Biology subjects

Darrow, M.

Publications and source records attributed to Darrow, M..

4 recordsLinked to original sources

bioRxiv2022

Transformation of Human Mesenchymal Stem Cells into High-Grade Sarcomas by YAP1 and K-RAS Reflects the Undifferentiated Pleomorphic Sarcoma-Myxofibrosarcoma Disease Spectrum

High-grade complex karyotype sarcomas are a heterogeneous group of more than seventy tumors that vary in histology, clinical course, and patient demographics. Despite these clear differences, these high-grade sarcomas are treated similarly with a uniformly high metastatic rate. Pre-clinical models that allow for rigorous comparisons of distinct human sarcoma subtypes would advance insights into the relationships between sarcomas and inform therapeutic decisions. We describe the robust transformation of human mesenchymal stem cells into multiple subtypes of high-grade sarcoma. Using a pooled genetic screening approach, we identified key drivers and potential modifiers of transformation. YAP1 and KRAS were validated as drivers of two distinct sarcoma subtypes, undifferentiated pleomorphic sarcoma (UPS) and myxofibrosarcoma (MFS), respectively. In addition, the pathology of tumors driven by CDK4 and PIK3CA reflected leiomyosarcoma (LMS) and osteosarcoma (OS) indicating that further iterations of this model could result in additional sarcoma subtypes. Histologically and phenotypically these tumors reflect human sarcomas including the pathognomonic complex karyotype. In addition, CDK4 and PIK3CA driven tumors demonstrated endogenous YAP1 amplification which is seen across a subset of human tumors. While all tumors overlapped transcriptionally with the TCGA sarcoma data, further analysis confirmed that YAP1 and KRAS tumors recapitulate the UPS and MFS subtypes. Co-analysis of TCGA and model tumors support that these sarcoma subtypes lie along a spectrum of disease and adds guidance for further transcriptome-based refinement of sarcoma subtyping. Within complex karyotype sarcomas, there are multiple genetic changes but identifying those that are clinically relevant has been challenging. Comparing differentially expressed genes in YAP1 and KRAS tumors to human UPS and MFS identified the enrichment of oxidative phosphorylation pathways in both YAP1 tumors and UPS. Treatment of a panel of sarcoma cell lines with the combination of an oxidative phosphorylation inhibitor and Hippo pathway inhibitor led to a significant impairment in growth identifying new therapeutic targets. A subset of human UPS tumors showed an even greater enrichment in these pathways indicating this model can be used to identify clinically relevant subtypes. This model can be used to begin to understand pathways and mechanisms driving human sarcoma development, the relationship between sarcoma subtypes and to identify and test new therapeutic vulnerabilities for this aggressive and heterogeneous disease. Statement of SignificanceWe have created the first model to study the development, growth, and metastasis of multiple human sarcoma subtypes. This system can be used as a platform to investigate sarcoma biology and identify new therapeutic targets across a heterogeneous disease.

cancer biology↗

bioRxiv2020

A massively multi-scale approach to characterising tissue architecture by synchrotron micro-CT applied to the human placenta

Multi-scale structural assessment of biological soft tissue is challenging but essential to gain insight into structure-function relationships of tissue/organ. Using the human placenta as an example, this study brings together sophisticated sample preparation protocols, advanced imaging, and robust, validated machine-learning segmentation techniques to provide the first massively multi-scale and multi-domain information that enables detailed morphological and functional analyses of both maternal and fetal placental domains. Finally, we quantify the scale-dependent error in morphological metrics of heterogeneous placental tissue, estimating the minimal tissue scale needed in extracting meaningful biological data. The developed protocol is beneficial for high-throughput investigation of structure-function relationships in both normal and diseased placentas, allowing us to optimise therapeutic approaches for pathological pregnancies. In addition, the methodology presented is applicable in characterisation of tissue architecture and physiological behaviours of other complex organs with similarity to the placenta, where an exchange barrier possesses circulating vascular and avascular fluid spaces.

bioengineering↗

bioRxiv2020

Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales

A host of new technologies are under development to improve the quality and reproducibility of cryoEM grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms, (the fastest reported to date), to interrogate particle behaviour at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimise. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic silver bullet for improving the quality of every cryoEM sample preparation.

biochemistry↗

bioRxiv2018

Solid immersion microscopy readily and inexpensively enables 12 nm resolution on plunge-frozen cells

Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryo-fixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. Here, simply by replacing the immersion fluid with a low-cost super-hemispherical solid immersion lens (superSIL), we effortlessly achieve <8 nm localisation precision and 12 nm resolution under cryogenic conditions in a low-cost, low-tech system. This is to our knowledge the best resolution yet attained in biological samples. Furthermore, we demonstrate multicolour imaging and show that the inexpensive setup outperforms 10-fold more costly super-resolution microscopes. By also removing the barrier to total internal reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy delivers a straightforward route to achieve unmatched nanoscale resolution on both bacterial and mammalian cell samples, which any laboratory can effortlessly and inexpensively implement.

biophysics↗