{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,4]],"date-time":"2026-05-04T17:59:35Z","timestamp":1777917575979,"version":"3.51.4"},"reference-count":40,"publisher":"Wiley","issue":"6","license":[{"start":{"date-parts":[[2026,2,1]],"date-time":"2026-02-01T00:00:00Z","timestamp":1769904000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,2,1]],"date-time":"2026-02-01T00:00:00Z","timestamp":1769904000000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Microscopy Res &amp; Technique"],"published-print":{"date-parts":[[2026,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>In spatial proteomics, multiplexed immunofluorescence (mIF) enables high\u2010plex visualization of protein expression in preserved tissue, offering insights into tumor heterogeneity and the microenvironment. While MACSima and PhenoCycler\u2010Fusion employ distinct strategies, direct comparisons under biologically controlled in\u00a0vivo conditions remain limited. We applied both platforms to sagittal formalin\u2010fixed, paraffin\u2010embedded (FFPE) sections from an orthotopic xenograft mouse model of human medulloblastoma (MB), featuring leptomeningeal seeding (LMS). These longitudinal sections spanning brain and spinal cord allowed simultaneous assessment of areas with distinct cellular architecture. Fifteen\u2010marker mIF was performed. MACSima utilized MICS technology with MACS iQ View for automated workflows; PhenoCycler\u2010Fusion used a DNA\u2010barcoded antibody system and QuPath for open\u2010ended image processing. Segmentation was evaluated using identical MACSima data. Both platforms enabled high\u2010plex imaging mIF while preserving tissue morphology. DAPI, Ki\u201067, and Actin were consistently detected across both systems. Ki\u201067 expression localized to densely packed tumor regions and was also observed in lower\u2010density areas. Analyzing MACSima data, MACS iQ View detected fewer cells but a higher Ki\u201067 positive rate in dense regions; conversely, QuPath detected more cells but with a lower positivity rate. In low\u2010density areas, both tools yielded similar results. These differences reflect distinct segmentation algorithms and thresholding strategies. This study confirms both platforms support mIF\u2010based spatial proteomic analysis in complex, heterogeneous tissues. However, analysis tools influence quantification. Therefore, standardization of algorithmic settings and additional validation are crucial for precise data interpretation. This research provides practical insights for platform selection in basic, translational, and clinical applications by directly evaluating staining, image acquisition, and analysis pipelines.<\/jats:p>","DOI":"10.1002\/jemt.70125","type":"journal-article","created":{"date-parts":[[2026,2,2]],"date-time":"2026-02-02T07:31:34Z","timestamp":1770017494000},"page":"959-971","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["An Evaluation of\n                    <scp>MACSima<\/scp>\n                    and PhenoCycler\u2010Fusion for Spatial Image Analysis in a Medulloblastoma Xenograft Model"],"prefix":"10.1002","volume":"89","author":[{"given":"Sokhoeun","family":"Heng","sequence":"first","affiliation":[{"name":"Division of Pediatric Neurosurgery Seoul National University Children's Hospital  Seoul Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8390-6670","authenticated-orcid":false,"given":"Taebok","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Research and Experiment, Biomedical Research Institute Seoul National University Hospital  Seoul Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Seung Ah","family":"Choi","sequence":"additional","affiliation":[{"name":"Division of Pediatric Neurosurgery Seoul National University Children's Hospital  Seoul Korea"},{"name":"Neuroscience Research Institute, Medical Research Center, Seoul National University College of Medicine  Seoul Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haneul","family":"Lee","sequence":"additional","affiliation":[{"name":"Division of Pediatric Neurosurgery Seoul National University Children's Hospital  Seoul Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Seung\u2010Ki","family":"Kim","sequence":"additional","affiliation":[{"name":"Division of Pediatric Neurosurgery Seoul National University Children's Hospital  Seoul Korea"},{"name":"Neuroscience Research Institute, Medical Research Center, Seoul National University College of Medicine  Seoul Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9603-5843","authenticated-orcid":false,"given":"Ji Hoon","family":"Phi","sequence":"additional","affiliation":[{"name":"Division of Pediatric Neurosurgery Seoul National University Children's Hospital  Seoul Korea"},{"name":"Neuroscience Research Institute, Medical Research Center, Seoul National University College of Medicine  Seoul Korea"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2026,2,1]]},"reference":[{"key":"e_1_2_11_2_1","unstructured":"Akoya Biosciences. n.d.\u201cPhenoCycler\u2010Fusion System.\u201dhttps:\/\/www.akoyabio.com\/phenocycler\/instrument\/phenocycler\u2010fusion\/."},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010017\u201017204\u20105"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.2174\/1874226200902010079"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41596\u2010021\u201000556\u20108"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41420\u2010023\u201001646\u20100"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.3390\/proteomes11020017"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12885\u2010016\u20102742\u2010y"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1768"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41592\u2010024\u201002533\u2010x"},{"key":"e_1_2_11_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2018.07.010"},{"issue":"10","key":"e_1_2_11_12_1","first-page":"1972","article-title":"Effects of Different Decalcification Protocols on Osteopontin and Osteocalcin Immunostaining in Rat Bone","volume":"6","author":"Gonz\u00e1lez\u2010Ch\u00e1vez S. 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