Comparison of imaging-based single-cell resolution spatial transcriptomics profiling platforms using formalin-fixed, paraffin-embedded tumor samples

Comparison of imaging-based single-cell resolution spatial transcriptomics profiling platforms using formalin-fixed, paraffin-embedded tumor samples | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparison of imaging-based single-cell resolution spatial transcriptomics profiling platforms using formalin-fixed, paraffin-embedded tumor samples Luisa Solis Soto, Nejla Ozirmak Lermi, Max Molina Ayala, Sharia Hernandez Ruiz, and 26 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5656204/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract 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. Biological sciences/Computational biology and bioinformatics/Data processing Biological sciences/Cancer/Cancer microenvironment Biological sciences/Biotechnology/Genomics/Transcriptomics Full Text Additional Declarations Yes there is potential Competing Interest. 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. T.C. has received over the past 24 months speaker fees/honoraria (including travel/meeting expenses) from ASCO Post, AstraZeneca, Bio Ascend, Bristol Myers Squibb, Clinical Care Options, IDEOlogy Health, Medical Educator Consortium, Medscape, OncLive, PEAK Medicals, PeerView, Physicians' Education Resource, Targeted Oncology; advisory role/consulting fees (including travel/meeting expenses) from AstraZeneca, Bristol Myers Squibb, Genentech, Merck, oNKo-innate, Pfizer, and RAPT Therapeutics; institutional research funding from AstraZeneca and Bristol Myers Squibb. All other authors declare no conflicts of interest related to the study. Supplementary Files SupplementaryTable1.xlsx Supplementary Table 1 SupplementaryTable2.xlsx Supplementary Table 2 SupplementaryTable3.xlsx Supplementary Table 3 SupplementaryFig1.pdf Supplementary Figure 1 SupplementaryFig2.pdf Supplementary Figure 2 Supplementary.Fig3.pdf Supplementary Figure 3 Cite Share Download PDF Status: Published Journal Publication published 26 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5656204","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":393701576,"identity":"7ad6010d-1986-4fc9-a330-da76ccd32c16","order_by":0,"name":"Luisa Solis 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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. T.C. has received over the past 24 months speaker fees/honoraria (including travel/meeting expenses) from ASCO Post, AstraZeneca, Bio Ascend, Bristol Myers Squibb, Clinical Care Options, IDEOlogy Health, Medical Educator Consortium, Medscape, OncLive, PEAK Medicals, PeerView, Physicians' Education Resource, Targeted Oncology; advisory role/consulting fees (including travel/meeting expenses) from AstraZeneca, Bristol Myers Squibb, Genentech, Merck, oNKo-innate, Pfizer, and RAPT Therapeutics; institutional research funding from AstraZeneca and Bristol Myers Squibb. All other authors declare no conflicts of interest related to the study.","formattedTitle":"Comparison of imaging-based single-cell resolution spatial transcriptomics profiling platforms using formalin-fixed, paraffin-embedded tumor samples","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5656204/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5656204/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImaging-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.\u003c/p\u003e","manuscriptTitle":"Comparison of imaging-based single-cell resolution spatial transcriptomics profiling platforms using formalin-fixed, paraffin-embedded tumor samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-17 04:20:05","doi":"10.21203/rs.3.rs-5656204/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fe5f3d36-0559-48b8-8f7a-a68802c0670f","owner":[],"postedDate":"January 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41962970,"name":"Biological sciences/Computational biology and bioinformatics/Data processing"},{"id":41962971,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":41962972,"name":"Biological sciences/Biotechnology/Genomics/Transcriptomics"}],"tags":[],"updatedAt":"2025-09-27T07:08:39+00:00","versionOfRecord":{"articleIdentity":"rs-5656204","link":"https://doi.org/10.1038/s41467-025-63414-1","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-09-26 04:00:00","publishedOnDateReadable":"September 26th, 2025"},"versionCreatedAt":"2025-01-17 04:20:05","video":"","vorDoi":"10.1038/s41467-025-63414-1","vorDoiUrl":"https://doi.org/10.1038/s41467-025-63414-1","workflowStages":[]},"version":"v1","identity":"rs-5656204","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5656204","identity":"rs-5656204","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}