Biology subjects

Calcutt, E.

Publications and source records attributed to Calcutt, E..

4 recordsLinked to original sources

bioRxiv2026

A platform for deep topographic multiomic mapping of the bone marrow environment in multiple myeloma

Multiple myeloma is a genetically complex plasma cell malignancy in which profound intratumoral heterogeneity shapes disease progression and therapeutic resistance. Genomic subclones dynamically emerge and evolve under treatment pressure, yet their spatial organization within intact bone marrow remains poorly defined. We hypothesized that genetically distinct myeloma subclones occupy spatially discrete ecological niches associated with unique molecular and microenvironmental states. To test this, we here demonstrate a proof-of-concept spatial tri-omics workflow combining laser capture microdissection-based genomics and deep LC-MS/MS proteomics with single-cell spatial transcriptomics, integrated by digital co-registration of adjacent tissue sections. Applied to a myeloma bone marrow trephine, this approach enabled coordinated profiling of matched anatomical regions demonstrating that genomic, transcriptomic and proteomic profiles converged to define clone-specific microenvironmental programmes. We show that genetically distinct myeloma subclones occupy mutually exclusive regions of the same bone marrow biopsy. Remarkably, an aggressive del(17p) clone and a gain(12q) clone occupied defined anatomical territories within the same trephine-biopsied section, each clone exhibiting distinct genomic, transcriptomic, proteomic and cellular ecosystems. The del(17p) clone localized to a fibrotic, osteolytic and angiogenic niche enriched for activated stromal cells, osteoblasts, osteoclasts and immunoregulatory myeloid populations, whereas the gain(12q) clone occupied a comparatively quiescent marrow niche with limited stromal remodelling. Together, these findings demonstrate that genetically distinct myeloma subclones establish spatially and biologically discrete ecological niches through coordinated remodelling of stromal, immune and bone compartments, providing a framework for understanding clonal evolution within the bone marrow microenvironment.

cancer biology↗

bioRxiv2025

Distinct 5' and 3' Coverage Biases Shape Transcriptome Interpretation in Nanopore Direct RNA versus PCR-cDNA Sequencing

Long-read RNA sequencing enables isoform-resolved transcriptomics, but library preparation introduces systematic biases that shape biological interpretation. We benchmarked Oxford Nanopores two protocols--PCR-cDNA and direct RNA--using SKMM2 myeloma cells stimulated with interleukin-6 (IL-6) and ERCC synthetic spike-ins. Direct RNA produced longer, higher-quality reads and more high-confidence isoforms, but showed pronounced 5' coverage loss. PCR-cDNA yielded shorter fragments with 3' underrepresentation, detecting more low-abundance transcripts at reduced confidence. Protocol-specific biases had major consequences: differential expression analysis revealed limited overlap in IL-6-responsive genes, and pathway enrichment was broader in direct RNA. At the isoform level, differential transcript usage was almost entirely protocol-specific, with case studies (e.g. RPL22L1, GRB2, RNF220) illustrating concordance and divergence. ERCC controls confirmed these biases as technical rather than biological. Together, our results show that while both methods provide accurate gene-level quantification, transcript-level conclusions depend critically on protocol choice, highlighting the need for careful selection in long-read transcriptomics.

cell biology↗

bioRxiv2025

ATF4-Mediated Metabolic Stress Response as a Therapeutic Vulnerability in Chordoma

Chordoma, a rare primary bone malignancy, currently lacks effective targeted therapies. Despite surgical resection and adjuvant radiotherapy, prognosis remains poor. Recent preclinical studies have highlighted potential therapeutic targets, including the transcription factor TBXT. However, clinical outcomes associated with therapies targeting TBXT remain underexplored or have been modest, warranting further investigation. In this study, we investigated the therapeutic potential of tRNA synthetase inhibitors in chordoma treatment. Focused compound screening identified distinct chemotypes targeting human glutamyl-prolyl-tRNA synthetase (EPRS) as effective in reducing cell viability in chordoma cell lines through an ATF4-mediated stress response rather than through TBXT regulation. Mechanistically significant upregulation of ATF4 and associated stress response genes was identified with consecutive pro-apoptotic DDIT3-mediated cell death. The prototypic EPRS inhibitor halofuginone demonstrated significant tumour growth inhibition in an in vivo patient-derived xenograft model. These results suggest that targeting metabolic stress pathways via ATF4 activation presents a novel therapeutic approach for chordoma, warranting further clinical investigation.

cancer biology↗

bioRxiv2024

Anchor-Enhanced Bead Design for Reduced Oligonucleotide Synthesis Errors in Single-cell sequencing

Single-cell transcriptomics, reliant on the incorporation of barcodes and unique molecular identifiers (UMIs) into captured polyA+ mRNA, faces a significant challenge due to synthesis errors in oligonucleotide capture sequences. These inaccuracies, which are especially problematic in long-read sequencing, impair the precise identification of sequences and result in inaccuracies in UMI deduplication. To mitigate this issue, we have modified the oligonucleotide capture design, which integrates an interposed anchor between the barcode and UMI, and a V base anchor adjacent to the polyA capture region. This configuration is devised to ensure compatibility with both short and long-read sequencing technologies, facilitating improved UMI recovery and enhanced feature detection, thereby improving the efficacy of droplet-based sequencing methods.

molecular biology↗