{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T13:01:35Z","timestamp":1784552495188,"version":"3.55.0"},"reference-count":31,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T00:00:00Z","timestamp":1777593600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"},{"start":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T00:00:00Z","timestamp":1777593600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100004704","name":"National Research Council of Thailand","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004704","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004156","name":"Mahidol University","doi-asserted-by":"publisher","award":["N42A680269"],"award-info":[{"award-number":["N42A680269"]}],"id":[{"id":"10.13039\/501100004156","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Biology of the Cell"],"published-print":{"date-parts":[[2026,5]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Therapeutic resistance is a major cause of treatment failure in glioblastoma (GBM), highlighting the need for physiologically relevant models to identify actionable resistance mechanisms. While two\u2010dimensional (2D) cultures are widely used for target discovery, they poorly represent the tumor microenvironment. In contrast, three\u2010dimensional (3D) spheroid cultures better recapitulate spatial heterogeneity, hypoxic gradients, and stress\u2010adaptive signaling observed in tumors.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We applied an integrated 2D\u20133D quantitative proteomic approach to identify microenvironment\u2010dependent regulators of chemoresistance in GBM. Proteomic profiling was performed in U87MG and U251MG cells grown as 2D monolayers or 3D spheroids. Differentially expressed proteins were validated by quantitative RT\u2010PCR, and functional studies were conducted using genetic depletion followed by assessment of temozolomide (TMZ) sensitivity.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Comparative analysis identified 13 proteins consistently differentially expressed between 2D and 3D cultures: NDUFB5, RNGTT, MLK4, SYN1, DDX5, EIF2AK2, ITGA1, ZNF33B, ZNF343, WDR19, JPH3, CCT8L2, and FNDC3A. Among these, Mixed Lineage Kinase 4 (MLK4) showed strong and reproducible upregulation in 3D spheroids in both GBM cell lines. Genetic depletion of MLK4 significantly increased TMZ sensitivity without affecting basal cell viability, suggesting a specific role in therapy response. Notably, MLK4 expression was induced only under 3D conditions.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>MLK4 functions as a microenvironment\u2010responsive regulator of chemoresistance in GBM. These findings demonstrate that 3D culture systems reveal clinically relevant resistance pathways not detectable in conventional 2D models and highlight 3D proteomic profiling as a powerful strategy for identifying therapeutically actionable targets.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1111\/boc.70068","type":"journal-article","created":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T05:20:58Z","timestamp":1777699258000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Integrated 2D\u20133D Proteomic Profiling Identifies MLK4 as a Microenvironment\u2010Responsive Regulator of Chemotherapeutic Resistance in Human Glioblastoma Cells"],"prefix":"10.1111","volume":"118","author":[{"given":"Wannawat","family":"Khotchawan","sequence":"first","affiliation":[{"name":"Siriraj Center of Excellence for Stem Cell Research (SiSCR), Faculty of Medicine, Siriraj Hospital Mahidol University  Bangkok 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