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

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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE305348 | GEO | 2026/08/29

REPOSITORIES: GEO

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Project description:Background and Aims Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly available form of archived clinical specimens, which are often stored as thin sections on glass slides. RNA isolated from such archived section (AS) of FFPE tissue is more degraded compared to freshly cut (FC) FFPE section because of prolonged air exposure. In this study, we evaluated performance of transcriptome profiling-based disease classification in AS-FFPE tissue. Methods Genome-wide gene-expression profiles of AS-FFPE tissues of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina), and compared with the profiles previously produced by using FC tissue sections from the same FFPE blocks. Quality of the profiles and performance of gene signature-based class prediction were systematically evaluated. Results RNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate ~ poor quality FC-FFPE samples (R2 as high as 0.93). Gene-expression signal was detected in lower number of probes in AS FFPE samples compared to FC-FFPE samples (proportion of probes with present signal (%P-call): 10%~60% and 70%~90% in AS- and FC-FFPE profiles, respectively). Based on %P-call quality threshold of 20%, 64/88 (77%) HCC and 37/48 (77%) liver profiles were judged as having relatively good quality data with comparable inter-sample correlation. Inter-sample correlation coefficient, as a measure to detect outlier profiles due to poor RNA quality, was also lower in AS-FFPE (0.4~0.9) compared to FC-FFPE (0.6~1.0). In the genome-wide profiling analysis, previously identified molecular subclasses of HCC tumors were reproduced in 67/83 (81%) samples, which was improved to 43/48 (90%) samples when we focused on statistically confident predictions (p<0.05). A 186-gene prognostic signature in liver cirrhosis was reproduced in 32/47 (68%) samples, which was slightly improved to 11/16 (69%) when focused on statistically significant predictions. Conclusions We observed decay of genome-wide transcriptional profiles in AS-FFPE tissues in quantitative manner. However, disease classification was still possible, which suggests potential of AS-FFPE material for clinical diagnosis and prognosis. FFPE tissue sections (10 micron-thick) sliced from 5~16-year-old FFPE blocks and archived for 6~7 years on glass slide Gene-expression profiles of archived section of formalin-fixed paraffin-embedded (AS-FFPE) liver tissues from HCC patients: 47 samples Gene-expression profiles of archived section of formalin-fixed paraffin-embedded (AS-FFPE) tumor tissues from HCC patients: 83 samples

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

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