start-ver=1.4 cd-journal=joma no-vol=12 cd-vols= no-issue=10 article-no= start-page=2373 end-page= dt-received= dt-revised= dt-accepted= dt-pub-year=2024 dt-pub=20241017 dt-online= en-article= kn-article= en-subject= kn-subject= en-title= kn-title=Development and Characterization of a Three-Dimensional Organotypic In Vitro Oral Cancer Model with Four Co-Cultured Cell Types, Including Patient-Derived Cancer-Associated Fibroblasts en-subtitle= kn-subtitle= en-abstract= kn-abstract=Background/Objectives: Cancer organoids have emerged as a valuable tool of three-dimensional (3D) cell cultures to investigate tumor heterogeneity and predict tumor behavior and treatment response. We developed a 3D organotypic culture model of oral squamous cell carcinoma (OSCC) to recapitulate the tumor–stromal interface by co-culturing four cell types, including patient-derived cancer-associated fibroblasts (PD-CAFs). Methods: A stainless-steel ring was used twice to create the horizontal positioning of the cancer stroma (adjoining normal oral mucosa connective tissue) and the OSCC layer (surrounding normal oral mucosa epithelial layer). Combined with a structured bi-layered model of the epithelial component and the underlying stroma, this protocol enabled us to construct four distinct portions mimicking the oral cancer tissue arising in the oral mucosa. Results: In this model, α-smooth muscle actin-positive PD-CAFs were localized in close proximity to the OSCC layer, suggesting a crosstalk between them. Furthermore, a linear laminin-γ2 expression was lacking at the interface between the OSCC layer and the underlying stromal layer, indicating the loss of the basement membrane-like structure. Conclusions: Since the specific 3D architecture and polarity mimicking oral cancer in vivo provides a more accurate milieu of the tumor microenvironment (TME), it could be crucial in elucidating oral cancer TME. en-copyright= kn-copyright= en-aut-name=AizawaYuka en-aut-sei=Aizawa en-aut-mei=Yuka kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=1 ORCID= en-aut-name=HagaKenta en-aut-sei=Haga en-aut-mei=Kenta kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=2 ORCID= en-aut-name=YoshibaNagako en-aut-sei=Yoshiba en-aut-mei=Nagako kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=3 ORCID= en-aut-name=YortchanWitsanu en-aut-sei=Yortchan en-aut-mei=Witsanu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=4 ORCID= en-aut-name=TakadaSho en-aut-sei=Takada en-aut-mei=Sho kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=5 ORCID= en-aut-name=TanakaRintaro en-aut-sei=Tanaka en-aut-mei=Rintaro kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=6 ORCID= en-aut-name=NaitoEriko en-aut-sei=Naito en-aut-mei=Eriko kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=7 ORCID= en-aut-name=AbéTatsuya en-aut-sei=Abé en-aut-mei=Tatsuya kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=8 ORCID= en-aut-name=MaruyamaSatoshi en-aut-sei=Maruyama en-aut-mei=Satoshi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=9 ORCID= en-aut-name=YamazakiManabu en-aut-sei=Yamazaki en-aut-mei=Manabu kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=10 ORCID= en-aut-name=TanumaJun-ichi en-aut-sei=Tanuma en-aut-mei=Jun-ichi kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=11 ORCID= en-aut-name=IgawaKazuyo en-aut-sei=Igawa en-aut-mei=Kazuyo kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=12 ORCID= en-aut-name=TomiharaKei en-aut-sei=Tomihara en-aut-mei=Kei kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=13 ORCID= en-aut-name=TogoShinsaku en-aut-sei=Togo en-aut-mei=Shinsaku kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=14 ORCID= en-aut-name=IzumiKenji en-aut-sei=Izumi en-aut-mei=Kenji kn-aut-name= kn-aut-sei= kn-aut-mei= aut-affil-num=15 ORCID= affil-num=1 en-affil=Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=2 en-affil=Division of Reconstructive Surgery for Oral and Maxillofacial Region, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=3 en-affil=Department of Oral Health and Welfare, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=4 en-affil=Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=5 en-affil=Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=6 en-affil=Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=7 en-affil=Division of Oral and Maxillofacial Surgery, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=8 en-affil=Division of Oral Pathology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=9 en-affil=Division of Oral Pathology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=10 en-affil=Division of Oral Pathology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=11 en-affil=Division of Oral Pathology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=12 en-affil=Neutron Therapy Research Center, Okayama University kn-affil= affil-num=13 en-affil=Division of Oral and Maxillofacial Surgery, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= affil-num=14 en-affil=Department of Respiratory Medicine, Graduate School of Medicine, Juntendo University kn-affil= affil-num=15 en-affil=Division of Biomimetics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University kn-affil= en-keyword=oral cancer kn-keyword=oral cancer en-keyword=cancer-associated fibroblasts kn-keyword=cancer-associated fibroblasts en-keyword=oral mucosa kn-keyword=oral mucosa en-keyword=patient-derived kn-keyword=patient-derived en-keyword=organotypic culture kn-keyword=organotypic culture en-keyword=3D in vitro model kn-keyword=3D in vitro model en-keyword=polarity kn-keyword=polarity END