Journal Article

Comparison of spatial transcriptomics technologies using tumor cryosections

Okonechnikov, Konstantin, Pajtler, Kristian W., Rippe, Karsten, Keitel, Denise, Wille, Sina Jasmin, Schumacher, Sabrina, Mallm, Jan-Philipp, Rademacher, Anne, Ghasemi, David R., Bortolomeazzi, Michele, Huseynov, Alik, Sant, Pooja

2025

Overview

Background Spatial transcriptomics technologies are revolutionizing our understanding of intra-tumor heterogeneity and the tumor microenvironment by revealing single-cell molecular profiles within their spatial tissue context. The rapid development of spatial transcriptomics methods, each with unique characteristics, makes it challenging to select the most suitable technology for specific research objectives. Here, we compare four imaging-based approaches—RNAscope HiPlex, Molecular Cartography, Merscope, and Xenium—alongside Visium, a sequencing-based method. These technologies were employed to study cryosections of medulloblastoma with extensive nodularity (MBEN), a tumor chosen for its distinct microanatomical features. Results Our analysis reveals that automated imaging-based spatial transcriptomics methods are well-suited to delineate the intricate MBEN microanatomy and capture cell-type-specific transcriptome profiles. We devise approaches to compare the sensitivity and specificity of different methods, along with their unique attributes, to guide method selection based on the research objective. Furthermore, we demonstrate how reimaging slides after the spatial transcriptomics analysis can significantly improve cell segmentation accuracy and integrate additional transcript and protein readouts, expanding the analytical possibilities and depth of insight. Conclusions This study underscores important distinctions between spatial transcriptomics technologies and offers a framework for evaluating their performance. Our findings support informed decisions regarding methods and outline strategies to improve the resolution and scope of spatial transcriptomic analyses, ultimately advancing spatial transcriptomics applications in solid tumor research.

• Publisher: BioMed Central,Springer Nature B.V,BMC
• Subject: Advances in Spatial Transcriptomics for Understanding Development and Disease / Animal Genetics and Genomics / Automation / Bioinformatics / Biomedical and Life Sciences / cartography / Design of experiments / Evolutionary Biology / Gene Expression Profiling - methods / Genes / genome / Genomics / Human Genetics / Humans / Hybridization / Life Sciences / Medulloblastoma / Medulloblastoma - genetics / Medulloblastoma - pathology / MERFISH / Merscope / Microbial Genetics and Genomics / Microscopy / Molecular Cartography / neoplasms / Plant Genetics and Genomics / RNAscope / Signal to noise ratio / Solid tumors / Spatial transcriptomics / Transcriptome / Transcriptomes / Transcriptomics / Tumor Microenvironment / Tumors / Visium