Biology subjects

Wragg, J. W.

Publications and source records attributed to Wragg, J. W..

3 recordsLinked to original sources

bioRxiv2024

A dual readout embryonic zebrafish xenograft model of rhabdomyosarcoma to assess clinically relevant multi-receptor tyrosine kinase inhibitors

BackgroundRhabdomyosarcoma (RMS) is a highly aggressive soft tissue sarcoma, affecting children and adolescents, with poor prognosis in some patient groups. Better therapeutic regimens and preclinical models to test them in are needed. Multi-receptor tyrosine kinase inhibitors (MRTKIs) are licensed for adult indications and explored in the clinic in sarcoma patients. The MRTKI Regorafenib is currently assessed in the relapse setting in patients with RMS (NCT04625907). Reliable biomarkers of response for MRTKIs are lacking. MRTKIs act not only against the cancer cell, but also the supporting stroma, particularly the vasculature. The embryonic zebrafish is translucent and allows assessment of this interaction with high-throughput in vivo imaging. MethodsA new preclinical embryo zebrafish xenograft model was developed using Tg(flk1:GFP) (blood vessel reporter) transgenic zebrafish embryos inoculated in the yolk with fluorescently labelled cells from 7 different RMS cell lines (fusion-positive (FP): Rh4, Rh30, Rh41, RMS-01, fusion-negative (FN): RD, JR1, SMS-CTR), and patient-derived cells IC-pPDX-104 at 50 hours post-fertilization and incubated at 34{degrees}C for up to 70 hours. Xenografts and vessel beds were imaged and analysed using custom FIJI pipelines. MRTKIs regorafenib and infigratinib were used at a concentration of 0.1uM added to the fish water 4 hours post cell inoculation. Pro-angiogenic growth factors VEFG-A, FGF-2 and PDGF-BB were measured in conditioned media of each cell line. ResultsAll 7 RMS cell lines and the patient-derived cells engrafted with tumour burden assessment by fluorescent imaging and direct cell counting indicating adequate growth and high cell viability during the observation period. RMS tumours induced neo-vascularisation towards the tumour and increased density of proximal vessel beds. MRTKI treatment revealed a greater tumour-intrinsic sensitivity of FP cells, but identified a significant blockade of neo-vascularisation across all RMS lines, with regorafenib response correlated with secretion of VEGF-A. ConclusionWe have developed an embryonic zebrafish xenograft model of RMS, which allows assessment of tumour growth, vascularisation initiation and therapeutic responses to clinically relevant MRTKIs. The identification of VEGF-A secretion as potential biomarker for Regorafenib response and the separation of therapeutic effects on tumour growth and neovascularisation suggests additional value of our model for response prediction to MRTKIs.

cancer biology↗

bioRxiv2021

Integrative annotation of genomic features reveal functional elements and epigenetic landscapes in the developing zebrafish

Zebrafish, a popular model for embryonic development and for modelling human diseases, has so far lacked a systematic functional annotation programme akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created the first central repository to store and process zebrafish developmental functional genomic data. Our Data Coordination Center (https://danio-code.zfin.org) combines a total of 1,802 sets of unpublished and reanalysed published genomics data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements in development, including novel classes with distinct features dependent on their activity in time and space. We delineated the distinction between regulatory elements active during zygotic genome activation and those active during organogenesis, identifying new aspects of how they relate to each other. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predict functional relationships between them beyond sequence similarity, extending the utility of zebrafish developmental genomics to mammals.

developmental biology↗

bioRxiv2019

Zebrafish embryonic tissue differentiation is marked by concurrent cell cycle dynamic and gene promoter regulatory changes

The core promoter, a stretch of DNA surrounding the transcription start site (TSS) is a major integration point for regulatory signals controlling gene transcription. The process of cell differentiation is accompanied by a marked divergence in transcriptional repertoire between cells of different fates, accompanied by changes in cellular behaviour, in particular their proliferative activity. Investigation of divergent core promoter architectures suggest distinct regulatory networks act on the core promoter, modulating cell behavior through transcriptional profile changes, which ultimately drives key transitions in cellular behaviour during embryonic development. The role that promoter-associated gene regulatory networks play in development associated transitions in cell cycle dynamics (e.g. during differentiation) however, is poorly understood. In this study we demonstrate in a developing in vivo model, how core promoter variations play a key role in defining transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. The FUCCI transgenic system, differentially marks cells in G1 and S/G2/M phases of the cell cycle and can therefore be used to separate rapidly and slowly cycling cells in vivo, by virtue of the cell cycle stage they primarily inhabit. Longitudinal assessment of cell proliferation rate during zebrafish embryo development, using this system, revealed a spatial and lineage-specific separation in cell cycling behaviour across post-gastrulation embryos. In order to investigate the role differential promoter usage plays in this process, cap analysis of gene expression (CAGE) was performed on fluorescent associated cell sorted (FACS) FUCCI zebrafish embryos going through somitogenesis, separating cells in accordance with the rate of their cell cycling. This analysis revealed a dramatic increase in lineage and tissue-specific gene expression, concurrent with a slowing of their cell cycling. Core promoters associated with rapidly cycling cells, showed broad distribution of transcription start site usage, featuring positionally constrained CCAAT-box, while slowly cycling cells favoured sharp TSS usage coupled with canonical TATA-box utilisation and enrichment of Sp1 binding sites. These results demonstrate the regulatory role of core promoters in cell cycle-dependent transcription regulation, during somitogenesis stages of embryo development.

developmental biology↗