Points2Regions: Fast, interactive clustering of imaging-based spatial transcriptomics data.

Imaging-based spatial transcriptomics techniques generate data in the form of spatial points belonging to different mRNA classes. A crucial part of analyzing the data involves the identification of regions with similar composition of mRNA classes. These biologically interesting regions can manifest at different spatial scales. For example, the composition of mRNA classes on a cellular scale corresponds to cell types, whereas compositions on a millimeter scale correspond to tissue-level structures. Traditional techniques for identifying such regions often rely on complementary data, such as pre-segmented cells, or lengthy optimization. This limits their applicability to tasks on a particular scale, restricting their capabilities in exploratory analysis. This article introduces "Points2Regions," a computational tool for identifying regions with similar mRNA compositions. The tool's novelty lies in its rapid feature extraction by rasterizing points (representing mRNAs) onto a pyramidal grid and its efficient clustering using a combination of hierarchical and k -means clustering. This enables fast and efficient region discovery across multiple scales without relying on additional data, making it a valuable resource for exploratory analysis. Points2Regions has demonstrated performance similar to state-of-the-art methods on two simulated datasets, without relying on segmented cells, while being several times faster. Experiments on real-world datasets show that regions identified by Points2Regions are similar to those identified in other studies, confirming that Points2Regions can be used to extract biologically relevant regions. The tool is shared as a Python package integrated into TissUUmaps and a Napari plugin, offering interactive clustering and visualization, significantly enhancing user experience in data exploration.

Cellular architecture of evolving neuroinflammatory lesions and multiple sclerosis pathology.

Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single-cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single-cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease-associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single-cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS.

TissUUmaps 3: Improvements in interactive visualization, exploration, and quality assessment of large-scale spatial omics data.

Background and objectives: Spatially resolved techniques for exploring the molecular landscape of tissue samples, such as spatial transcriptomics, often result in millions of data points and images too large to view on a regular desktop computer, limiting the possibilities in visual interactive data exploration. TissUUmaps is a free, open-source browser-based tool for GPU-accelerated visualization and interactive exploration of 107+ data points overlaying tissue samples. Methods: Herein we describe how TissUUmaps 3 provides instant multiresolution image viewing and can be customized, shared, and also integrated into Jupyter Notebooks. We introduce new modules where users can visualize markers and regions, explore spatial statistics, perform quantitative analyses of tissue morphology, and assess the quality of decoding in situ transcriptomics data. Results: We show that thanks to targeted optimizations the time and cost associated with interactive data exploration were reduced, enabling TissUUmaps 3 to handle the scale of today's spatial transcriptomics methods. Conclusion: TissUUmaps 3 provides significantly improved performance for large multiplex datasets as compared to previous versions. We envision TissUUmaps to contribute to broader dissemination and flexible sharing of largescale spatial omics data.

Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin.

The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5-12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.

A topographic atlas defines developmental origins of cell heterogeneity in the human embryonic lung.

The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

Visualization and quality control tools for large-scale multiplex tissue analysis in TissUUmaps3.

Large-scale multiplex tissue analysis aims to understand processes such as development and tumor formation by studying the occurrence and interaction of cells in local environments in, for example, tissue samples from patient cohorts. A typical procedure in the analysis is to delineate individual cells, classify them into cell types, and analyze their spatial relationships. All steps come with a number of challenges, and to address them and identify the bottlenecks of the analysis, it is necessary to include quality control tools in the analysis workflow. This makes it possible to optimize the steps and adjust settings in order to get better and more precise results. Additionally, the development of automated approaches for tissue analysis requires visual verification to reduce skepticism with regard to the accuracy of the results. Quality control tools could be used to build users' trust in automated approaches. In this paper, we present three plugins for visualization and quality control in large-scale multiplex tissue analysis of microscopy images. The first plugin focuses on the quality of cell staining, the second one was made for interactive evaluation and comparison of different cell classification results, and the third one serves for reviewing interactions of different cell types.

Automated detection of vascular remodeling in tumor-draining lymph nodes by the deep-learning tool HEV-finder.

Vascular remodeling is common in human cancer and has potential as future biomarkers for prediction of disease progression and tumor immunity status. It can also affect metastatic sites, including the tumor-draining lymph nodes (TDLNs). Dilation of the high endothelial venules (HEVs) within TDLNs has been observed in several types of cancer. We recently demonstrated that it is a premetastatic effect that can be linked to tumor invasiveness in breast cancer. Manual visual assessment of changes in vascular morphology is a tedious and difficult task, limiting high-throughput analysis. Here we present a fully automated approach for detection and classification of HEV dilation. By using 12,524 manually classified HEVs, we trained a deep-learning model and created a graphical user interface for visualization of the results. The tool, named the HEV-finder, selectively analyses HEV dilation in specific regions of the lymph nodes. We evaluated the HEV-finder's ability to detect and classify HEV dilation in different types of breast cancer compared to manual annotations. Our results constitute a successful example of large-scale, fully automated, and user-independent, image-based quantitative assessment of vascular remodeling in human pathology and lay the ground for future exploration of HEV dilation in TDLNs as a biomarker. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Morphological Features Extracted by AI Associated with Spatial Transcriptomics in Prostate Cancer.

Prostate cancer is a common cancer type in men, yet some of its traits are still under-explored. One reason for this is high molecular and morphological heterogeneity. The purpose of this study was to develop a method to gain new insights into the connection between morphological changes and underlying molecular patterns. We used artificial intelligence (AI) to analyze the morphology of seven hematoxylin and eosin (H&E)-stained prostatectomy slides from a patient with multi-focal prostate cancer. We also paired the slides with spatially resolved expression for thousands of genes obtained by a novel spatial transcriptomics (ST) technique. As both spaces are highly dimensional, we focused on dimensionality reduction before seeking associations between them. Consequently, we extracted morphological features from H&E images using an ensemble of pre-trained convolutional neural networks and proposed a workflow for dimensionality reduction. To summarize the ST data into genetic profiles, we used a previously proposed factor analysis. We found that the regions were automatically defined, outlined by unsupervised clustering, associated with independent manual annotations, in some cases, finding further relevant subdivisions. The morphological patterns were also correlated with molecular profiles and could predict the spatial variation of individual genes. This novel approach enables flexible unsupervised studies relating morphological and genetic heterogeneity using AI to be carried out.

Glandular Segmentation of Prostate Cancer: An Illustration of How the Choice of Histopathological Stain Is One Key to Success for Computational Pathology.

Digital pathology offers the potential for computer-aided diagnosis, significantly reducing the pathologists' workload and paving the way for accurate prognostication with reduced inter-and intra-observer variations. But successful computer-based analysis requires careful tissue preparation and image acquisition to keep color and intensity variations to a minimum. While the human eye may recognize prostate glands with significant color and intensity variations, a computer algorithm may fail under such conditions. Since malignancy grading of prostate tissue according to Gleason or to the International Society of Urological Pathology (ISUP) grading system is based on architectural growth patterns of prostatic carcinoma, automatic methods must rely on accurate identification of the prostate glands. But due to poor color differentiation between stroma and epithelium from the common stain hematoxylin-eosin, no method is yet able to segment all types of glands, making automatic prognostication hard to attain. We address the effect of tissue preparation on glandular segmentation with an alternative stain, Picrosirius red-hematoxylin, which clearly delineates the stromal boundaries, and couple this stain with a color decomposition that removes intensity variation. In this paper we propose a segmentation algorithm that uses image analysis techniques based on mathematical morphology and that can successfully determine the glandular boundaries. Accurate determination of the stromal and glandular morphology enables the identification of the architectural pattern that determine the malignancy grade and classify each gland into its appropriate Gleason grade or ISUP Grade Group. Segmentation of prostate tissue with the new stain and decomposition method has been successfully tested on more than 11000 objects including well-formed glands (Gleason grade 3), cribriform and fine caliber glands (grade 4), and single cells (grade 5) glands.