{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T19:02:29Z","timestamp":1790708549855,"version":"4.1.0"},"reference-count":50,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2025,4,3]],"date-time":"2025-04-03T00:00:00Z","timestamp":1743638400000},"content-version":"vor","delay-in-days":2,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2025,4,1]],"date-time":"2025-04-01T00:00:00Z","timestamp":1743465600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2025,4]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Fresh tumor cytometric profiling is essential for interrogating the tumor microenvironment (TME) and identifying potential therapeutic targets to enhance antitumor immunity. Challenges arise due to the limited number of cells in clinical biopsies and inter\u2010patient variability. To maximize data derived from a single biopsy, spectral cytometry was leveraged, enabling extensive profiling with significantly fewer cells than mass cytometry. Furthermore, the utilization of multiple markers within one tube can potentially reveal novel and extensive dynamic immune characteristics in cancer, thereby aiding treatment strategies and enhancing patient outcomes. Here, we introduce a customized 39\u2010color panel for in\u2010depth phenotyping of exhausted T cells (T\n                    <jats:sub>EX<\/jats:sub>\n                    ), which are dysfunctional T\u2010cell subsets that arise during cancer progression. This study aims to investigate profiles of CD4 T, CD8 T, regulatory T (Treg), and \u03b3\u03b42 cells while exploring the heterogeneity of CD8\n                    <jats:sup>+<\/jats:sup>\n                    T\n                    <jats:sub>EX<\/jats:sub>\n                    subsets. Given the rarity and heterogeneity of tumor biopsies, we evaluated the effects of tissue dissociation enzymes on staining protocols using cryopreserved peripheral blood mononuclear cells (PBMCs). This is vital for the development of high\u2010dimensional cytometry panels, especially since collagenases may cleave markers in dissociated tumor cells (DTCs). Our protocol also optimizes intracellular marker staining, enhancing insights into T\n                    <jats:sub>EX<\/jats:sub>\n                    function and biology, ultimately identifying potential therapeutic targets.\n                  <\/jats:p>","DOI":"10.1002\/cyto.a.24929","type":"journal-article","created":{"date-parts":[[2025,4,4]],"date-time":"2025-04-04T21:41:16Z","timestamp":1743802876000},"page":"271-280","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Diving Deep: Profiling Exhausted T Cells in the Tumor Microenvironment Using Spectral Flow Cytometry"],"prefix":"10.1002","volume":"107","author":[{"given":"Karen Wei Weng","family":"Teng","sequence":"first","affiliation":[{"name":"Quantitative Biosciences MSD  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weng Hua","family":"Khoo","sequence":"additional","affiliation":[{"name":"Quantitative Biosciences MSD  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas Ching Wei","family":"Ho","sequence":"additional","affiliation":[{"name":"Quantitative Biosciences MSD  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S. Jasemine","family":"Yang","sequence":"additional","affiliation":[{"name":"Quantitative Biosciences Merck &amp; Co. Inc.  South San Francisco California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Douglas C.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Quantitative Biosciences Merck &amp; Co. Inc.  South San Francisco California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edmond","family":"Chua","sequence":"additional","affiliation":[{"name":"Research and Development Cytek Biosciences  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shu Wen Samantha","family":"Ho","sequence":"additional","affiliation":[{"name":"Quantitative Biosciences MSD  Singapore Singapore"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,4,3]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.03.030"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2013.07.012"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrc3239"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2018.10.038"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.24213"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22938"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.22983"},{"key":"e_1_2_9_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2020.04.014"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2019.09.013"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ccell.2021.04.004"},{"key":"e_1_2_9_12_1","first-page":"279","article-title":"Bystander CD8+ T Cells Are Abundant and Phenotypically Distinct in Human Tumour Infiltrates","volume":"12","author":"Simoni Y.","year":"2021","journal-title":"Nature"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010021\u201023324\u20104"},{"key":"e_1_2_9_14_1","first-page":"139","article-title":"Coregulation of CD8+ T Cell Exhaustion During Chronic Viral Infection by Multiple Inhibitory Receptors","volume":"176","author":"Mackie K.","year":"2017","journal-title":"Physiology & 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(\u03b3\u03b4) T Cells: Friend or Foe in Cancer Development","volume":"16","author":"Zhao Y.","year":"2018","journal-title":"Journal of Translational Medicine"},{"key":"e_1_2_9_47_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20180684"},{"key":"e_1_2_9_48_1","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.23331"},{"key":"e_1_2_9_49_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2023.1107483"},{"key":"e_1_2_9_50_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0154253"},{"key":"e_1_2_9_51_1","first-page":"86","article-title":"CD11c Is Upregulated in CD8+ T Cells of Patients With Beh\u00e7et's Disease","volume":"34","author":"Park Y. J.","year":"2016","journal-title":"Clinical and Experimental Rheumatology"}],"container-title":["Cytometry Part A"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cyto.a.24929","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cyto.a.24929","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cyto.a.24929","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T18:15:02Z","timestamp":1790705702000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/cyto.a.24929"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4]]},"references-count":50,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2025,4]]}},"alternative-id":["10.1002\/cyto.a.24929"],"URL":"https:\/\/doi.org\/10.1002\/cyto.a.24929","archive":["Portico"],"relation":{},"ISSN":["1552-4922","1552-4930"],"issn-type":[{"value":"1552-4922","type":"print"},{"value":"1552-4930","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4]]},"assertion":[{"value":"2024-07-15","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-03-12","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-04-03","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}