well as their place of activation. Free VLCFAs preferentially partition into LDs in molecular simulations and in vitro reconstitutions, whereas Fat1 targets the LD surface in cells via a N-terminal amphipathic helix. Structural predictions identify an essential hydrophobic cavity in Fat1 that connects the active site to the membrane, suggesting that VLCFAs directly shuttle from the LD core into the enzymatic pocket. Our model explains how the organelle targeting of a fatty acid activator evolved to match the biophysical properties and consequently the subcellular localization of its highly hydrophobic substrate.

biochemistry↗

bioRxiv2023

Anti-cancer compound screening identifies Aurora Kinase A inhibition as a means to favor CRISPR/Cas9 gene correction over knock-out

CRISPR gene therapy holds the potential to cure a variety of genetic diseases by targeting causative mutations and introducing double stranded DNA breaks, subsequently allowing the host DNA repair mechanisms to introduce mutations. One option to introduce precise gene corrections is via the homology-directed repair (HDR) pathway. HDR can introduce a range of desired mutations dictated by a DNA template which holds a corrected DNA sequence which is written into the targeted gene. The problem in utilizing this pathway is that CRISPR-induced double stranded DNA breaks are repaired more often through the non-homologous end joining (NHEJ) pathway, which does not use a designed template and introduces random DNA damage in the form of insertions and deletions at the cut site. Since HDR activation depends on many interconnected processes in the cell, we aimed to screen a small library of drug compounds in clinical use or clinical development for cancer, to steer the DNA repair process towards preferential HDR activation. We included compounds in our screen based on three relevant mechanisms in CRISPR gene editing: the cell cycle, DNA repair processing and chromosomal packing. We included forty compounds, based on these criteria, screened their toxicity and dosed them in sub-toxic concentrations in cells during genome editing. Of these forty compounds we identified nine potential hits to have an effect on preferential activation of the HDR pathway over NHEJ. Alisertib, rucaparib and belinostat revealed a significant and major effect on gene editing pathway selection in further validation. Alisertib, an Aurora kinase A inhibitor, showed a particularly strong effect towards improving HDR over NHEJ. We subsequently investigated this effect at the genetic level and in a murine hepatoma cell line, which corroborated the initial findings. Alisertib led to an over 4-fold increase in preferential gene correction over gene knock-out, at a dose of 0.3 micromolar. However, the observations that Aurora kinase A inhibitors show considerable cytotoxicity (<50% cell viability) and can induce morphological changes at this concentration pose a limitation for the direct use of these inhibitors as HDR enhancers. However these findings do implicate that the pathways mediated by Aurora kinase A strongly influence HDR outcomes, which warrants further investigation into the downstream pathways driving this effect.

pharmacology and toxicology↗

bioRxiv2023

Multi-dimensional profiling of hepatoblastomas and patient-derived tumor organoids uncovers tumor subpopulations with divergent WNT activation profiles and identifies pan-hepatoblastoma drug sensitivities

Hepatoblastoma, the most prevalent pediatric liver cancer, almost always carries a WNT-activating CTNNB1 mutation, yet exhibits notable molecular heterogeneity. To characterize this heterogeneity and identify novel targeted therapies, we performed comprehensive analysis of hepatoblastomas and tumor-derived organoids using single-cell RNA-seq, spatial transcriptomics, single-cell ATAC-seq and high throughput drug profiling. We identified two distinct tumor epithelial signatures: hepatic fetal-like and WNT-high embryonal-like signatures, displaying divergent WNT signaling patterns. The liver-specific WNT targets were enriched in the fetal-like group, while the embryonal-like group was enriched in canonical WNT target genes. Gene regulatory network analysis revealed enrichment of regulons related to hepatic function such as bile acid, lipid and xenobiotic metabolism in the fetal-like subgroup but not in the embryonal-like subgroup. In addition, the dichotomous expression pattern of the transcription factors HNF4A and LEF1 allowed for a clear distinction between the fetal- and embryonal-like tumors. We also performed high-throughput drug screening using patient-derived tumor organoids and identified sensitivity to multiple inhibitor classes, most notably HDAC inhibitors. Intriguingly, embryonal-like tumor organoids, but not fetal-like tumor organoids, were sensitive to FGFR inhibitor treatments, suggesting a dependency on FGFR signaling. In summary, our data uncover the molecular and drug sensitivity landscapes of hepatoblastoma and pave the way for the development of targeted therapies.

cancer biology↗

bioRxiv2023

Complex Sphingolipid Profiling and Identification of an Inositol Phosphorylceramide Synthase in Dictyostelium discoideum

Dictyostelium discoideum is a professional phagocyte frequently used as experimental model to study cellular processes underlying the recognition, engulfment and infection course of microbial pathogens. Sphingolipids are abundant components of the plasma membrane that bind cholesterol, control vital membrane properties, participate in signal transmission and serve as adhesion molecules in recognition processes relevant to immunity and infection. While the pathway of sphingolipid biosynthesis has been well characterized in plants, animals and fungi, the identity of sphingolipids produced in D. discoideum, an organism at the crossroads between uni- and multicellular life, is not known. Combining lipidomics with a bioinformatics-based cloning strategy for key sphingolipid biosynthetic enzymes, we show here that D. discoideum produces phosphoinositol-containing sphingolipids with predominantly phytoceramide backbones. Cell-free expression of candidate inositol-phosphorylceramide (IPC) synthases from D. discoideum in defined lipid environments enabled identification of an enzyme that selectively catalyses the transfer of phosphoinositol from phosphatidylinositol onto ceramide. The corresponding IPC synthase, DdIPCS1, is non-homologous to but shares multiple sequence motifs with yeast IPC and human sphingomyelin synthases and localizes to the Golgi apparatus as well as the contractile vacuole of D. discoideum. Collectively, these findings open up important opportunities for exploring a role of sphingolipids in phagocytosis and infection across major evolutionary boundaries.

cell biology↗

bioRxiv2022

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here we describe the generation of a collection of pediatric RMS tumor organoid (tumoroid) models comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within four to eight weeks, indicating the feasibility of personalized drug screening. Molecular, genetic and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to six months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.

cancer biology↗

bioRxiv2021

Loss of p16INK4a in neuroblastoma cells induces shift to an immature state with mesenchymal characteristics and increases sensitivity to EGFR inhibitors

Homozygous inactivation of the CDKN2A locus is one of the most common genomic aberrations in human cancer. The locus codes for two unrelated and distinctly regulated proteins: p14ARF and p16INK4a, which inhibit MDM2 and CDK4/6, respectively. Loss of CDKN2A is also a recurrent event in relapsed neuroblastoma, a childhood tumour that arises from neural crest cells. To examine the consequences of the loss of the two distinct gene transcripts in neuroblastoma, we used the CRISPR-Cas9 system to knockout p14, p16 and p14+p16 in SY5Y cells. RNA sequencing of the transcriptome revealed a striking shift towards an immature Schwann cell precursor-like phenotype with mesenchymal characteristics, specifically in the p16 and p14+p16 knockouts. High-throughput drug screening of p16 and p14+p16 knockout clones identified a large increase in sensitivity to EGFR inhibitors. On protein level, we were able to confirm that EGFR pathway activation is higher in p14+p16 knockout cells and that treatment with the EGFR inhibitor afatinib resulted in higher levels of apoptosis. Afatinib also reduced tumour growth in vivo in xenografts transplanted with p14+p16 knockout SY5Y cells. Overall, our study suggests that CDKN2A deletion in neuroblastoma relates to a phenotypic shift towards a more progenitor like state and increases sensitivity to EGFR inhibitors.

cancer biology↗

bioRxiv2021

A yeast lysosomal biogenesis map uncovers the cargo spectrum of lysosomal protein targeting pathways

The lysosome is the major catabolic organelle and a key metabolic signaling center of the cell. Mutations in lysosomal proteins can have catastrophic effects, causing neurodegeneration, cancer, and age-related diseases. The vacuole is the lysosomal analog of Saccharomyces cerevisiae that harbors many conserved proteins. Vacuolar proteins reach their destination via the endosomal vacuolar protein sorting (VPS) pathway, via the alkaline phosphatase (ALP or AP-3) pathway, and via the cytosol-to-vacuole transport (CVT) pathway. While these pathways have been extensively studied, a systematic understanding of the cargo spectrum of each pathway is completely lacking. Here we combine quantitative proteomics of purified vacuoles with mutant analyses to generate the lysosomal biogenesis map. This dataset harbors information on the cargo-receptor relationship of virtually all vacuolar proteins. We map binding motifs of Vps10 and the AP-3 complex and identify a novel cargo of the CVT pathway under nutrient-rich conditions. Our data uncover how organelle purification and quantitative proteomics can uncover fundamental insights into organelle biogenesis.

cell biology↗

bioRxiv2020

Multiple roles for the ESCRT machinery in maintaining plasma membrane homeostasis

The endosomal sorting complexes required for transport (ESCRT) execute evolutionary conserved membrane remodeling processes. Here we used budding yeast to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. In response to reduced membrane tension and inhibition of the target of rapamycin complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help to maintain membrane integrity. Conversely, the growth of ESCRT mutants strongly depends on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This is caused by calcineurin phosphatase activity which causes Orm2 to accumulate at the endoplasmic reticulum (ER) in ESCRT mutants. Orm2 is a repressor of SL biosynthesis and its accumulation provokes increased membrane stress. This necessitates TORC2 signaling through its downstream kinase Ypk1 to control Orm2 protein levels and prevent a detrimental imbalance of SL metabolism. Our findings reveal new aspects of antagonistic calcineurin/TORC2 signaling for the regulation of SL biosynthesis and the maintenance of PM homeostasis, and suggest that the ESCRT machinery contributes directly and indirectly to these processes.

cell biology↗

bioRxiv2018

A Systematic Approach to Identify Recycling Endocytic Cargo Depending on the GARP Complex

Proteins and lipids of the plasma membrane underlie constant remodeling via a combination of the secretory- and the endocytic pathway. In the yeast endocytic pathway, cargo is sorted for recycling to the plasma membrane or degradation in vacuoles. In a previous paper we have shown a role for the GARP complex in sphingolipid sorting and homeostasis (Frohlich et al. 2015). However, the majority of cargo sorted in a GARP dependent process remain largely unknown. Here we use auxin induced degradation of GARP combined with mass spectrometry based vacuolar proteomics and lipidomics to show that recycling of two specific groups of proteins, the amino-phospholipid flippases and cell wall synthesis proteins depends on a functional GARP complex. Our results suggest that mis-sorting of flippases and remodeling of the lipid composition are the first occurring defects in GARP mutants. Our assay can be adapted to systematically map cargo of the entire endocytic pathway.

cell biology↗