Comparison of Imaging-Based Single-Cell Resolution Spatial Transcriptomics Profiling Platforms Using Formalin-Fixed, Paraffin-Embedded Tumor Samples

Imaging-based spatial transcriptomics (ST) is evolving rapidly as a pivotal technology in studying the biology of tumors and their associated microenvironments. However, the strengths of the commercially available ST platforms in studying spatial biology have not been systematically evaluated using rigorously controlled experiments. In this study, we used serial 5-μm sections of formalin-fixed, paraffin-embedded surgically resected lung adenocarcinoma and pleural mesothelioma tumor samples in tissue microarrays to compare the performance of the single cell ST platforms CosMx, MERFISH, and Xenium (uni/multi-modal) platforms in reference to bulk RNA sequencing, multiplex immunofluorescence, GeoMx Digital Spatial Profiler, and hematoxylin and eosin staining data for the same samples. In addition to objective assessment of automatic cell segmentation and phenotyping, we performed pixel-resolution manual evaluation of phenotyping to carry out pathologically meaningful comparison between ST platforms. Our study detailed the intricate differences between the ST platforms, revealed the importance of parameters such as tissue age and probe design in determining the data quality, and suggested reliable workflows for accurate spatial profiling and molecular discovery. ### Competing Interest Statement Acknowledgements This study was supported by Lung SPORE grant P50 CA070907 from the NIH, the Aileen M. Dillon & Lee M. Bourg Mesothelioma Fund, the Re & RM Kennedy Lung Cancer Fund, the Fleming Endowed Fund, the MD Anderson Rare Tumor Initiative, the MD Anderson Translational Molecular Pathology-Immunoprofiling Lab (TMP-IL) Moon Shots Platform, NCI grant U24CA224285 (to the MD Anderson Cancer Center CIMAC) and the NIH/NCI under award number P30CA016672 and used the Research Histology Core Lab and the Advanced Technology Genomics Core. We thank the MD Anderson Department of Thoracic and Cardiovascular Surgery nurses and staff for their support of this study. The authors acknowledge Donald Norwood, members of the Editing Services, Research Medical Library for editing this manuscript. Fig. 1 was created in BioRender. Ozirmak lermi, N. (2025) https://BioRender.com/ y68w242. Competing interests C.H. declares research funding to institution from Sanofi, BTG, Iovance, Obsidian, KSQ, EMD Serono, Takeda, Genentech, BMS, Summit Therapeutics, Artidis, Immunogenesis and Novartis; scientific advisory board member of Briacell with stock options; personal fees from Regeneron outside the scope of the submitted work. LS declares travel support for participation in 10x Genomic Pathology Day event and participation in NanoString Roadshow event, both unrelated to this work. M.A. declares research funding to institution from Genentech, Nektar Therapeutics, Merck, GlaxoSmithKline, Novartis, Jounce Therapeutics, Bristol Myers Squibb, Eli Lilly, Adaptimmune, Shattuck Lab, Gilead, Verismo therapeutics, Lyell; scientific advisory board member of GlaxoSmithKline, Shattuck Lab, Bristol Myers Squibb, AstraZeneca, Insightec, Regeneron, Genprex; personal fees from AstraZeneca, Nektar Therapeutics, SITC; participation of safety review committee for Nanobiotix-MDA Alliance, Henlius outside the scope of the submitted work. J.Z. declares research funding from Johnson and Johnson, Helius, Merck, Novartis and Summit, honoraria and consulting fees from AstraZeneca, BeiGene, Catalyst, GenePlus, Helius, Innovent, Johnson and Johnson, Novartis, Takeda and Varian outside the submitted work. D.L.G. has served on scientific advisory committees for Sanofi, Menarini Ricerche, Onconova, and Eli Lilly, and has received research support from Takeda, NGM Biopharmaceuticals, Boehringer Ingelheim and AstraZeneca. All other authors declare no conflicts of interest related to the study.