A laser capture microdissection-based method for high-sensitivity transcriptomics from archived FFPE tissue slides with single-cell resolution using LCM-FFPEseq

Callens, E.; Syryn, H.; Deserranno, K.; Cattebeke, G.; Couckuyt, H.; Van De Velde, J.; Berrevoet, D.; Verplancke, A.; Deforce, D.; De Spiegelaere, W.; Van de Vijver, K.; Cools, M.; Van Nieuwerburgh, F.

doi:10.1101/2025.08.13.669096

bioRxiv · 10.1101/2025.08.13.669096

A laser capture microdissection-based method for high-sensitivity transcriptomics from archived FFPE tissue slides with single-cell resolution using LCM-FFPEseq

Callens, E.·Syryn, H.·Deserranno, K.·Cattebeke, G.·Couckuyt, H.·Van De Velde, J.·Berrevoet, D.·Verplancke, A.·Deforce, D.·De Spiegelaere, W.·Van de Vijver, K.·Cools, M.·Van Nieuwerburgh, F.

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Abstract

Understanding gene expression within its spatial context is essential for unravelling biological processes. Laser Capture Microdissection (LCM) has emerged as a transformative technology, enabling targeted isolation of individual cells or regions from tissue sections while preserving spatial context. However, its application to formalin-fixed, paraffin-embedded (FFPE) tissues has been limited by RNA degradation, leaving the vast repository of clinical FFPE samples underutilized. To address this, we introduce LCM-FFPEseq, a novel method combining LCM with the advanced Smart-seq3xpress protocol and FFPE-specific adaptations for spatial transcriptomics of FFPE sections. Unlike traditional protocols requiring thousands of cells to generate high-quality libraries, LCM-FFPEseq achieves high sensitivity, reproducibility, and transcript coverage. With as few as 30 FFPE-embedded K562 cells and Sertoli cells, we detected over 14,000 protein-coding genes per sample, with no substantial gains when a higher number of cells were isolated. Even individual LCM-isolated cells yielded an average of 7,353 or 6,490 protein-coding genes per K562 or Sertoli single cell, respectively. To demonstrate its clinical utility, we applied LCM-FFPEseq to archived testicular FFPE samples from transgender females receiving gender-affirming hormone therapy. Transcriptomic profiling of isolated seminiferous tubules revealed tubular hyalinization to be associated with greater upregulation of extracellular matrix remodelling and inflammatory pathways, alongside stronger downregulation of spermatogenesis-associated pathways. These findings suggest that testicular fibrosis and/or tubular hyalinization may contribute to germ cell loss following inappropriate hormonal exposure. By enabling high-resolution transcriptomics in archived FFPE samples, LCM-FFPEseq unlocks new possibilities for investigating rare cell types, spatial heterogeneity, and therapy-induced tissue remodelling in vast FFPE repositories.

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Callens, E., Syryn, H., Deserranno, K., Cattebeke, G., Couckuyt, H., Van De Velde, J., Berrevoet, D., Verplancke, A., Deforce, D., De Spiegelaere, W., Van de Vijver, K., Cools, M., Van Nieuwerburgh, F.. 2025-08-15. A laser capture microdissection-based method for high-sensitivity transcriptomics from archived FFPE tissue slides with single-cell resolution using LCM-FFPEseq. https://doi.org/10.1101/2025.08.13.669096

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