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The five-year overall survival rates remain low, around 50% in advanced stages. Cell line models are essential tools to understand the complexity of TSCC and develop new therapies. However, most commercially available TSCC cell lines are derived from patients with a history of tobacco use or have unknown exposure backgrounds. We established two novel TSCC cell lines, LMSCC03 and LMSCC16, derived from non-smoking and treatment na\u00efve Brazilian patients. These cell lines were characterized by doubling time, 3D culture (spheroids and organoids), tumorigenicity through xenotransplantation in nude mice, immunohistochemical expression of key OSCC biomarkers and drug response. Genetic identity was confirmed by STR profiling. LMSCC03 and LMSCC16 displayed epithelial morphology and pan-cytokeratin staining. They demonstrated in vitro capacity to form spheroids and organoids, and generated tumors in vivo. STR profiling confirmed their novelty relative to existing cell lines in the DSMZ database. Protein analysis revealed high p53 nuclear levels in LMSCC16 cells. Interestingly, CD44 and c-Myc expression were observed only in fibroblast-enriched cultures, but not in the epithelial LMSCC03 and LMSCC16 cells. LMSCC16 also harbored two\n                    <jats:italic>TP53<\/jats:italic>\n                    mutations and showed increased resistance to Cisplatin and Paclitaxel compared to established TSCC cell lines. Cisplatin treatment in spheroids reduced\n                    <jats:italic>OCT4<\/jats:italic>\n                    ,\n                    <jats:italic>CCND1<\/jats:italic>\n                    ,\n                    <jats:italic>CCNB1<\/jats:italic>\n                    , and\n                    <jats:italic>CDH1<\/jats:italic>\n                    gene expression in LMSCC03. In contrast, LMSCC16 showed significant modulation of\n                    <jats:italic>OCT4<\/jats:italic>\n                    , increased\n                    <jats:italic>CCND1<\/jats:italic>\n                    and\n                    <jats:italic>CCNB1<\/jats:italic>\n                    expression, and reduced\n                    <jats:italic>CDH1<\/jats:italic>\n                    levels following treatment. We established two novel TSCC cell lines that represent clinically relevant models to explore TSCC carcinogenesis and therapeutic responses, particularly in non-smoking populations.\n                  <\/jats:p>","DOI":"10.1007\/s12032-026-03311-9","type":"journal-article","created":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T07:20:50Z","timestamp":1782717650000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Novel patient-derived tongue squamous cell carcinoma cell lines from non-smokers: 3D and in vivo models for drug response studies"],"prefix":"10.1007","volume":"43","author":[{"given":"Graziella Ribeiro","family":"de Sousa","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guilherme","family":"da Silva Carvalho","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bruna Miyoko Ikenaga","family":"de Brito","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriel","family":"da Silva","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria Clara Rezende Barbosa","family":"Lopes","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pablo Shimaoka","family":"Chagas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elvis Terci","family":"Valera","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Graziela Vieira","family":"Cavalcanti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luiz Carlos Conti","family":"de Freitas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8313-4754","authenticated-orcid":false,"given":"Andr\u00e9ia Machado","family":"Leopoldino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,6,29]]},"reference":[{"key":"3311_CR1","doi-asserted-by":"publisher","first-page":"229","DOI":"10.3322\/caac.21834","volume":"74","author":"F Bray","year":"2024","unstructured":"Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. 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