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Three\u2010dimensional (3D) tissue\u2010engineered models can better reproduce the tumor microenvironment than monolayer cultures; however, their routine use is often constrained by low\u2010throughput workflows and reliance on destructive analytical endpoints. Here, we present\u00a0FOS3D, a fluorescence\u2010enabled OS model based on gelatin methacryloyl hydrogels incorporating stable green fluorescent protein (GFP)\u2010expressing tumor cells. By varying polymer concentration and photo\u2010crosslinking duration, hydrogels spanning compressive moduli of approximately 5\u201350\u00a0kPa were generated, covering stiffness ranges reported for OS stromal environments. Whole\u2010well fluorescence scanning enabled rapid, non\u2010destructive quantification of proliferation in intact constructs and showed correlation with DNA and metabolic assays. Integration with light\u2010sheet microscopy further enabled volumetric imaging of tumor organization, revealing time\u2010dependent changes in spatial distribution. Transcriptional profiling and immunohistochemistry confirmed microenvironment\u2010driven adaptations associated with extracellular matrix remodeling, stemness, and drug\u2010resistance pathways. Finally, GFP\u2010based longitudinal monitoring enabled chemotherapeutic screening of cisplatin and doxorubicin across two OS cell lines, capturing dose\u2010dependent responses and the increased treatment tolerance characteristic of 3D cultures. Collectively, FOS3D provides a scalable fluorescence\u2010enabled toolkit for high\u2010content characterization and drug\u2010response profiling of OS in physiologically relevant 3D environments.<\/jats:p>","DOI":"10.1002\/advs.76031","type":"journal-article","created":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T08:27:01Z","timestamp":1781166421000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["FOS3D: A Fluorescence\u2010Enabled Toolkit for Characterizing a Three\u2010dimensional Osteosarcoma Model"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-2057-4789","authenticated-orcid":false,"given":"William","family":"Humble","sequence":"first","affiliation":[{"name":"Aikenhead Centre for Medical Discovery (ACMD) St Vincent's Hospital Melbourne  Fitzroy Victoria Australia"},{"name":"Department of Surgery The University of Melbourne St Vincent's Hospital Melbourne  Fitzroy Victoria Australia"},{"name":"Department of Biomedical Engineering The Graeme Clark Institute The University of Melbourne  Parkville Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wiktor","family":"Zywicki","sequence":"additional","affiliation":[{"name":"Aikenhead Centre for Medical Discovery (ACMD) St Vincent's Hospital Melbourne  Fitzroy Victoria Australia"},{"name":"Department of Biomedical Engineering The Graeme Clark Institute The University of Melbourne  Parkville Victoria Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Enrico","family":"Lucarelli","sequence":"additional","affiliation":[{"name":"Osteoncology, Bone and Soft Tissue Sarcomas and Innovative Therapies Unit IRCCS Istituto Ortopedico Rizzoli  Bologna Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2163-6476","authenticated-orcid":false,"given":"Ania Naila","family":"Guerrieri","sequence":"additional","affiliation":[{"name":"Osteoncology, Bone and Soft Tissue Sarcomas and Innovative Therapies Unit IRCCS Istituto Ortopedico Rizzoli  Bologna Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesca","family":"Taraballi","sequence":"additional","affiliation":[{"name":"Center For Musculoskeletal Regeneration Houston Methodist Research Institute  Houston Texas USA"},{"name":"Orthopaedics and Sports Medicine Houston Methodist Hospital  Houston Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9445-6994","authenticated-orcid":false,"given":"Gianluca","family":"Cidonio","sequence":"additional","affiliation":[{"name":"Department of Mechanical and Aerospace Engineering University of Rome La Sapienza  Rome Italy"},{"name":"Center For Life Nano\u2010 &amp; 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