Biology subjects

Malcolm, J. R.

Publications and source records attributed to Malcolm, J. R..

4 recordsLinked to original sources

bioRxiv2026

Adapting the OpenFlexure Microscope for Affordable Live-Cell Imaging

Live-cell imaging (LCI) captures dynamic cellular behaviours missed in fixed samples. Researchers in resource-limited settings are underrepresented in the adoption of LCI due to the prohibitive cost of dedicated systems. We adapted the 3D-printed OpenFlexure Microscope (OFM) for LCI in humid tissue culture incubators. We relocated electronics to prevent corrosion, and printed in acrylonitrile styrene acrylate (ASA) to improve image stability in prolonged experiments. Current-limiting resistors reduced heat buildup inside sealed incubators, while our simple graphical user interface (GUI)-based application enabled rapid setup of timelapse experiments. Using a 48-hour docetaxel treatment of breast cancer cells, we validated how the LCI-OFM seamlessly fits into established bioimaging pipelines to generate biologically meaningful data. We further demonstrated broad applicability of the LCI-OFM by imaging Leishmania-infected macrophages in a containment level 3 laboratory and gut fungi in an anaerobic environment. This accessible platform expands opportunities for resource-limited researchers to study locally-relevant health challenges.

cell biology↗

bioRxiv2025

An ERα-Dependent Hypoxia Response Defines EMT-Adjacent Tumour Regions and Suppresses the Pro-survival Effects of Amiloride in Estrogen Receptor-Positive Breast Cancer

Estrogen receptor-positive (ER+) breast cancer carries a lifelong risk of recurrence and disease progression, with hypoxia-associated transcriptional signatures linked to poor prognosis and therapy resistance. While the effects of hypoxia on tumour progression are well studied, the impact on ER epigenomic regulation remains poorly characterised. Here, we demonstrate that activation of hypoxia-inducible factors (HIFs) dramatically remodels ER chromatin localisation in ER+ breast cancer cells. Transcripts of genes located near hypoxia-induced ER binding sites are significantly associated with reduced recurrence-free survival in breast cancer patients. Transcriptomic profiling under hypoxic conditions (1% oxygen), with and without ER depletion by fulvestrant, revealed a hypoxia-induced, ER-dependent gene expression programme, including upregulation of epithelial sodium channel (ENaC) regulatory subunits that results in acquired sensitivity to the ENaC inhibitor amiloride. Notably, this transcriptional response is spatially correlated with the epithelial-to-mesenchymal hallmark in patient tumours. Our findings establish an interdependence between ER signalling and the hypoxic response, and present functional evidence that ER reprogramming offers novel therapeutic opportunities that bypass the need to directly target the hypoxic response.

cancer biology↗

bioRxiv2025

Characterising cancer-stroma interactions through high-content phenotyping from microscopy time-lapses

Understanding how cancer-stromal interactions shape cancer progression requires tools that can capture dynamic phenotypic changes in physiologically relevant conditions. Traditional approaches for studying co-culture interactions, such as transcriptomics and flow cytometry, provide valuable insights but are limited by their static nature and reliance on fixed or dissociated cells. In contrast, label-free time-lapse microscopy preserves temporal and spatial context, enabling observation of live-cell behaviours over time. A major challenge, however, lies in the analysis of the resulting high-dimensional datasets. Using co-cultures of breast cancer cells and cancer-associated fibroblasts (CAFs) as a model system, we show that the CellPhe toolkit enables label-free identification and phenotypic characterisation of different cell types within complex live-cell imaging datasets. Our analysis shows that exposure to CAFs drives marked phenotypic shifts in breast cancer cells, including elongation, loss of cell-cell adhesion, and redistribution of intracellular components - hallmarks of epithelial-mesenchymal transition (EMT). To probe the underlying mechanisms, we performed a Luminex immunoassay on CAF-conditioned media and identified secreted analytes strongly associated with EMT induction. Together, these results highlight how automated phenotyping can be integrated with molecular profiling to identify and characterise cellular processes shaped by stromal interactions and reveal the signalling mediators that drive them.

cancer biology↗

bioRxiv2024

Identification of robust RT-qPCR reference genes for studying changes in gene expression in response to hypoxia in breast cancer cell lines

Hypoxia is common in breast tumours and is linked to therapy resistance and advanced disease. To understand hypoxia-driven breast cancer progression, RT-qPCR quantifies transcriptional changes important for malignant development. Reference genes (RGs) are endogenous RT-qPCR controls used to normalise mRNA levels, allowing accurate assessment of transcriptional changes. However, hypoxia reprograms transcription and post-transcriptional processing of RNA such that favoured RGs including GAPDH or PGK1 are unsuitable for this purpose. To address the need for robust RGs to study hypoxic breast cancer cell lines, we identified 10 RG candidates by analysing public RNA-seq data of MCF-7, T-47D, MDA-MB-231 and MDA-MB-468 cells cultured in normoxia or hypoxia. RT-qPCR determined RG candidate levels in normoxic breast cancer cells, removing TBP and EPAS1 from downstream analysis due to insufficient transcript abundance. Assessing primer efficiency further removed ACTB, CCSER2 and GUSB from consideration. Following culture in normoxia, or acute or chronic hypoxia, we ascertained robust non-variable RGs using RefFinder. Here we present RPLP1 and RPL27 as optimal RGs for breast cancer cell lines cultured in normoxia or hypoxia. Our result enables accurate evaluation of gene expression in hypoxic breast cancer cell lines and provides an essential resource for assessing hypoxias impact in breast cancer progression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/606329v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@188a16forg.highwire.dtl.DTLVardef@11eb9aborg.highwire.dtl.DTLVardef@2f914eorg.highwire.dtl.DTLVardef@ecad85_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗