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Biomed. Eng"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Multiplex immunostaining analysis remains fragmented, underperforming and labour intensive despite tissue proteomic methodologies achieving ever-increasing marker complexity. Here we propose an open-source, user-guided automated pipeline that streamlines start-to-finish, single-cell resolution analysis of whole-slide tissue, named multiplex-imaging analysis, registration, quantification and overlaying (MARQO). MARQO integrates elastic image registration, iterative nuclear segmentation, unsupervised clustering with mini-batch\n                    <jats:italic>k<\/jats:italic>\n                    -means and user-guided cell classification through a graphical interface. We compare and validate MARQO using multiplexed immunohistochemical consecutive staining on a single slide using human tumour and adjacent normal tissue samples. Performance is compared with manually curated pathologist determinations and quantification of multiple markers. We optimize MARQO to analyse diverse tissue sizes from whole tissue, biopsy, and tissue microarray and staining approaches, such as singleplex immunohistochemistry and 20-colour multiplex immunofluorescence, to determine marker co-expression patterns in multiple human solid cancer types. Lastly, we validate CD8\n                    <jats:sup>+<\/jats:sup>\n                    T cell enrichment in hepatocellular carcinoma responders to neoadjuvant cemiplimab in a phase 2 clinical trial, further showing the ability of MARQO to identify spatially resolved in situ mechanisms by providing multiplex whole-slide single-cell resolution data.\n                  <\/jats:p>","DOI":"10.1038\/s41551-025-01475-9","type":"journal-article","created":{"date-parts":[[2025,8,25]],"date-time":"2025-08-25T09:03:11Z","timestamp":1756112591000},"page":"517-531","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Multiparametric cellular and spatial organization in cancer tissue lesions with a streamlined pipeline"],"prefix":"10.1038","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6938-3474","authenticated-orcid":false,"given":"Mark","family":"Buckup","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Igor","family":"Figueiredo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7956-7605","authenticated-orcid":false,"given":"Giorgio","family":"Ioannou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sinem","family":"Ozbey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rafael","family":"Cabal","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra","family":"Tabachnikova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2529-9267","authenticated-orcid":false,"given":"Leanna","family":"Troncoso","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica","family":"Le Berichel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhen","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen C.","family":"Ward","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clotilde","family":"Hennequin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7032-8085","authenticated-orcid":false,"given":"Guray","family":"Akturk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steve","family":"Hamel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Isabel","family":"Fiel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachel","family":"Brody","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9113-886X","authenticated-orcid":false,"given":"Myron","family":"Schwartz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5903-8191","authenticated-orcid":false,"given":"Thomas U.","family":"Marron","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0192-4400","authenticated-orcid":false,"given":"Seunghee","family":"Kim-Schulze","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vladimir","family":"Roudko","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edgar","family":"Gonzalez-Kozlova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6776-1635","authenticated-orcid":false,"given":"Pauline","family":"Hamon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4481-7827","authenticated-orcid":false,"given":"Miriam","family":"Merad","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5643-9520","authenticated-orcid":false,"given":"Sacha","family":"Gnjatic","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,8,25]]},"reference":[{"key":"1475_CR1","doi-asserted-by":"publisher","first-page":"3802","DOI":"10.1038\/s41596-021-00556-8","volume":"16","author":"S Black","year":"2021","unstructured":"Black, S. et al. 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M.M. serves on the scientific advisory board of Innate Pharma, DBV and Genenta. M.M. receives funding for contracted research from Regeneron and Boehringer Ingelheim. T.U.M. has served on advisory and\/or data safety monitoring boards for Rockefeller University, Regeneron Pharmaceuticals, Abbvie, Bristol Meyers Squibb, Boehringer Ingelheim, Atara, AstraZeneca, Genentech, Celldex, Chimeric, Glenmark, Simcere, Surface, G1 Therapeutics, NGMbio, DBV Technologies, Arcus and Astellas, and has research grants from Regeneron, Bristol Myers Squibb, Merck and Boehringer Ingelheim. S.G. reports past consultancy or advisory roles for Merck and OncoMed; research funding from Regeneron Pharmaceuticals related to this study, and research funding from Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Genentech, EMD Serono, Pfizer and Takeda, unrelated to the current work. S.G. is a named co-inventor on an issued patent (US20190120845A1) for multiplex immunohistochemistry to characterize tumours and treatment responses. The technology is filed through Icahn School of Medicine at Mount Sinai and is currently unlicensed. This technology was used to evaluate tissue in this study and the results could impact the value of this technology.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}]}}