Identification of the stress granule transcriptome via RNA-editing in single cells and in vivo.

Stress granules are phase-separated assemblies formed around RNAs. So far, the techniques available to identify these RNAs are not suitable for single cells and small tissues displaying cell heterogeneity. Here, we used TRIBE (target of RNA-binding proteins identified by editing) to profile stress granule RNAs. We used an RNA-binding protein (FMR1) fused to the catalytic domain of an RNA-editing enzyme (ADAR), which coalesces into stress granules upon oxidative stress. RNAs colocalized with this fusion are edited, producing mutations that are detectable by VASA sequencing. Using single-molecule FISH, we validated that this purification-free method can reliably identify stress granule RNAs in bulk and single S2 cells and in Drosophila neurons. Similar to mammalian cells, we find that stress granule mRNAs encode ATP binding, cell cycle, and transcription factors. This method opens the possibility to identify stress granule RNAs and other RNA-based assemblies in other single cells and tissues.

High-throughput total RNA sequencing in single cells using VASA-seq.

Most methods for single-cell transcriptome sequencing amplify the termini of polyadenylated transcripts, capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts and hinders alternative splicing analysis. We, therefore, developed VASA-seq to detect the total transcriptome in single cells, which is enabled by fragmenting and tailing all RNA molecules subsequent to cell lysis. The method is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to more than 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. Analyzing the dynamics of the total single-cell transcriptome, we discovered cell type markers, many based on non-coding RNA, and performed in vivo cell cycle analysis via detection of non-polyadenylated histone genes. RNA velocity characterization was improved, accurately retracing blood maturation trajectories. Moreover, our VASA-seq data provide a comprehensive analysis of alternative splicing during mammalian development, which highlighted substantial rearrangements during blood development and heart morphogenesis.

Spatial deconvolution of HER2-positive breast cancer delineates tumor-associated cell type interactions.

In the past decades, transcriptomic studies have revolutionized cancer treatment and diagnosis. However, tumor sequencing strategies typically result in loss of spatial information, critical to understand cell interactions and their functional relevance. To address this, we investigate spatial gene expression in HER2-positive breast tumors using Spatial Transcriptomics technology. We show that expression-based clustering enables data-driven tumor annotation and assessment of intra- and interpatient heterogeneity; from which we discover shared gene signatures for immune and tumor processes. By integration with single cell data, we spatially map tumor-associated cell types to find tertiary lymphoid-like structures, and a type I interferon response overlapping with regions of T-cell and macrophage subset colocalization. We construct a predictive model to infer presence of tertiary lymphoid-like structures, applicable across tissue types and technical platforms. Taken together, we combine different data modalities to define a high resolution map of cellular interactions in tumors and provide tools generalizing across tissues and diseases.

Identification and transfer of spatial transcriptomics signatures for cancer diagnosis.

BACKGROUND: Distinguishing ductal carcinoma in situ (DCIS) from invasive ductal carcinoma (IDC) regions in clinical biopsies constitutes a diagnostic challenge. Spatial transcriptomics (ST) is an in situ capturing method, which allows quantification and visualization of transcriptomes in individual tissue sections. In the past, studies have shown that breast cancer samples can be used to study their transcriptomes with spatial resolution in individual tissue sections. Previously, supervised machine learning methods were used in clinical studies to predict the clinical outcomes for cancer types. METHODS: We used four publicly available ST breast cancer datasets from breast tissue sections annotated by pathologists as non-malignant, DCIS, or IDC. We trained and tested a machine learning method (support vector machine) based on the expert annotation as well as based on automatic selection of cell types by their transcriptome profiles. RESULTS: We identified expression signatures for expert annotated regions (non-malignant, DCIS, and IDC) and build machine learning models. Classification results for 798 expression signature transcripts showed high coincidence with the expert pathologist annotation for DCIS (100%) and IDC (96%). Extending our analysis to include all 25,179 expressed transcripts resulted in an accuracy of 99% for DCIS and 98% for IDC. Further, classification based on an automatically identified expression signature covering all ST spots of tissue sections resulted in prediction accuracy of 95% for DCIS and 91% for IDC. CONCLUSIONS: This concept study suggest that the ST signatures learned from expert selected breast cancer tissue sections can be used to identify breast cancer regions in whole tissue sections including regions not trained on. Furthermore, the identified expression signatures can classify cancer regions in tissue sections not used for training with high accuracy. Expert-generated but even automatically generated cancer signatures from ST data might be able to classify breast cancer regions and provide clinical decision support for pathologists in the future.

A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart.

The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.

High-definition spatial transcriptomics for in situ tissue profiling.

Spatial and molecular characteristics determine tissue function, yet high-resolution methods to capture both concurrently are lacking. Here, we developed high-definition spatial transcriptomics, which captures RNA from histological tissue sections on a dense, spatially barcoded bead array. Each experiment recovers several hundred thousand transcript-coupled spatial barcodes at 2-µm resolution, as demonstrated in mouse brain and primary breast cancer. This opens the way to high-resolution spatial analysis of cells and tissues.

Barcoded solid-phase RNA capture for Spatial Transcriptomics profiling in mammalian tissue sections.

Spatial resolution of gene expression enables gene expression events to be pinpointed to a specific location in biological tissue. Spatially resolved gene expression in tissue sections is traditionally analyzed using immunohistochemistry (IHC) or in situ hybridization (ISH). These technologies are invaluable tools for pathologists and molecular biologists; however, their throughput is limited to the analysis of only a few genes at a time. Recent advances in RNA sequencing (RNA-seq) have made it possible to obtain unbiased high-throughput gene expression data in bulk. Spatial Transcriptomics combines the benefits of traditional spatially resolved technologies with the massive throughput of RNA-seq. Here, we present a protocol describing how to apply the Spatial Transcriptomics technology to mammalian tissue. This protocol combines histological staining and spatially resolved RNA-seq data from intact tissue sections. Once suitable tissue-specific conditions have been established, library construction and sequencing can be completed in ~5-6 d. Data processing takes a few hours, with the exact timing dependent on the sequencing depth. Our method requires no special instruments and can be performed in any laboratory with access to a cryostat, microscope and next-generation sequencing.

Spatial maps of prostate cancer transcriptomes reveal an unexplored landscape of heterogeneity.

Intra-tumor heterogeneity is one of the biggest challenges in cancer treatment today. Here we investigate tissue-wide gene expression heterogeneity throughout a multifocal prostate cancer using the spatial transcriptomics (ST) technology. Utilizing a novel approach for deconvolution, we analyze the transcriptomes of nearly 6750 tissue regions and extract distinct expression profiles for the different tissue components, such as stroma, normal and PIN glands, immune cells and cancer. We distinguish healthy and diseased areas and thereby provide insight into gene expression changes during the progression of prostate cancer. Compared to pathologist annotations, we delineate the extent of cancer foci more accurately, interestingly without link to histological changes. We identify gene expression gradients in stroma adjacent to tumor regions that allow for re-stratification of the tumor microenvironment. The establishment of these profiles is the first step towards an unbiased view of prostate cancer and can serve as a dictionary for future studies.

Gene expression profiling of periodontitis-affected gingival tissue by spatial transcriptomics.

Periodontitis is a highly prevalent chronic inflammatory disease of the periodontium, leading ultimately to tooth loss. In order to characterize the gene expression of periodontitis-affected gingival tissue, we have here simultaneously quantified and localized gene expression in periodontal tissue using spatial transcriptomics, combining RNA sequencing with histological analysis. Our analyses revealed distinct clusters of gene expression, which were identified to correspond to epithelium, inflamed areas of connective tissue, and non-inflamed areas of connective tissue. Moreover, 92 genes were identified as significantly up-regulated in inflamed areas of the gingival connective tissue compared to non-inflamed tissue. Among these, immunoglobulin lambda-like polypeptide 5 (IGLL5), signal sequence receptor subunit 4 (SSR4), marginal zone B and B1 cell specific protein (MZB1), and X-box binding protein 1 (XBP1) were the four most highly up-regulated genes. These genes were also verified as significantly higher expressed in gingival tissue of patients with periodontitis compared to healthy controls, using reverse transcription quantitative polymerase chain reaction. Moreover, the protein expressions of up-regulated genes were verified in gingival biopsies by immunohistochemistry. In summary, in this study, we report distinct gene expression signatures within periodontitis-affected gingival tissue, as well as specific genes that are up-regulated in inflamed areas compared to non-inflamed areas of gingival tissue. The results obtained from this study may add novel information on the genes and cell types contributing to pathogenesis of the chronic inflammatory disease periodontitis.

Spatial detection of fetal marker genes expressed at low level in adult human heart tissue.

Heart failure is a major health problem linked to poor quality of life and high mortality rates. Hence, novel biomarkers, such as fetal marker genes with low expression levels, could potentially differentiate disease states in order to improve therapy. In many studies on heart failure, cardiac biopsies have been analyzed as uniform pieces of tissue with bulk techniques, but this homogenization approach can mask medically relevant phenotypes occurring only in isolated parts of the tissue. This study examines such spatial variations within and between regions of cardiac biopsies. In contrast to standard RNA sequencing, this approach provides a spatially resolved transcriptome- and tissue-wide perspective of the adult human heart, and enables detection of fetal marker genes expressed by minor subpopulations of cells within the tissue. Analysis of patients with heart failure, with preserved ejection fraction, demonstrated spatially divergent expression of fetal genes in cardiac biopsies.

Spatially resolved transcriptome profiling in model plant species.

Understanding complex biological systems requires functional characterization of specialized tissue domains. However, existing strategies for generating and analysing high-throughput spatial expression profiles were developed for a limited range of organisms, primarily mammals. Here we present the first available approach to generate and study high-resolution, spatially resolved functional profiles in a broad range of model plant systems. Our process includes high-throughput spatial transcriptome profiling followed by spatial gene and pathway analyses. We first demonstrate the feasibility of the technique by generating spatial transcriptome profiles from model angiosperms and gymnosperms microsections. In Arabidopsis thaliana we use the spatial data to identify differences in expression levels of 141 genes and 189 pathways in eight inflorescence tissue domains. Our combined approach of spatial transcriptomics and functional profiling offers a powerful new strategy that can be applied to a broad range of plant species, and is an approach that will be pivotal to answering fundamental questions in developmental and evolutionary biology.

ST Pipeline: an automated pipeline for spatial mapping of unique transcripts.

MOTIVATION: In recent years we have witnessed an increase in novel RNA-seq based techniques for transcriptomics analysis. Spatial transcriptomics is a novel RNA-seq based technique that allows spatial mapping of transcripts in tissue sections. The spatial resolution adds an extra level of complexity, which requires the development of new tools and algorithms for efficient and accurate data processing. RESULTS: Here we present a pipeline to automatically and efficiently process RNA-seq data obtained from spatial transcriptomics experiments to generate datasets for downstream analysis. AVAILABILITY AND IMPLEMENTATION: The ST Pipeline is open source under a MIT license and it is available at https://github.com/SpatialTranscriptomicsResearch/st_pipeline. CONTACT: jose.fernandez.navarro@scilifelab.se. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An automated approach to prepare tissue-derived spatially barcoded RNA-sequencing libraries.

Sequencing the nucleic acid content of individual cells or specific biological samples is becoming increasingly common. This drives the need for robust, scalable and automated library preparation protocols. Furthermore, an increased understanding of tissue heterogeneity has lead to the development of several unique sequencing protocols that aim to retain or infer spatial context. In this study, a protocol for retaining spatial information of transcripts has been adapted to run on a robotic workstation. The method spatial transcriptomics is evaluated in terms of robustness and variability through the preparation of reference RNA, as well as through preparation and sequencing of six replicate sections of a gingival tissue biopsy from a patient with periodontitis. The results are reduced technical variability between replicates and a higher throughput, processing four times more samples with less than a third of the hands on time, compared to the standard protocol.

Massive and parallel expression profiling using microarrayed single-cell sequencing.

Single-cell transcriptome analysis overcomes problems inherently associated with averaging gene expression measurements in bulk analysis. However, single-cell analysis is currently challenging in terms of cost, throughput and robustness. Here, we present a method enabling massive microarray-based barcoding of expression patterns in single cells, termed MASC-seq. This technology enables both imaging and high-throughput single-cell analysis, characterizing thousands of single-cell transcriptomes per day at a low cost (0.13 USD/cell), which is two orders of magnitude less than commercially available systems. Our novel approach provides data in a rapid and simple way. Therefore, MASC-seq has the potential to accelerate the study of subtle clonal dynamics and help provide critical insights into disease development and other biological processes.

Visualization and analysis of gene expression in tissue sections by spatial transcriptomics.

Analysis of the pattern of proteins or messengerRNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.