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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. [ABSTRACT FROM AUTHOR]

Development of effective in vitro human lip models, specific to the vermilion epithelium, has not progressed as much as that of skin and oral mucosa/gingiva models in vitro. Our histologic examination demonstrated that a Japanese macaque (male, 7 years and 9 months old) had vermilion in the lip distinct from adjacent skin and oral mucosa, resembling histological characteristics of the human lip. Therefore, in this study, we examined the gene expression profile of the three distinct epithelia (skin/vermilion/oral mucosa) within the lip of a Japanese macaque to explore a single potential marker of human vermilion epithelium. Six pairwise comparisons in the skin/vermilion/oral mucosa epithelium in vitro and in vivo revealed 69 differentially up-regulated genes in vermilion epithelium in vivo, in which a few unique genes were highly expressed when compared with both skin and oral mucosa epithelium in vivo using clustering analysis. However, we could not detect a single marker specific to vermilion epithelium supported by the gene expression profile of a Japanese macaque. Instead, the pair of keratin 10 and small proline-rich protein 3 resulted in a potential marker of vermilion epithelium in the human lip (female, 53-year-old) via a double-immunostaining technique. Nonetheless, our result may provide further clues leading to other potential markers of the vermilion epithelium. [ABSTRACT FROM AUTHOR]