Biology subjects

Brackenbury, W. J.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Conventional dendritic cells type I with an enhanced type-I-IFN signaling underpin anti-tumor immune responses in brain metastases

Brain metastases are associated with poor prognosis. A better understanding of anti-tumor immune responses in the context of immune specialized microenvironment of the brain is required to develop improved therapeutic strategies for this disease. We demonstrate that the conventional dendritic cells type 1 (cDC1) gene signature positively correlates with a prolonged BrM-dependent survival in melanoma and breast cancer patients. Furthermore, intracranial anti-tumor immune responses in preclinical BrM models consistently rely on cDC1s for tumor growth control, BrM-dependent survival and maintenance of the intra-tumoral CD8+ T cell pool, in contrast to variable, cancer type-dependent cDC1 roles in extracranial tumors. This is underpinned by tumor site-specific cDC1 molecular profiles with distinct Toll like receptor repertoires, upregulation of co-stimulatory molecules and IL-12, and enhanced type-I-IFN signaling in intracranial cDC1s, with the latter driving increased cDC1 activation. cDC1s also promote the conversion of progenitor exhausted CD8+ T cells to transient effectors, which is further enhanced by immune checkpoint blockade therapy. These findings pinpoint cDC1s as a major cell population of interest in the development of future immunotherapies for BrM. Significance statementCharacteristics of immune cells and tumor microenvironment in brain metastases (BrM) are distinct to extracranial tumor sites and characterized by special immunological barriers and suppressive stroma. Conventional dendritic cells type 1 (cDC1s), which are key regulators of anti-tumor immunity, remain largely unexplored in this context. This study deepens our understanding of how anti-tumor immune responses differ between BrM and extracranial tumors by revealing BrM-specific phenotype and function of cDC1s, with the latter being critical for anti-tumor immunity in BrM even in cancer models where extracranial cDC1s demonstrate pro-tumorigenic function, highlighting cDC1s as universal regulators of intracranial anti-tumor immunity. Our study suggests that tumor site-specific augmentation of cDC1s should be pursued to enhance the efficacy of immunotherapies in BrM.

cancer biology↗

bioRxiv2026

Breast cancer cohort study identifies an effective, non-invasive, breath-based diagnostic linked to cellular environment-dependent, novel methylation metabolisms

Volatile organic compounds (VOCs) demonstrate promise as non-invasive diagnostic tools. However, lack of mechanistically linked VOCs with biomarker discovery platforms limit delivery to the clinic. In this cohort-based study, we observed significant alterations of chloride-containing volatile fluxes, inclusive of methyl chloride (MeCl), in the breath of cancer patients that are consistent with our newly described metabolic model, derived through in vitro cellular assays and in vivo mice models. The diagnostic accuracy of this cohort study (60 patients) is equivalent to mammogram approaches. This newly identified and novel metabolism along with associated diagnostic biomarkers were initially identified through headspace studies of breast cancer cell lines which were deprived of serum, glucose or oxygen, similar to cellular conditions in tumors. In these cellular assays MeCl was consistently informative of cellular stress. Under resource limited conditions cellular production of MeCl was significantly reduced, and in several cases, cellular metabolism shifted to consumption. We present a new "push-pull" model in which cellular production of MeCl is linked to cellular methylation potential and methyl-transferase activity while consumption of MeCl is associated with methionine generation. Neither consumption nor production metabolisms have been described or quantified in humans or human tissues previously. The cellular headspace-derived model was tested using xenograft tumour bearing mice, which demonstrated reduced MeCl production, consistent with this model This work therefore presents a potentially powerful breath biomarker for cancer that translates from cellular and mice models through to human subjects. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/726956v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@71fcdeorg.highwire.dtl.DTLVardef@1905726org.highwire.dtl.DTLVardef@149bb10org.highwire.dtl.DTLVardef@1981c33_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LI(Poly)chloromethane compounds, including methyl chloride and chloroform, are indicative of cancer status in breath sampled from breast cancer clinic patients C_LIO_LIGlucose, serum and oxygen starvation induce significant changes in cellular metabolic fluxes C_LIO_LIReduced methyl chloride production is indicative of cellular stress in vitro C_LIO_LIMethyl chloride production is linked to cellular methylation activity C_LIO_LIMethyl chloride consumption is linked to methionine synthesis, cellular enrichment in chloride concentration, and enhanced chloroform fluxes C_LIO_LIMethyl chloride fluxes in in vitro cell cultures under pathophysiologically relevant conditions translate to the breath of tumour bearing mice C_LI

molecular biology↗

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

EGFR-targeted and MMP-activated membranolytic peptides derived from Polybia paulista MP1 kill cancer cells specifically in vitro and reduce tumour growth in vivo

Membranolytic peptides have demonstrated potential as cancer therapeutics, although targeting to cancer cells and reducing toxicity associated with activity in normal cells remain unmet challenges. We have investigated the membranolytic peptide MP1, from Polybia paulista, in order to assess ways to reduce its non-specific toxicity and thereby improve its characteristics as a cancer therapeutic. Using a panel of human breast cell lines and cell survival assays, we show that C-terminal addition of an EGFR binding sequence, with or without a linking MMP-2 cleavage sequence, generally reduced the efficacy of the peptides relative to wildtype MP1, as determined by increases in IC50 values. Critically, cell lines that show the highest sensitivities to these fusion peptides (MDA-MB-468, MDA-MB-231) expressed the highest EGFR and/or MMP-2 levels, demonstrating that these additions direct the cytotoxic activity to cells expressing these biomarkers. MMP-2 inhibition significantly reduced the cell-killing activity of peptides containing MMP-2 cleavage sites, further demonstrating targeting to this biomarker. Fusion peptides significantly induced apoptosis and reduced survival in EGFR/MMP-2 high cancer cells, while sparing EGFR/MMP-2 low cells in the context of standard tissue culture and 3D-spheroids. Finally, systemic treatment with the EGFR and MMP-2 cleavage fusion significantly reduced tumour size in MDA-MB-468 xenograft models, confirming in vivo efficacy against cancer cells and acceptable systemic toxicity. We present this EGFR-MMP-MP1 peptide as a novel cancer therapeutic for further pre-clinical and clinical development.

cancer 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↗

bioRxiv2025

Determinants of membrane sensitivity to the peptide MP-1 (Polybia paulista)

The membrane-disrupting peptide Mastoparan-1 (MP-1), derived from the wasp Polybia paulista is known to possess antimicrobial properties, and exhibits enhanced activity against a number of cancer cell lines relative to healthy cells. Due to the mechanism of action of MP-1 it is likely that differences in plasma membrane lipid composition arising from cancer associated mutations, such as localisation of phosphatidylserine (PS) lipids to the outer leaflet of the plasma membrane, are involved in driving that enhanced activity. Rapid screening of MP-1 mutants in a combined approach using model membrane and cell-based biological assays, has led to the identification of a number of derivative peptides with enhanced selectivity for cancer-like membrane models and breast cancer cell lines and provided insights into the mechanism of membrane disruption and cell death. Notably, the morphology of the membrane perturbations observed by Atomic Force Microscopy (AFM) and activity in cell model systems can change considerably in response to single-point mutations in the MP-1 sequence, indicating a complex structure-activity relationship.

biophysics↗

bioRxiv2025

Artemether and Euphorbia Factor L9 suppress kynurenine production through distinct effects on Tryptophan metabolism.

L-Tryptophan (Trp) is an essential amino acid, catabolised through the kynurenine pathway, which is mediated by the enzymes indoleamine-2,3-dioxygenase 1 (IDO1), IDO2, or Trp-2,3-deoxygenase (TDO). Therapeutic targeting of Trp metabolism could be relevant to several pathologies. In cancer, IDO1 acts as an immune checkpoint suppressing effector T cell function. Yet, direct inhibition of IDO1 has had limited success in clinical trials. Therefore, alternative approaches to Trp metabolism therapeutic targeting are needed. We screened a library of 597 natural products (NPs) or NP derivatives for their effect on kynurenine production in triple negative breast cancer cells. This revealed 24 candidate inhibitors of kynurenine production. Amongst them, artemether, a member of the artemisinin family of anti-malarial drugs, suppressed kynurenine production, likely via an endoperoxide bridge-dependent mechanism. The Euphorbia factor L9 (EFL9) inhibited kynurenine production likely via a C7-benzoylation-dependent mechanism. Neither artemether nor EFL9 affected JAK/STAT signaling or IDO1 levels. Targeted metabolomics analyses demonstrated that artemether suppressed kynurenine production through heme sequestration, a mechanism that would affect all members of the IDO/TDO family of metalloenzymes. EFL9 affected purine and amino acid metabolism and the cellular redox balance. Comparisons to the effects of ouabain, a NP regulator of IDO1 levels, and Linrodostat, a clinically used small molecule IDO1 inhibitor, revealed distinct metabolic profiles, with ouabain and EFL9 showing the largest overlap. Importantly, the kynurenine-suppressing activity of artemether and EFL9 is not cancer cell-specific. Overall, our findings set the foundation for the use of derivatives of artemether or EFL9 as novel Trp metabolism-targeting therapeutics.

cancer biology↗

bioRxiv2025

Spatial profiling of central nervous system cellular markers in murine breast cancer to brain metastases

BackgroundBrain metastasis occurs in approximately 16% of metastatic breast cancer patients, and incidence is increasing. Patients with breast cancer brain metastases frequently develop neurological problems including cognitive impairments and seizures. We previously demonstrated accumulation of activated microglia around the tumour site in a breast cancer brain metastasis model, which co-localised with spontaneously occurring local field potential events that resembled interictal epileptic discharges. However, the mechanisms underlying these spatially restricted effects on neuronal excitability are poorly understood. MethodsTo better understand the underlying cellular changes and potential mechanisms, we used digital spatial profiling of brain cell subtype-specific markers to chart the spatial organisation of neurons, oligodendrocytes, astrocytes, and microglia in relation to metastatic lesion site. We developed a novel chordline-based approach to analyse these spatial changes in biomarker distribution. ResultsSeveral protein markers associated with proliferation (Ki67), astrocytes (GFAP) and microglia (CD11b, IBA1, CD45, MSR1) were upregulated in the tumour compared to healthy contralateral brain parenchyma. In contrast, some neuroglial markers (MAP2, Neurofilament light, NeuN, S100B and TMEM119) were lower in the lesion compared to normal tissue. ConclusionsOverall, the protein marker changes in the lesion are indicative of neuronal cell loss and changes in the immune microenvironment of the metastatic lesion involving activated microglia, astrocytes and recruitment of peripheral immune cells. Such cellular changes may contribute to the changes in electrical activity resulting from brain metastases observed in mice and patients.

neuroscience↗

bioRxiv2025

Selection of pre-leukemic hematopoietic stem cells driven by distinct extracellular matrix molecules

Despite rapid advances in mapping genetic drivers and gene expression changes in hematopoietic stem cells (HSCs), there is a relative paucity of studies at the protein level. Here, we perform a deep, multi-omic characterization (epigenome, transcriptome and proteome) of HSCs carrying a loss-of-function mutation in Tet2, a key driver of increased self-renewal in blood cancers. Using state-of-the-art, multiplexed, low-input mass spectrometry (MS)-based proteomics, we profile wildtype (WT) and TET2-deficient (Tet2-/-) HSCs and show that the proteome captures previously unrecognized molecular processes which define the pre-leukemic HSC molecular landscape. Specifically, we obtain more accurate stratification of WT and Tet2-/- HSCs than transcriptomic approaches and identify extracellular matrix (ECM) molecules as novel points of dysregulation upon TET2 loss. HSC expansion assays using ECM-functionalized hydrogels confirm a selective effect on the expansion of Tet2-mutant HSCs. Taken together, our study represents a comprehensive molecular characterization of Tet2-mutant HSCs and identifies a previously unanticipated role of ECM molecules in regulating self-renewal of disease-driving HSCs.

cell biology↗

bioRxiv2025

Chloroform release from ageing cells and Drosophila DJ-1 mutants

Volatile Organic Compounds (VOCs) offer potential for non-invasive diagnosis as biomarkers of disease and metabolism. In complex biological matrices, such as breath however, identifying useful biomarkers from hundreds, or even thousands of VOCs can be challenging. Models of disease, such as cellular or animal models, offer a means to elucidate VOC metabolisms, for accurate targeted studies in patient samples. Neurodegenerative conditions, such as parkinsons have been associated with changed VOCs, offering a potential for early diagnostics and interventions improving treatments and outcomes for patients. Here, three separate models including; human HEK-293t cells, isolated primary rat glial cells, Drosophila flies (wild type and a mutant of the parkinsons associated gene, DJ-1) were grown for an extended period and levels of the VOC chloroform (CHCl3) investigated using custom static headspace sampling chambers. Samples were analysed using targeted gas chromatography mass spectroscopy over time to generate metabolic flux values and chloroform shown to dramatically increase in all models as they aged. HEK-293t cells revealed a 60-fold increase after 10 weeks, glial cells revealed a 10-fold increase after 3 to 4 weeks and DJ-1 mutant flies revealed significant increases compared to control flies at 4 weeks. These results, taken together, indicate that chloroform release is related to ageing in these models and may provide a target for neurodegenerative studies moving forward. We present here the first evidence of chloroform being actively produced by human and rat cells and the first observation of volatile metabolisms in Drosophila.

biochemistry↗

bioRxiv2025

CellPhePy: a Python implementation of the CellPhe toolkit for automated cell phenotyping from microscopy time-lapse videos

We previously developed the CellPhe toolkit1, an open-source R package for automated cell phenotyping from ptychography time-lapse videos. To align with the growing adoption of python-based image analysis tools and to enhance interoperability with widely used software for cell segmentation and tracking, we developed a python implementation of CellPhe, named CellPhePy. CellPhePy preserves all of the core functionality of the original toolkit, including single-cell phenotypic feature extraction, time-series analysis, feature selection and cell type classification. In addition, CellPhePy introduces significant enhancements, such as an improved method for identifying features that differentiate cell populations and extended support for multiclass classification, broadening its analytical capabilities. Notably, the CellPhePy package supports CellPose segmentation and TrackMate tracking, meaning that a set of microscopy images are the only required input with segmentation, tracking and feature extraction fully automated for downstream analysis, without reliance on external applications. The workflows increased flexibility and modularity make it adaptable to different imaging modalities and fully customisable to address specific research questions. CellPhePy can be installed via PyPi or GitHub, and we also provide a CellPhePy GUI to aid user accessibility.

cell 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↗

bioRxiv2023

Exploring the impact of variability in cell segmentation and tracking approaches

Segmentation and tracking are essential preliminary steps in the analysis of almost all live cell imaging applications. Although the number of open-source software systems that facilitate automated segmentation and tracking continue to evolve, many researchers continue to opt for manual alternatives for samples that are not easily auto-segmented, tracing cell boundaries by hand and re-identifying cells on consecutive frames by eye. Such methods are subject to inter-user variability, introducing idiosyncrasies into the results of downstream analysis that are a result of subjectivity and individual expertise. Such methods are also susceptible to intra-user variability, meaning findings are challenging to reproduce. Here we demonstrate and quantify the degree of intra- and inter-user variability in manual cell segmentation and tracking by comparing the phenotypic metrics extracted from cells segmented and tracked by different members of our research team. Furthermore, we compare the segmentation results for a ptychographic cell image obtained using different automated software and demonstrate the high dependence of performance on their imaging modality optimisation. Our results show that choice of segmentation and tracking methods should be considered carefully in order to enhance the quality and reproducibility of results.

cell biology↗

bioRxiv2023

An Nav1.5-dependent feedback mechanism driving glycolytic acidification in breast cancer metastasis

Solid tumours have abnormally high intracellular [Na+]. The activity of various Na+ channels may underlie this Na+ accumulation. Voltage-gated Na+ channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Nav1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knock down of Nav1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Nav1.5 activity increases glycolysis in breast cancer cells, likely by up-regulating activity of the Na+/K+ ATPase, thus promoting H+ production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na+ influx through Nav1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and movement of Na+ into cancer cells which can facilitate invasion. These results highlight the clinical significance of Nav1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment.

cancer biology↗

bioRxiv2023

Hypoxic volatile metabolic markers in the MDA-MB-231 breast cancer cell line

Hypoxia in disease describes persistent low oxygen conditions, observed in a range of pathologies, including cancer. In the discovery of biomarkers in biological models, pathophysiological traits present a source of translatable metabolic products for the diagnosis of disease in humans. Part of the metabolome is represented by its volatile, gaseous fraction; the volatilome. Human volatile profiles, such as those found in breath, are able to diagnose disease, however accurate volatile biomarker discovery is required to target reliable biomarkers to develop new diagnostic tools. Using custom chambers to control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast cancer cell line was exposed to hypoxia (1% oxygen) for 24 hours. The maintenance of hypoxic conditions in the system was successfully validated over this time period. Targeting and non-targeting gas chromatography mass spectrometry approaches revealed four significantly altered volatile organic compounds when compared to control cells. Three compounds were actively consumed by cells: methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced significant amounts of styrene. This work presents a novel methodology for identification of volatile metabolisms under controlled gas conditions with novel observations of volatile metabolisms by breast cancer cells.

cell biology↗

bioRxiv2022

Sodium regulates PLC and IP3R-mediated calcium signalling in invasive breast cancer cells

Intracellular Ca2+ signalling and Na+ homeostasis are inextricably linked via ion channels and co-transporters, with alterations in the concentration of one ion having profound effects on the other. Evidence indicates that intracellular Na+ concentration ([Na+]i) is elevated in breast tumours, and that aberrant Ca2+ signalling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effects of Na+ depletion on intracellular Ca2+ handling in metastatic breast cancer cell lines. The relationship between Na+ and Ca2+ was probed using fura-2 and SBFI fluorescence imaging and replacement of extracellular Na+ with equimolar N-methyl-D-glucamine (0Na+/NMDG) or choline chloride (0Na+/ChoCl). In triple-negative MDA-MB-231 and MDA-MB-468 cells and Her2+ SKBR3 cells, but not ER+ MCF-7 cells, 0Na+/NMDG and 0Na+/ChoCl resulted in a slow, sustained depletion in [Na+]i that was accompanied by a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+]i). Application of La3+ in nominal Ca2+-free conditions had no effect on this response, ruling out reverse-mode NCX activity and Ca2+ entry channels. Moreover, the Na+-linked [Ca2+]i increase was independent of membrane potential hyperpolarisation (NS-1619), but was inhibited by pharmacological blockade of IP3 receptors (2-APB), phospholipase C (PLC, U73122) or following depletion of endoplasmic reticulum Ca2+ stores (cyclopiazonic acid). Thus, Na+ is linked to PLC/IP3-mediated activation of endoplasmic reticulum Ca2+ release in metastatic breast cancer cells and this may have an important role in breast tumours where [Na+]i is perturbed.

cancer biology↗

bioRxiv2021

Subcellular dynamics and functional activity of the cleaved Na+ channel β1 subunit intracellular domain

The voltage-gated Na+ channel {beta}1 subunit, encoded by SCN1B, regulates cell surface expression and gating of subunits, and participates in cell adhesion. {beta}1 is cleaved by /{beta} and {gamma}-secretases, releasing an extracellular domain and intracellular domain (ICD) respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on {beta}1 function in breast cancer cells. Using a series of GFP-tagged {beta}1 constructs, we show that {beta}1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal {beta}1-GFP levels occurred following {gamma}-secretase inhibition, implicating endosomes, and/or the preceding plasma membrane, as important sites for secretase processing. Using live-cell imaging, we report {beta}1ICD-GFP accumulation in the nucleus. Furthermore, {beta}1-GFP and {beta}1ICD-GFP both increased Na+ current, whereas {beta}1STOP-GFP, which lacks the ICD, did not, thus highlighting that the {beta}1-ICD was necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is TTX-resistant (carried by Nav1.5), the Na+ current increased by {beta}1-GFP or {beta}1ICD-GFP was TTX-sensitive. In addition, {beta}1-GFP increased mRNA levels of the TTX-sensitive subunits SCN1A/Nav1.1 and SCN9A/Nav1.7. Taken together, this work suggests that the {beta}1-ICD is a critical regulator of subunit function in cancer cells. Our data further highlight that {gamma}-secretase may play a key role in regulating {beta}1 function in breast cancer.

cell biology↗