Biology subjects

Boyne, J. R.

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

5 recordsLinked to original sources

bioRxiv2026

Isolation Strategy Shapes the Matrisome Landscape of Cancer-Associated Fibroblast Extracellular Vesicles

Cancer-associated fibroblasts (CAFs) secrete small extracellular vesicles (sEVs) that mediate stromal remodelling, tumour progression, and pre-metastatic niche formation. A foundational assumption in EV research is that MISEV-compliant preparations from the same conditioned medium are biologically equivalent. Here, we directly challenged this assumption through a side-by-side comparison of ultracentrifugation (UC), size exclusion chromatography (SEC), and the EXODUS nanofiltration platform using breast CAF-conditioned media, characterised in accordance with MISEV2023 guidelines using nanoparticle tracking analysis, cryogenic transmission electron microscopy (Cryo-TEM), and quantitative proteomics. EXODUS and SEC recovered approximately 7-fold more particles per mL than UC. While Cryo-TEM confirmed intact vesicle morphology across all methods, UC preparations exhibited substantial non-vesicular background, with gene ontology analysis revealing significant enrichment of ribosomal, mitochondrial, and ER-derived proteins absent from EXODUS and SEC. Matrisome profiling further uncovered method-dependent divergence in the composition of core versus matrisome-associated proteins, highlighting differences extending beyond standard purity metrics. These findings demonstrate that MISEV2023 compliance is necessary but insufficient for methodological equivalence. The isolation method should be treated as a biological variable and selected according to the EV subpopulation or cargo class under investigation.

cancer biology↗

bioRxiv2026

Stress-responsive enhancer RNAs couple chromatin reprogramming to post-transcriptional control of senescence

BackgroundCellular senescence is accompanied by extensive epigenomic reprogramming leading to changes in enhancer RNA levels, yet how enhancer activity is translated into functional RNA-level regulation remains unclear. Here we investigate how enhancer reprogramming during senescence impacts functional RNA-level regulation by eRNAs. ResultsBy integrating time-resolved transcriptomic analyses across multiple primary human cell types, we identify a set of recurrently dysregulated senescence-associated enhancer RNAs (SAeRs). We focus on one of these transcripts, EN526, which is reproducibly repressed during senescence while its locus remains broadly stable across cell states. EN526 eRNA exhibits cytoplasmic localisation and extensive eRNA-mRNA interactions, and cytoplasmic depletion of EN526 recapitulates its senescence-associated loss and alters the stability and translation of the cell-cycle regulator CDKN2C. EN526 perturbation further mediates stress responses, cellular survival, and extracellular remodelling associated with the senescence phenotype. ConclusionTogether, these findings show that SAeRs changes accompanying enhancer reprogramming in senescence are not merely passive events but can act as functional intermediates linking enhancer dynamics to post-transcriptional regulatory networks that phenocopy key senescence-associated cellular features. Extending this model, genetic associations at the EN526 locus further connect this regulatory axis to age-related traits and circulating protein phenotypes, supporting its broader relevance to human ageing and disease.

genomics↗

bioRxiv2026

Reconstructing the human enhancer RNA transcriptome

Transcript-resolved models of RNA enable functional interrogation of RNA biology by linking processing, structure, localisation, and regulatory interactions to specific RNA molecules. Across coding and noncoding transcriptomes, such models have been essential for defining RNA-level mechanisms relevant to physiology and disease. Enhancer RNAs (eRNAs), however, remain largely characterised without transcript-level definitions, and no widely adopted transcript-resolved reference exists, limiting investigation of how individual eRNAs are processed, localised, and participate in transcriptional regulation or their emerging post-transcriptional functions. Here, we reconstruct a transcript-resolved catalogue of human eRNAs by pan-transcriptome assembly across diverse tissues, cell types and compartments, defining 36,536 transcripts, including a subset with multi-exonic structure. We show that eRNA splice junctions are reproducible features that exhibit cell-type specificity, subcellular localisation bias, and sensitivity to spliceosome perturbation. In perturbation experiments, eRNA splice junction usage responded to SF3B1 mutation, nuclear-cytoplasmic partitioning, and pharmacological inhibition of RNA export, demonstrating regulation across multiple layers of RNA biology. In head and neck squamous cell carcinoma, a subset of these junctions showed altered usage between tumour and matched normal tissue, indicating that processing varies in disease contexts. Across three validation contexts, nearly one-fifth of reconstructed junctions were detectable, with some showing regulated usage, supporting biological reproducibility. Motivated by these observations, we provide both the GTF annotation and junctions BED file, as a framework for studying eRNAs, enabling RNA-centric investigation of their potential functions. The annotations have been incorporated into the eRNAkit database, available at https://github.com/AneneLab/eRNAkit.

genomics↗

bioRxiv2025

Prioritising Functional Noncoding Variants via eRNA Post-transcriptional Interaction Maps in Human Samples

Noncoding variants and mutations outnumber their coding counterparts but remain challenging to interpret functionally. We present TranCi, a method that prioritises human genetic variations by integrating enhancer RNA (eRNA) expression with eRNA-mRNA interactome maps. By linking variant-associated changes in eRNA to downstream gene regulation, TranCi captures functional effects missed by sequence-based or chromatin-centric approaches. In esophageal squamous cell cancer, TranCi identifies noncoding mutations with roles in disease initiation and progression. A personalised mode enables analysis at single-patient resolution, uncovering potential individual-specific regulatory variants. TranCi thus provides a mechanistic framework for interpreting noncoding variations and uniquely identifies their downstream targets, where standard methods often fall short. TranCi is available as a module within the eRNAkit R package (https://github.com/AneneLab/eRNAkit), leveraging its database of eRNA expression and interactions for functional variant interpretation.

genomics↗

bioRxiv2025

Extracellular vesicles from a model of melanoma cancer-associated fibroblasts induce changes in brain microvascular cells consistent with pre-metastatic niche priming.

Malignant melanoma has one of the lowest 5-year survival rates of any cancer, and is recognised for being particularly invasive and metastatic, with the poorest survival outcomes in brain metastases patients. A key characteristic of these tumours is crosstalk between melanoma cells and cells of the tumour microenvironment (TME), such as cancer associated fibroblasts (CAFs). The role of melanoma-derived small extracellular vesicles (sEVs) in potentiating CAFs has been studied extensively, however the role of CAF sEVs in regulation of the local TME and distal pre-metastatic niche (PMN) is less clear. Here, we demonstrate that sEVs derived from an in vitro model of melanoma CAFs alter melanoma to promote oncogenic parameters within models of the TME and target a model of the brain PMN to promote changes associated with melanoma extravasation. Cargo profiling of these sEVs found significant differential expression of proteins, and RNA associated with pre-metastatic niche remodelling and unfavourable outcomes in patients. Together these data suggest a role for CAF sEVs in local and distal PMN formation, highlighting a potential therapeutic target for metastatic melanoma and identifying prospective liquid biomarker reservoirs.

cancer biology↗