Biology subjects

Stelzer, E. H. K.

Publications and source records attributed to Stelzer, E. H. K..

7 recordsLinked to original sources

bioRxiv2024

Inhibition of IAPs induces programmed cell death and inflammatory signaling in patient-derived metastatic breast cancer organoids

Breast cancer (BC) is the most common type of cancer among women worldwide and underlies relapse, disease progression and metastasis. Resistance to chemotherapy and programmed cell death (PCD), including apoptosis, strongly affects therapy success and remains a major challenge. Representative and translational models to understand, manipulate and cultivate advanced BC and to model PCD resistance are therefore urgently required. Smac mimetics are promising compounds to circumvent apoptosis resistance and are able to induce caspase-independent necroptosis, a lytic and inflammatory mode of PCD. Here, we apply primary, patient-derived human mammary organoids (hMOs) to investigate alternative forms of PCD to overcome apoptosis resistance. Using time lapse brightfield with immunofluorescent confocal microscopy, biochemistry and gene expression analysis, we demonstrate that Smac mimetics induce apoptosis in primary hMOs. By mimicking apoptosis resistance via caspase inhibition, hMOs undergo necroptosis, associated with expression and secretion of inflammatory mediators. Inhibition of linear ubiquitination by the LUBAC inhibitor HOIPIN-8 prevents necroptosis, as well as the expression and release of inflammatory mediators in hMOs. Our findings demonstrate that primary hMOs are effective models to model, study and manipulate PCD responses and inflammation in in primary BC organoids and open new therapeutic screening options for chemotherapy-resistant BC.

cancer biology↗

bioRxiv2024

Spatio-temporal segmentation of contraction waves in the extra-embryonic membranes of the red flour beetle

In this paper, we introduce an image analysis approach for spatio-temporal segmentation, quantification and visualization of movement or contraction patterns in 2D+t or 3D+t microscopy recordings of biological tissues. The imaging pipeline is applied to time lapse images of the embryonic development of the red flour beetle Tribolium castaneum recorded with light-sheet fluorescence microscopy (LSFM). We are particularly interested in the dynamics of extra-embryonic membranes, and provide quantitative evidence of the existence of contraction waves during late stages of development. These contraction waves are a novel observation of which neither origin, nor function are yet known. The proposed pipeline relies on particle image velocimetry (PIV) for quantitative movement analysis, surface detection, tissue cartography, and an algorithmic approach to detect characteristic movement dynamics. This approach locates contraction waves in 2D+t and 3D+t reliably and efficiently and allows the automated quantitative analysis, such as the area involved in the contractile behavior, contraction wave duration and frequency, path of contractile area, or the relation to the spatio-temporal velocity distribution. The pipeline will be used in the future to conduct a large-scale characterization and quantification of contraction wave behavior in Tribolium castaneum development and can be adapted easily to the identification and segmentation of characteristic tissue dynamics in other systems of interest.

bioinformatics↗

bioRxiv2024

Open-hardware solutions for light sheet microscopy specimen chambers

Light sheet fluorescence microscopy (LSFM) is a powerful tool for imaging large three-dimensional biological samples. However, the design and fabrication of specimen chambers for these systems present significant challenges, particularly in maintaining water-tight seals, preventing contamination, and ensuring the flexibility needed for precise positioning of the objective and sample. This study introduces open-hardware solutions to address these challenges, utilising a combination of 3D printing, silicone injection moulding, and FEP-foil thermoforming. We describe the development of custom, highly flexible silicone seals and connectors through a laboratory-scale injection moulding process. These components enable precise, low-resistance movement of imaging objectives and specimen holders, which is crucial for maintaining imaging accuracy. Additionally, we introduce a novel "optical window" design that isolates the objective lens from the immersion medium, significantly reducing the risk of contamination and facilitating easy exchange of chambers and lenses without compromising sterility. The practicality of these designs is demonstrated through their application in long-term live imaging of Tribolium castaneum embryos, honey bee embryos, and human mesenchymal stem cell spheroids. By providing open-source CAD and 3D printing files, this work promotes accessibility and customization in microscopy, enabling researchers to easily replicate and adapt these solutions to their specific needs.

developmental biology↗

bioRxiv2021

Bioprinting the Tumor Microenvironment with an Upgraded Consumer Stereolithographic 3D Printer

A widespread application of three-dimensional (3D) bioprinting in basic and translational research requires the accessibility to affordable printers able to produce physiologically relevant tissue models. To facilitate the use of bioprinting as a standard technique in biology, an open-source device based on a consumer-grade 3D stereolithographic (SL) printer was developed. This SL bioprinter can produce complex constructs that preserve cell viability and recapitulate the physiology of tissues. The detailed documentation of the modifications apported to the printer as well as a throughout performance analysis allow for a straightforward adoption of the device in other labs and its customization for specific applications. Given the low cost, several modified bioprinters could be simultaneously operated for a highly parallelized tissue production. To showcase the capability of the bioprinter, we produced constructs consisting of patient-derived cholangiocarcinoma organoids encapsulated in a gelatin methacrylate (GelMA)/polyethylene glycol diacrylate (PEGDA) hydrogel. A thorough characterization of different GelMA/PEGDA ratios revealed that the mechanical properties of the bioprinted tumor model can be accurately fine-tuned to mimic a specific tumor micro-environment. Immunofluorescence and gene expression analyses of tumor markers confirmed that the bioprinted synthetic hydrogel provides a flexible and adequate replacement of animal-derived reconstituted extracellular matrix.

bioengineering↗

bioRxiv2019

p63 sets the threshold for induction of apoptosis using a kinetically encoded ‘doorbell-like’ mechanism

Cell fate decisions such as apoptosis require cells to translate signaling input into a binary yes/no response. A tight control of the process is required to avoid loss of cells by accidental activation of cell death pathways. One particularly critical situation exists in primary oocytes because their finite number determines the reproductive capacity of females. On the one hand a stringent genetic quality control is necessary to maintain the genetic integrity of the entire species; on the other hand an overly stringent mechanism that kills oocytes with even minor DNA damage can deplete the whole primary oocyte pool leading to infertility. The p53 homolog TAp63 is the key regulator of genome integrity in oocytes. After DNA damage TAp63 is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer. By measuring activation kinetics in ovaries and single site phosphorylation kinetics in vitro with peptides and full length protein we show that TAp63 phosphorylation follows a biphasic behavior. While the first two CK1 phosphorylation events are fast, the third one that constitutes the decisive step to form the active conformation is slow. We reveal the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63 substrate interaction and demonstrate by quantitative simulation that the slow phosphorylation phase determines the threshold of DNA damage required for induction of apoptosis.

biochemistry↗

bioRxiv2019

Ultra-thin fluorocarbon foils optimize multiscale imaging of three-dimensional native and optically cleared specimens

In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast, and achieve an optimal resolution is minimizing the impact of changes of the refractive index along an optical path. Many light sheet fluorescence microscopes operate with a large chamber that contains an aqueous immersion medium and an inner specimen holder that contains the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimize the impact of the specimen holder on the optical quality, we use multi-facetted cuvettes fabricated with vacuum-formed ultra-thin fluorocarbon (FEP) foils The ultra-thin FEP-foil cuvettes have a wall thickness of about 12 {micro}m. They are resilient to fluidic exchanges, durable, mechanically stable and yet flexible. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs, thick tissue sections and dense organoid clusters. The cuvettes outperform many other sample-mounting techniques in terms of full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding, as well as their applicability for multiple-view imaging and automated segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The ultra-thin cuvettes allow the fixation and staining of the specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.

cell biology↗

bioRxiv2017

Three-dimensional cell neighbourhood impacts differentiation in the inner mass cells of the mouse blastocyst

During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterised by the transcription factors NANOG and GATA6, respectively. Here, we present quantitative three-dimensional single cell-based neighbourhood analyses to investigate the spatial distribution of NANOG and GATA6 expression in the ICM of the mouse blastocyst. The cell neighbourhood is characterised by the expression levels of the fate markers in the surrounding cells, together with the number of surrounding cells and cell position. We find that cell neighbourhoods are established in early blastocysts and different for cells expressing different levels of NANOG and GATA6. Highest NANOG expressing cells occupy specific positions within the ICM and are surrounded by 9 neighbours, while GATA6 expressing cells cluster according to their GATA6 levels. The analysis of mutants reveals that NANOG local neighbourhood is regulated by GATA6.\n\nSummary statementThree-dimensional cell neighbourhood, which includes fate marker levels, number of neighbouring cells and cell position, determines cell fate decision in early mouse embryos.

developmental biology↗