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Formalin-fixed paraffin-embedded (FFPE) tissues constitute a vast and valuable patient material bank for clinical history and follow-up data. It is still challenging to achieve single cell/nucleus RNA (sc/snRNA) profile in FFPE tissues. Here, we develop a droplet-based snRNA sequencing technology (snRandom-seq) for FFPE tissues by capturing full-length total RNAs with random primers. snRandom-seq shows a minor doublet rate (0.3%), a much higher RNA coverage, and detects more non-coding RNAs and nascent RNAs, compared with state-of-art high-throughput scRNA-seq technologies. snRandom-seq detects a median of >3000 genes per nucleus and identifies 25 typical cell types. Moreover, we apply snRandom-seq on a clinical FFPE human liver cancer specimen and reveal an interesting subpopulation of nuclei with high proliferative activity. Our method provides a powerful snRNA-seq platform for clinical FFPE specimens and promises enormous applications in biomedical research. Formalin-fixed paraffin-embedded (FFPE) tissues constitute a vast and valuable patient material bank, but single nucleus RNAseq using such tissues is challenging. Here the authors develop a droplet-based method called snRandom-seq for high-throughput and sensitive single nucleus RNA-seq of FFPE samples.

Hydrogel-based tissue expansion combined with mass spectrometry (MS) offers an emerging spatial proteomics approach. Here, we present a filter-aided expansion proteomics (FAXP) strategy for spatial proteomics analysis of archived formalin-fixed paraffin-embedded (FFPE) specimens. Compared to our previous ProteomEx method, FAXP employed a customized tip device to enhance both the stability and throughput of sample preparation, thus guaranteeing the reproducibility and robustness of the workflow. FAXP achieved a 14.5-fold increase in volumetric resolution. It generated over 8 times higher peptide yield and a 255% rise in protein identifications while reducing sample preparation time by 50%. We also demonstrated the applicability of FAXP using human colorectal FFPE tissue samples. Furthermore, for the first time, we achieved bona fide single-subcellular proteomics under image guidance by integrating FAXP with laser capture microdissection. Hydrogel-based tissue expansion proteomics represents an emerging spatial proteomics approach. Here, the authors develop the filter-aided expansion proteomics (FAXP) strategy, enabling proteomic analysis of single cells and nuclei in formalin-fixed paraffin-embedded (FFPE) tissue sections.

Whole genome sequencing (WGS) provides comprehensive, individualised cancer genomic information. However, routine tumour biopsies are formalin-fixed and paraffin-embedded (FFPE), damaging DNA, historically limiting their use in WGS. Here we analyse FFPE cancer WGS datasets from England’s 100,000 Genomes Project, comparing 578 FFPE samples with 11,014 fresh frozen (FF) samples across multiple tumour types. We use an approach that characterises rather than discards artefacts. We identify three artefactual signatures, including one known (SBS57) and two previously uncharacterised (SBS FFPE, ID FFPE), and develop an “FFPEImpact” score that quantifies sample artefacts. Despite inferior sequencing quality, FFPE-derived data identifies clinically-actionable variants, mutational signatures and permits algorithmic stratification. Matched FF/FFPE validation cohorts shows good concordance while acknowledging SBS, ID and copy-number artefacts. While FF-derived WGS data remains the gold standard, FFPE-samples can be used for WGS if required, using analytical advancements developed here, potentially democratising whole cancer genomics to many. Formalin fixation is commonly used in tissue storage; however, this process has traditionally limited downstream whole genome sequencing usage. Here, the authors identify artefactual signatures in FFPE-derived sequencing data and demonstrate the preservation of clinical utility, thus enabling FFPE whole genome sequencing when required.

A major issue facing the field of cellular imaging, immunofluorescence (IF), and immunohistochemistry (IHC) microscopy is antibody quality. One of the main methods of antibody validation is testing on positive and negative control tissues with known expression levels of a given antigen. However, this approach is reliant on availability of tissues and reliable protein expression datasets, which are not always available. In contrast, cultured cell lines often have more extensive and reproducible protein expression data available, are relatively inexpensive to maintain, and can be used to produce knockout lines for more robust and functional validation. Due to the difference in staining protocols between formalin-fixed paraffin-embedded (FFPE) tissues and cultured cell lines, an antibody that works well in cultured cells does not always produce the same results in FFPE tissues. For this reason, there is a need for methods to embed cultured cells in paraffin for antibody testing. Previous methods have been published, but many involve use of sharps, which introduces risk of cuts to the investigator, or embedded in agarose first, which results in a lower density of cells. This paper introduces a method of embedding cultured cells using custom designed silicone molds. These molds allow an easy, risk-free embedding process that results in high density cell pellet blocks which can be used for IF and IHC experiments, as well as creation of cell microarrays. Additionally, the silicone molds can be used to embed organoids for IF and IHC analysis.

The ability to analyze intratumoral heterogeneity is of great interest for both diagnostic and basic research purposes. However, currently available dissection techniques are unsuitable for routine use and hard to access financially. Recently, a novel microcore-based dissection technique has been developed by the company Excilone for studying tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) microcore samples. The use of FFPE biological samples for transcriptomic studies, coupled with their small size, remains a real barrier to dissection applications. The efficacy of five commercially available RNA extraction kits were analyzed on microcores collected from human and mice FFPE tissues. Thirty microcore samples of healthy tissue (human uterus and stomach, and murine liver and spleen) were collected and distributed equally and randomly to the five kits assessed. Microcores were collected directly from paraffin blocks using 200 µm inner diameter needles with a sample depth, variable regarding to tissue type, ranging from 450 to 600 µm. Overall RNA yield and RNA fragmentation were evaluated, and RT-qPCR analyses were carried out after deparaffinization and compared to non-deparaffinization protocols. RNA yields and RNA fragmentation varied considerably between kits and FFPE tissues analyzed. Although the main limitation of this technique is the small initial sample size, differences in qPCR efficiency were also observed. Interestingly, no significant differences were observed between deparaffinized and non-deparaffinized microcore samples. Ultimately, we demonstrate the feasibility of using FFPE microcore samples for sensitive molecular biology applications, both with and without deparaffinization. The importance of setting up an optimized workflow was emphasized by significant differences observed in outcomes of the different protocols.

Formalin fixation and paraffin embedding is a timeless, cost-efficient, and widely adopted method of preserving human tissue biospecimens that has resulted in a substantial reservoir of formalin-fixed, paraffin-embedded blocks that represent both the pathology and preanalytical handling of the biospecimen. This reservoir of specimens is increasingly being used for DNA, RNA, and proteomic analyses. To evaluate the impact of preanalytical factors associated with the formalin fixation and paraffin embedding process on downstream morphological and molecular endpoints. We surveyed the existing literature using the National Cancer Institute's Biospecimen Research Database for published reports investigating the potential influence of preanalytical factors associated with the formalin fixation and paraffin embedding process on DNA, RNA, protein, and morphological endpoints. Based on the literature evidence, the molecular, proteomic, and morphological endpoints can be altered in formalin-fixed, paraffin-embedded specimens by suboptimal processing conditions. While the direction and magnitude of effects associated with a given preanalytical factor were dependent on the analyte (DNA, RNA, protein, and morphology) and analytical platform, acceptable conditions are highlighted, and a summary of conditions that could preclude analysis is provided.

The histopathological examination of radical prostatectomy specimens is essential for assessing critical tumor characteristics, including stage, grade, and margins, all of which impact patient prognosis. However, the extent of embedding the prostate has long been a subject of debate, with some advocating partial/selective embedding and others favoring complete embedding. This study establishes a standardized and time-efficient protocol for processing radical prostatectomy specimens with limited embedding while maintaining diagnostic accuracy. Two hundred twenty-six prostatectomy specimens were analyzed, and the results of a highly standardized selective embedding protocol, systematically embedding the apex, the base, the transition to the seminal vesicles, and selected horizontal sections, were compared with full embedding as the gold standard. Non-inferiority testing was conducted by one-sided binomial tests and Pearson-Clopper confidence intervals. Selective embedding provided consistent and accurate diagnostic information with up to 90–98% concordance in pT, margins, ISUP-grade groups, and presence of IDC-P and cribriform tumor growth. In summary, this study establishes an economical standardized protocol for selective embedding of radical prostatectomy specimens with only minimal loss of information.

This review covers the advancements of optical super-resolution microscopy (SRM) on formalin-fixed paraffin-embedded (FFPE) histological samples. We cover the implementation of various SRM strategies in histology, including wide field methods such as structured illumination microscopy, single-molecule localization microscopy and fluorescence fluctuations-based SRM, as well as the point-scanning stimulated emission depletion microscopy. We also cover the recent developments in FFPE-based expansion microscopy. The review highlights the advantages and challenges of these SRM methods in FFPE histology, and provides insights into emerging optical and computational techniques that can potentially open avenues for understanding disease mechanisms, tailoring treatments, and advancing personalized medicine across disciplines. This review article is intended for a broad audience, including histopathologists, biologists, physiologists, and physicists. The review covers cutting-edge advancements in optical super-resolution microscopy (SRM) on formalin-fixed paraffin-embedded (FFPE) histological samples.

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Formalin-fixed paraffin-embedded (FFPE) samples represent a vast, untapped resource for epigenomic research, yet molecular tools for deep analysis of these specimens remain limited. We introduce spatial FFPE-ATAC-seq, an approach for in situ profiling chromatin accessibility within archived tissues. This approach overcomes formalin-induced crosslinking challenges, allowing high-resolution mapping of chromatin landscapes while preserving tissue architecture. Applying spatial FFPE-ATAC-seq to mouse and human tissues, including brain and thymus, reveals intricate spatial organization and distinct cell types in alignment with tissue morphology. Integration with single-cell RNA sequencing validates the precision of our chromatin profiles in identifying key cell types and regulatory elements. We further apply this method to human melanoma, comprehensively characterizing chromatin accessibility across both tumor and non-tumor regions. This method significantly expands the toolkit for epigenomic research, unlocking the potential of an extensive collection of archived FFPE samples for studying gene regulation and disease mechanisms with spatial context. FFPE tissues are widely used to preserve clinical specimens, but formalin-induced crosslinking limits their use in epigenomic profiling. Here, the authors present spatial FFPE-ATACseq, enabling in situ chromatin accessibility mapping while preserving tissue architecture in archived samples.