Human Formalin-fixed Paraffin-embedded Research Articles

Abstract 6840: Automated preparation of single nuclei from FFPE samples for snRNA-Seq

Abstract S2 Genomics developed the Singulator™ 100 and 200 Systems to automate the processing of fresh, frozen, or OCT tissues into single cell or single nuclei suspensions using disposable cartridges, reagents, and customizable protocols. The systems have been used to prepare cells or nuclei from a wide range of tissues and organisms, including human, mouse, chicken, insects, snails, zebrafish, and plants. Formalin-fixed, paraffin-embedded (FFPE) tissues are the preferred format for pathologists and as many as one billion FFPE slides are in repositories. FFPE tissue samples have traditionally been difficult to study with molecular techniques due to the chemical modifications caused by the formalin fixation, which can lead to degradation and sequestration of RNA and DNA. Recent advances in single cell/nuclei RNA-Seq library preparation technology have made it possible to overcome these challenges and generate high-quality single cell or nuclei genomic data from FFPE samples. Protocols have been released for processing previously deparaffinized and rehydrated FFPE samples on the Singulator 100 or 200 into single nuclei. We now report the development of a fully automated workflow for the deparaffinization and rehydration of FFPE tissues in about 40 min, with an optional enzymatic treatment of 45 min, and processing of FFPE tissues into singulated cells or nuclei on prototype Singulator 200 systems. Data will be presented for the optimization of process parameters for cell or nuclei recovery and RNA quality for six tissues. Finally, single nuclei sequencing data will be presented for snRNA-Seq libraries from healthy and cancerous human FFPE samples including glioblastomas and colorectal cancer. These data demonstrate the successful recovery of genomic materials from FFPE samples using an automated sample preparation system for single cell/nuclei transcriptomics. Citation Format: Stevan Jovanovich, Nate Pereira, Danielle Meyer, Catherine Snopkowski, Ignas Masilionis, Brigita Meškauskaitė, Meril Takizawa, Zaki Abou-Mrad, Kenny Yu, Viviane Tabar, Kathleen Luckett, Karuna Ganesh, Nabiha Khan, Ronan Chaligne. Automated preparation of single nuclei from FFPE samples for snRNA-Seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6840.

Abstract 5354: High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach

Abstract The immune checkpoint involving PD-1 and PD-L1 generates suppressive signals in T cells, which help to prevent autoimmunity by inducing a state of immune exhaustion. In the context of the tumor microenvironment (TME), however, cancer cells can manipulate these pathways to camouflage themselves from an immune attack. Blocking these checkpoints has thus emerged as a key immunotherapeutic tactic. Nonetheless, the success rate of immune checkpoint inhibitors (ICIs) has been mixed, even for patients who test positive for relevant diagnostic biomarkers. Patient stratification currently depends on immunohistochemical staining for checkpoint proteins like PD-L1, but these tests do not provide adequate stratification. A more comprehensive stratification, one that includes immune profiling of the TME plus an evaluation of immune checkpoint interactions, might provide better patient stratification and ICI responsiveness. Here we describe mIF that is augmented with a PD-1 and PD-L1 protein-protein interaction assay. Human formalin-fixed, paraffin-embedded (FFPE) tissue sections were subjected to standard histological processing and then incubated with primary antibodies against PD1 and PD-L1. Sections were then treated with oligonucleotide-modified secondary probes suitable for in situ Proximity Ligation Assay (isPLA), enabling the detection of PD1 and PD-L1 interactions. The PLA protocol utilized a fluorescent probe complementary to amplified nucleic acid to enhance the visibility of the protein interactions. The sample was then mounted in a closed microfluidic chamber and imaged on the CellScape™ platform for Precise Spatial Multiplexing. After imaging of the isPLA signal, the CellScape cyclic multiplex mIF staining approach was used to iteratively stain immune and structural markers on the tissue sample utilizing the VistaPlex™ Spatial Immune Profiling Assay Kit. This proof-of-concept study demonstrates that high-plex mIF can be augmented with an isPLA on human FFPE samples. The combined application of both methods allows visualization of PD1/PD-L1 protein-protein interactions and integrates this interaction within the spatial context of the surrounding cell populations. This approach allows for a more comprehensive insight into the interplay of different immune cell and non-immune cell populations during checkpoint activation processes in normal and neoplastic tissues. Citation Format: Arne Christians, Jannik Boog, Charles E. Jackson, Matthew H. Ingalls, J Spencer Schwarz, Sara Bodbin, Agata Zieba Wicher, Subham Basu, Oliver Braubach. High-resolution analysis of immune checkpoint activation utilizing a combined PD1/PD-L1 in situ proximity ligation assay (isPLA) and multiplex immunofluorescence (mIF) imaging approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5354.

A Hitchhiker's guide to high-dimensional tissue imaging with multiplexed ion beam imaging.

Advancements in multiplexed tissue imaging technologies are vital in shaping our understanding of tissue microenvironmental influences in disease contexts. These technologies now allow us to relate the phenotype of individual cells to their higher-order roles in tissue organization and function. Multiplexed Ion Beam Imaging (MIBI) is one of such technologies, which uses metal isotope-labeled antibodies and secondary ion mass spectrometry (SIMS) to image more than 40 protein markers simultaneously within a single tissue section. Here, we describe an optimized MIBI workflow for high-plex analysis of Formalin-Fixed Paraffin-Embedded (FFPE) tissues following antigen retrieval, metal isotope-conjugated antibody staining, imaging using the MIBI instrument, and subsequent data processing and analysis. While this workflow is focused on imaging human FFPE samples using the MIBI, this workflow can be easily extended to model systems, biological questions, and multiplexed imaging modalities.

Protocol Development for Yielding High Quality DNA and RNA from Archived Formalin-Fixed Paraffin Embedded Tissues

Introduction: While genetic analysis of archived formalin-fixed paraffin embedded (FFPE) tissue specimens would be a significant research resource, extraction of high-quality DNA and RNA from these specimens is a significant challenge. Storage duration, tissue handling and tissue preservation processes as well as their interactions with the nucleic acids could influence the quality and integrity of the DNA or RNA required for genetic analysis.
 Objectives: The goal of this study was to establish a protocol to extract high quality DNA and RNA from the FFPE tissues for further use in quantitative PCR analysis.
 Methods: For the purposes of this study, human FFPE biopsy tissues from lung, liver, colon and kidney were obtained from a single center biorepository. GeneJET Genomic DNA Purification Kit (Catalog # K0722) obtained from Thermo Fisher Scientific and RecoverAll Total Nucleic Acid Isolation Kit obtained from Life Technologies were modified and used for extraction of DNA and RNA, respectively. Briefly, modifications involved extending reagent incubation times, increasing sample volumes and wash steps, and increased final nucleic acids recovery and concentration steps. Eight sections around 8-10 ?m thick were microtomed for each tissue sample and used for extraction. The purity of the nucleic acids obtained was verified using Nanodrop Spectrophotometer.
 Results: The average DNA yield from eight sections for each of the tissues was 270±184 ng/?l and for RNA was 296±188 ng/?l. Nucleic acid quality was assessed by measuring the 260nm/280nm absorbance ratio for protein contamination as well as the 260nm/230nm absorbance ratio for salt contamination. Both were found to be within acceptable ranges. RNA was reverse transcribed to cDNA and qPCR was successfully performed on both DNA and cDNA samples.
 Conclusions: These results indicate that protocols using the silica-based membrane technology can yield high quality DNA and RNA that can be successfully used for downstream genetic analysis.

A high-throughput single-cell RNA expression profiling method identifies human pericyte markers.

We sought to identify and optimise a universally available histological marker for pericytes in the human brain. Such a marker could be a useful tool for researchers. Further, identifying a gene expressed relatively specifically in human pericytes could provide new insights into the biological functions of this fascinating cell type. We analysed single-cell RNA expression profiles derived from different human and mouse brain regions using a high-throughput and low-cost single-cell transcriptome sequencing method called EasySci. Through this analysis, we were able to identify specific gene markers for pericytes, some of which had not been previously characterised. We then used commercially (and therefore universally) available antibodies to immunolabel the pericyte-specific gene products in formalin-fixed paraffin-embedded (FFPE) human brains and also performed immunoblots to determine whether appropriately sized proteins were recognised. In the EasySci data sets, highly pericyte-enriched expression was notable for SLC6A12 and SLC19A1. Antibodies against these proteins recognised bands of approximately the correct size in immunoblots of human brain extracts. Following optimisation of the immunohistochemical technique, staining for both antibodies was strongly positive in small blood vessels and was far more effective than a PDGFRB antibody at staining pericyte-like cells in FFPE human brain sections. In an exploratory sample of other human organs (kidney, lung, liver, muscle), immunohistochemistry did not show the same pericyte-like pattern of staining. The SLC6A12 antibody was well suited for labelling pericytes in human FFPE brain sections, based on the combined results of single-cell RNA-seq analyses, immunoblots and immunohistochemical studies.

Feasibility and safety of synchrotron-based X-ray phase contrast imaging as a technique complementary to histopathology analysis.

X-ray phase contrast imaging (X-PCI) is a powerful technique for high-resolution, three-dimensional imaging of soft tissue samples in a non-destructive manner. In this technical report, we assess the quality of standard histopathological techniques performed on formalin-fixed, paraffin-embedded (FFPE) human tissue samples that have been irradiated with different doses of X-rays in the context of an X-PCI experiment. The data from this study demonstrate that routine histochemical and immunohistochemical staining quality as well as DNA and RNA analyses arenot affected by previous X-PCI on human FFPE samples. From these data we conclude it is feasible and acceptable to perform X-PCI on FFPE human biopsies.

Quantitative super-resolution microscopy reveals the differences in the nanoscale distribution of nuclear phosphatidylinositol 4,5-bisphosphate in human healthy skin and skin warts.

Introduction: Imaging of human clinical formalin-fixed paraffin-embedded (FFPE) tissue sections provides insights into healthy and diseased states and therefore represents a valuable resource for basic research, as well as for diagnostic and clinical purposes. However, conventional light microscopy does not allow to observe the molecular details of tissue and cell architecture due to the diffraction limit of light. Super-resolution microscopy overcomes this limitation and provides access to the nanoscale details of tissue and cell organization. Methods: Here, we used quantitative multicolor stimulated emission depletion (STED) nanoscopy to study the nanoscale distribution of the nuclear phosphatidylinositol 4,5-bisphosphate (nPI(4,5)P2) with respect to the nuclear speckles (NS) marker SON. Results: Increased nPI(4,5)P2 signals were previously linked to human papillomavirus (HPV)-mediated carcinogenesis, while NS-associated PI(4,5)P2 represents the largest pool of nPI(4,5)P2 visualized by staining and microscopy. The implementation of multicolor STED nanoscopy in human clinical FFPE skin and wart sections allowed us to provide here the quantitative evidence for higher levels of NS-associated PI(4,5)P2 in HPV-induced warts compared to control skin. Discussion: These data expand the previous reports of HPV-induced increase of nPI(4,5)P2 levels and reveal for the first time the functional, tissue-specific localization of nPI(4,5)P2 within NS in clinically relevant samples. Moreover, our approach is widely applicable to other human clinical FFPE tissues as an informative addition to the classical histochemistry.

Application of Real-Time PCR Assays for the Diagnosis of Histoplasmosis in Human FFPE Tissues Using Three Molecular Targets.

Histoplasmosis is a fungal infection caused by the thermally dimorphic fungus Histoplasma capsulatum. This infection causes significant morbidity and mortality in people living with HIV/AIDS, especially in countries with limited resources. Currently used diagnostic tests rely on culture and serology but with some limitations. No molecular assays are commercially available and the results from different reports have been variable. We aimed to evaluate quantitative real-time PCR (qPCR) targeting three protein-coding genes of Histoplasma capsulatum (100-kDa, H and M antigens) for detection of this fungus in formalin-fixed paraffin-embedded (FFPE) samples from patients with proven histoplasmosis. The sensitivity of 100-kDa, H and M qPCR assays were 93.9%, 91% and 57%, respectively. The specificity of 100-kDa qPCR was 93% when compared against samples from patients with other mycoses and other infections, and 100% when samples from patients with non-infectious diseases were used as controls. Our findings demonstrate that real-time PCR assays targeting 100-kDa and H antigen showed the most reliable results and can be successfully used for diagnosing this mycosis when testing FFPE samples.

Immunohistochemistry for acetylcholinesterase and butyrylcholinesterase in the dorsal motor nucleus of the vagus (DMNV) of formalin-fixed, paraffin-embedded tissue: comparison with reported literature.

The majority of research regarding the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the brain has been conducted using histochemistry to identify enzymatic activity in frozen fixed tissue. However, retrospective human neurochemistry studies are generally restricted to formalin-fixed, paraffin-embedded (FFPE) tissues that are not suitable for histochemical procedures. The availability of commercially available antibody formulations provides the means to study such tissues by immunohistochemistry (IHC). In this study, we optimised IHC conditions for evaluating the expression of AChE and BuChE in the brainstem, focusing on the dorsal motor nucleus of the vagus, in human and piglet FFPE tissues, using commercially available antibodies. Our results were compared to published reports of histochemically determined AChE and BuChE expression. We varied antibody concentrations and antigen retrieval methods, and evaluated different detection systems, with the overall aim to optimise immunohistochemical staining. The primary findings, consistent across both species, are: (1) AChE and BuChE expression dominated in the neuronal somata, specifically in the neuronal cytoplasm; and (2) no change in the protocol resulted in axonal/neuropil expression of AChE. These results indicate that IHC is a suitable tool to detect AChE and BuChE in FFPE tissue using commercial antibodies, albeit the staining patterns obtained differed from those using histochemistry in frozen tissue. The underlying cause(s) for these differences are discussed in detail and may be associated with the principal components of the staining method, the antibody protein target and/or limitations to the detection of epitopes by tissue fixation.

497 Protein biomarkers in the paraffine-archived human melanoma samples

Since there are only a few tissue markers used in the routine histopathology of melanoma, there is an unmet need for novel protein discovery for both prognostic and predictive implications. Our introduction of formalin-fixed paraffin-embedded (FFPE) melanoma samples provides new opportunities to understand the progression of melanoma. The study was based on the previously archived 175 human FFPE primary and metastatic melanoma samples which were annotated by an enhanced pathology validation for mutated BRAF protein and analyzed by high-resolution mass spectrometry.

A multispectral immunohistochemistry panel to investigate γδ T cells and butyrophilin molecules in the tumour microenvironment

Conventional immunohistochemistry methods though once fundamental for the individual staining of cell markers, have now been superseded by multispectral immunohistochemistry (mIHC). mIHC enables simultaneous detection of multiple cell markers in situ using single formalin-fixed paraffin-embedded (FFPE) tissue sections. In addition to conserving patient tissue specimens, the ability to visualise more than one marker on individual cells allows for further refining of cell phenotypes, and provides insight into cell-to-cell interactions and spatial arrangements across single tissue sections. Here, a comprehensive protocol is described for the in situ interrogation of γδ T cells and phosphoantigen-presenting butyrophilin (BTN) molecules (BTN2A1 and BTN3A1) in human FFPE tissue using Opal™ tyramide signal amplification (TSA)-based mIHC. It is demonstrated that an effectively optimised Opal™-TSA 7-marker [CD3, Pan-γδ T cell receptor (TCR), granzyme B, BTN2A1, BTN3A1, tumour marker, 4’,6-diamidino-2-phenylindole (DAPI)] mIHC panel can be used to define the presence, localisation, and activation status of γδ T cells and the BTN2A1 and BTN3A1 ligands.

Abstract 3865: Spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the RNAscope™ HiPlex v2 in situ hybridization assay

Abstract The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. Understanding spatial interactions between various cell types and their activation states in the TME is crucial for implementing successful immunotherapy strategies against various types of cancer. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in human lung, breast, cervical and ovarian FFPE tumor tissues. We have expanded our current RNAscope HiPlex assay capability of iteratively multiplexing up to 12 targets in fixed and fresh frozen samples to include formalin fixed paraffin embedded (FFPE) tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. We have leveraged this technology to investigate spatial expression of 12 oncology and immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokine RNA expression profile within the TME. The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction. We visualized T cell infiltration and identified T cell subsets within tumors using CD3 and CD8 expression and activated T cells by IFNG expression. We further identified subsets of pro- and anti-inflammatory macrophages by CD68 and CD163 expression as well effector cells which secrete chemokines and cytokine. We also detected the hypoxia markers HIF1A and VEGF to elucidate the immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis software showed higher copy numbers in the lung tumor as compared to the other tumors, demonstrating the sensitivity of the assay through differential expression. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME. Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, we have demonstrated the capability of this technique to spatially resolve 12 targets in four different tumor types. The FFPE reagent efficiently quenched background autofluorescence in the tissues and identified immune cell signatures within the TME. Quantification of immunosuppressive markers further depicted differential expression among various tumors. This technology is highly beneficial for investigating complex and spatial tumor-stroma interactions in basic science and translational research. The assay can also provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues. Citation Format: Anushka Dikshit, Sayantani Basak, Ching-Wei Chang, Michaeline Bunting, Kim Collins. Spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the RNAscope™ HiPlex v2 in situ hybridization assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3865.

Abstract 3814: Multiomic characterization of the tumor microenvironment in FFPE tissue by simultaneous protein and gene expression profiling

Abstract The tumor microenvironment (TME) is composed of highly heterogeneous structures and cell types that dynamically influence and communicate with each other. The constant interaction between a tumor and its microenvironment plays a critical role in how the cancer develops, progresses, and responds to therapies. Traditionally, Hematoxylin and Eosin (H&E) staining has been used to annotate and characterize tissues and associated pathologies. Recent single analyte approaches spatially interrogate targeted or transcriptome-wide expression of RNA in tissue sections, while others capture phenotypes using a limited number of protein markers. However, for a more comprehensive understanding of the unique characteristics of cell types, cell states, and cell-cell interactions within the TME, multiple layers of information are needed and must be studied together. Here we demonstrate a novel, streamlined multiomic spatial assay that integrates histological staining and imaging with simultaneous transcriptome-wide gene expression and highly multiplexed protein expression profiling from the same formalin-fixed paraffin embedded (FFPE) tissue section. In short, tissue sections from archived FFPE samples were placed on slides containing arrayed capture oligos with unique positional barcodes. The H&E or immunofluorescence stained tissues were then imaged, followed by incubation with transcriptome-wide probes and a high-plex DNA-barcoded antibody panel containing intra- and extracellular markers. Transcriptome probes and antibody-barcodes were then spatially captured on the slide and converted into sequencing-ready libraries. Our data analysis and interactive visualization software enable interrogation of all data layers (H&E/immunofluorescence, RNA, protein) from the same tissue section. We apply this method to simultaneously measure gene and protein expression within the TME of human breast cancer and melanoma FFPE samples using whole transcriptome probes and an immune-oncology antibody panel. The data enables comparison and correlation of multiple analytes and their patterns within the same sample section. In addition, this simultaneous detection enables marker-guided regional selection and differential gene expression analysis on the defined regions. Taken together, our data demonstrates that a spatially resolved, multiomic approach provides a more comprehensive understanding of cellular behavior in and around tumors, yielding new insights into disease progression, predictive biomarkers, drug response and resistance, and therapeutic development. Citation Format: Cedric Uytingco, Jennifer Chew, Naishitha Anaparthy, Jun D. Chiang, Christina Galonska, Karthik Ganapathy, Ryo Hatori, Alexander Hermes, Layla Katiraee, Anna-Maria Katsori, William Nitsch, Patrick Roelli, Joe Shuga, Rapolas Spalinskas, Mesruh Turkekul, Benton Veire, Dan Walker, Neil Weisenfeld, Stephen R. Williams, Zachary Bent, Marlon Stoeckius. Multiomic characterization of the tumor microenvironment in FFPE tissue by simultaneous protein and gene expression profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3814.

Tumor Microenvironment Characterization using a Spatial Multiomic Assay to Simultaneously Profile Protein and Gene Expression in FFPE Tumors

The tumor microenvironment (TME) is composed of highly heterogeneous structures and cell types that dynamically influence and communicate with each other. The constant interaction between a tumor and its microenvironment plays a critical role in how the cancer develops, progresses, and responds to therapies. Traditionally, Hematoxylin and Eosin (H&E) and immunohistochemistry staining have been used to annotate and characterize tissues and associated pathologies. Recent single analyte approaches spatially interrogate targeted or transcriptome‐wide expression of RNA in tissue sections, while others capture phenotypes using a limited number of protein markers. However, for a more comprehensive understanding of the unique characteristics of cell types, cell states, and cell‐cell interactions within the TME, analysis of multiple analytes is necessary.Here we demonstrate a novel, streamlined multiomic spatial assay that integrates histological staining and imaging with simultaneous transcriptome‐wide gene expression and highly multiplexed protein expression profiling from the same formalin‐fixed paraffin embedded (FFPE) tissue section. In short, tissue sections from archived FFPE samples were placed on slides containing arrayed capture oligos with unique positional barcodes. The H&E stained tissues were then imaged, followed by incubation with transcriptome‐wide probes and a high‐plex DNA‐barcoded antibody panel containing intra‐ and extracellular markers. Transcriptome probes and antibody‐barcodes were then spatially captured on the slide and converted into sequencing‐ready libraries. Our data analysis and interactive visualization software enable interrogation of all data layers (H&E morphology, RNA, protein) from the same tissue section.We apply this method to simultaneously measure gene and protein expression within the TME of human breast cancer and melanoma FFPE samples using whole transcriptome probes and an immune‐oncology antibody panel. The data enables comparison and correlation of multiple analytes and their patterns within the same sample section. In addition, this simultaneous detection enables marker‐guided regional selection and differential gene expression analysis on the defined areas. Taken together, our data demonstrates that a spatially resolved, multiomic approach provides a more comprehensive understanding of cellular behavior in and around tumors, yielding new insights into disease progression, predictive biomarkers, drug response and resistance, and therapeutic development.

Spatially Resolved Neuropeptide Characterization from Neuropathological Formalin-Fixed, Paraffin-Embedded Tissue Sections by a Combination of Imaging MALDI FT-ICR Mass Spectrometry Histochemistry and Liquid Extraction Surface Analysis-Trapped Ion Mobility Spectrometry-Tandem Mass Spectrometry.

To make the vast collections of well-documented human clinical samples archived in biobanks accessible for mass spectrometry imaging (MSI), recent developments have focused on the label-free top-down MS analysis of neuropeptides in sections of formalin-fixed, paraffin-embedded (FFPE) tissues. In analogy to immunohistochemistry (IHC), this variant of MSI has been designated MSHC (mass spectrometry histochemistry). Besides the detection and localization of neuropeptide and other biomolecular MS signals in these FFPE samples, there is great interest in their molecular identification and full characterization. We here used matrix assisted laser desorption ionization (MALDI) MSI employing ultrahigh-resolution FT-ICR MS on 2,5-dihydroxybenzoic acid (DHB) coated five-micron sections of human FFPE pituitary to demonstrate clear isotope patterns and elemental composition assignment of neuropeptides (with ∼1 ppm mass accuracy). Besides tandem MS fragmentation pattern analysis to deduce or confirm amino acid sequence information (Arg-vasopressin for the case presented here), there is a need for orthogonal primary structure characterization of the peptide-like MS signals of biomolecules desorbed directly off FFPE tissue sections. In the present work, we performed liquid extraction surface analysis (LESA) extractions on consecutive (uncoated) tissue slices. This enables the successful characterization by ion mobility MS of vasopressin present in FFPE material. Differences in sequence coverage are discussed on the basis of the mobility selected collision induced dissociation (CID), electron capture dissociation (ECD), and UV photodissociation (UVPD) MS/MS. Using Arg-vasopressin as model case (a peptide with a disulfide bridged ring structure), we illustrate the use of LESA in combination with a reduction agent for effective sequencing using mobility selected CID, ECD, and UVPD MS/MS.

Chemical capping improves template switching and enhances sequencing of small RNAs.

Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5′ cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5′ cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA—a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&E) annotations are used to target small tumor areas using a blade that is 250 µm2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 µm in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Precise morphological mapping of high plex neurodegenerative and neuroinflammation protein targets in human and mouse FFPE brain tissue with the GeoMx™ digital spatial profiler

AbstractBackgroundNeurodegenerative diseases, including Alzheimer’s, Parkinson’s, and amytrophic lateral sclerosis (ALS), affect millions of people worldwide and represent an increasing burden in terms of healthcare economics and societal impact. The investigation and characterization of the abundance, specific spatial distribution and co‐localization of neurodegenerative and neuroinflammatory targets within the human brain are critical for the advancement of our understanding of progressive degenerative disease.MethodNanoString’s GeoMx Digital Spatial Profiler (DSP) platform allows for simultaneous analysis of >70 proteins from discrete regions of interest (ROI) in FFPE tissue sections, providing morphological context to high plex molecular analysis. The assay relies upon antibody probes coupled to photocleavable oligonucleotide tags. After hybridization of probes to slide‐mounted formalin fixed paraffin‐embedded (FFPE) tissue sections, the oligonucleotide tags are released from discrete regions of the tissue via UV exposure. The size and shape of the UV exposure pattern is adjustable allowing for precise investigation into rare or specific cell populations, disease microenvironments, or neural sub‐regions. Released tags are quantitated on the standard nCounter® platform, and counts are mapped back to tissue location, yielding a spatially‐resolved digital profile of analyte abundance.ResultsIn this study, we validate the GeoMx Human Neural Cell Profiling Core panel (∼20‐plex, RUO) and five add‐on Modules (∼10‐plex each, RUO) covering Alzheimer’s Pathology, Parkinson’s Pathology, Autophagy, and Glial Cell Subtyping on GeoMx DSP platform. In addition, diseased human brain tissue was mapped with fluorescent morphology markers highlighting amyloid beta plaques, microglia, and astrocytes to select ROIs for high plex profiling using the validated antibody set. In comparable studies, mouse neurodegenerative disease models were evaluated with the GeoMx Mouse Neural Cell Profiling Core panel and Alzheimer’s and Parkinson’s Pathology Modules in both FFPE and fresh frozen samples.ConclusionSpecific cell population sampling was demonstrated by masking for multiple or individual microglia, neurons, and astrocytes. Concentric masks around plaques (Contour Profiling) allowed for spatially distinct sampling of the amyloid beta plaque microenvironments. Ongoing efforts will expand the neuroscience‐specific human and mouse antibody catalogues validated for the GeoMx DSP platform.

Targeted metabolomics in formalin-fixed paraffin-embedded tissue specimens: Liquid chromatography-tandem mass spectrometry determination of acidic metabolites in cancer research

Formalin-fixed paraffin-embedded (FFPE) tissues play an irreplaceable role in cancer research. Although extensive research has been conducted for the detection of DNA, RNA and proteins in FFPE samples, literature dealing with the FFPE determination of small molecules is scarce. In this study, we aimed to explore the potential of targeted metabolomics in FFPE specimens. For that purpose, we developed a LC-MS/MS method for the quantification of acidic metabolites in FFPE samples. The method involves trimming tissue slices from FFPE blocks, deparaffinization, lysis of the tissue, o-benzyl hydroxylamine derivatization and LC-MS/MS detection. Deparaffinization and lysis steps were optimized to maximize the analytes extraction and to minimize the effect of the ubiquitous presence of some metabolites in the paraffin. Two validation approaches were applied: (i) using blank paraffin as matrix and (ii) using actual human FFPE tissue samples by standard additions. The method quantified 40 metabolites with appropriate accuracy (commonly 80–120%) and precision (CV 2–19%) in both validation approaches. LLOQs ranging 0.88–2001 pg mg−1 with low-moderate matrix effects (commonly 85–115%) were obtained. FFPE samples from 15 patients with colorectal cancer were analyzed and metabolites concentrations in tumor vs matched normal FFPE tissues were compared. Results show that tumor tissues have a well-established fingerprint including an increase in ketogenesis, a decrease in lipogenesis and an imbalance in the tricarboxylic acid cycle.

Removing Formaldehyde-Induced Peptidyl Crosslinks Enables Mass Spectrometry Imaging of Peptide Hormone Distributions from Formalin-Fixed Paraffin-Embedded Tissues.

Linking molecular and chemical changes to human disease states depends on the availability of appropriate clinical samples, mostly preserved as formalin-fixed paraffin-embedded (FFPE) specimens stored in tissue banks. Mass spectrometry imaging (MSI) enables the visualization of the spatiotemporal distribution of molecules in biological samples. However, MSI is not effective for imaging FFPE tissues because of the chemical modifications of analytes, including complex crosslinking between nucleophilic moieties. Here we used an MS-compatible inorganic nucleophile, hydroxylamine hydrochloride, to chemically reverse inter- and intra-crosslinks from endogenous molecules. The analyte restoration appears specific for formaldehyde-reactive amino acids. This approach enabled the MSI-assisted localization of pancreatic peptides expressed in the alpha, beta, and gamma cells. Pancreatic islet-like distributions of islet hormones were observed in human FFPE tissues preserved for more than five years, demonstrating that samples from biobanks can effectively be investigated with MSI.