Background

Clinical tissue specimens are predominantly preserved using the formalin-fixed paraffin-embedded (FFPE) technique, a universally established method for the long-term conservation of biological tissues. In the United States alone, pathology laboratories process between 10,000 and 100,000 FFPE blocks annually, underscoring the immense scale and significance of this resource for clinical diagnostics and translational research [1]. These FFPE samples serve as a cornerstone for retrospective studies, enabling molecular and histopathological analyses that drive advancements in precision medicine and biomedical discovery [1]. Advances in molecular technologies, including genomics, transcriptomics, and proteomics, have expanded the utility of FFPE samples for molecular characterization [2,3]. However, these applications are traditionally conducted at the bulk tissue level, which fails to resolve the intricate molecular heterogeneity inherent in cancers and other complex biological settings. Technologies enabling single-cell transcriptomic profiling of FFPE tissues thus hold immense potential for advancing human health research.

Previously, our team successfully conducted single-nucleus (sn) transcriptomic profiling using clinical human FFPE tissue samples, demonstrating the feasibility of isolating intact nuclei from FFPE samples for molecular characterization [4]. However, extending this technology to transcriptomics poses a significant challenge due to RNA fragmentation caused by formalin fixation, heat exposure, and paraffin embedding [2]. RNA degradation poses a significant challenge to the performance of widely used single-cell/nucleus RNA sequencing (sc/sn RNA-seq) methods, such as the 10× Genomics 3' assay and SMART-seq, which rely on poly(dT) probes to selectively capture and reverse-transcribe intact mRNA molecules [5,6]. RNA-binding probes enable gene expression profiling in FFPE samples and are used in spatial methods like MERFISH, 10× Visium, and Xenium. While resilient to RNA fragmentation, these methods lack transcriptome-wide or single-cell resolution [7–9]. To address this limitation, 10× Genomics introduced Flex chemistry, which employs RNA-targeting probes capable of capturing short RNA fragments, significantly improving compatibility with FFPE-derived material [10].

To address challenges in analyzing FFPE samples, we developed snPATHO-seq, a nuclei isolation protocol optimized for FFPE tissues. This workflow integrates rehydration, enzyme-based dissociation, and nuclei isolation (an example of good-quality nuclei is shown in Figure 1), followed by gene expression analysis using the 10× Genomics Flex assay. We validated snPATHO-seq on three human breast cancer FFPE samples, demonstrating strong agreement with standard 10× 3' and Flex chemistries, previously restricted to fresh or frozen tissues.

Figure 1. Representative example of good-quality nuclei preparation. Brightfield image was taken with a 20× objective on the ZEISS Primovert.

In parallel, 10× Genomics introduced a single-cell fixation protocol for single-cell RNA sequencing [11]. 10× Genomics’ scFFPE protocol does not include a defined nuclei isolation step, leading to the isolation of a mixture of intact cells and nuclei from FFPE samples. This incomplete separation can compromise data quality by introducing variability in transcript capture efficiency and downstream interpretation. In contrast, snPATHO-seq incorporates dedicated membrane lysis and nuclei isolation steps, enabling consistent enrichment for nuclei and minimizing contamination from partially digested cells. Our direct comparison of the two workflows demonstrated that snPATHO-seq delivers more robust and reproducible gene expression profiles, better suited for high-quality single-nucleus transcriptomics from FFPE tissues [12]. This technical advancement strengthens snPATHO-seq as a more mature and reliable protocol for FFPE single-nucleus studies.

In summary, snPATHO-seq unlocks the potential of archival FFPE samples, offering a robust and mature protocol for single-nucleus transcriptomic analysis. By overcoming the technical challenges of RNA degradation and enabling single-cell resolution profiling, snPATHO-seq bridges the gap between traditional pathology and molecular research, fostering new opportunities for clinical and translational studies.