Abstract
Squamous cell carcinoma (SCC) is the most major malignant tumor of the tongue. The tongue exists at the air–liquid interface and is covered with saliva. In addition, the tongue constituent cells and tongue cancer are present under fluid flow stimulation due to the abundant capillary network and contraction of muscle tissue. Therefore, replicating both cell–cell interactions (the cellular microenvironment) and the aforementioned physical microenvironment is very important for understanding the kinetics of tongue SCC. To elucidate the effects of the cellular and physical microenvironment on tongue SCC and to investigate the relationships between these factors, we developed a collagen cell disc, with double dish under a rotational culture method to generate cancer–stroma interactions and to create fluid flow stimulation. Mesenchymal cells, NIH-3T3 cells and tongue-derived fibroblasts influenced the proliferative potential. Extracellular signal-regulated kinase and p38 signaling were regulated either synergistically or independently by cellular interactions and fluid flow stimulation, depending on the SCC cell type. The cell–cell interactions and fluid flow stimulation independently, synergistically or contradictorily affected the behavior of tongue SCC. Fluid flow stimulation synergistically enhanced the antiproliferative effect of cis-diamminedichloroplatinum on tongue SCC cells, but mesenchymal cells abolished the synergistic antiproliferative effect related to fluid flow stimulation. In conclusion, a reconstructed model was established to investigate the cellular and physical microenvironments of tongue SCC in vitro. The newly established system is a promising model for the development of further regimes to treat general oral cancer.
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Data availability
The data that support the findings of this study are available from the corresponding authors, SA upon reasonable request.
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Acknowledgements
We thank M. Nishida, F. Mutoh, S. Nakahara and S. Nishimura for excellent technical assistance.
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This work was supported by grants from JSPS KAKENHI Grant Number 19K18468 (to MN).
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SI: investigation, writing—original draft. MN: investigation, methodology, writing—reviewing. MK, SM, TS: investigation. YY, ST: supervision. SA: conceptualization, methodology, investigation, writing—reviewing and editing, project administration.
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Supplementary file1 Fig. S1. Phenotypic characteristics of PFs. PFs were positive for Vimentin. αSMA-positive cells were found in 15.20 ± 3.29% of the total population. Data are presented as means ± SD of 3 determinations. PFs exhibited no positivity to CD34, S100 or CK AE1/AE3. PFs, primary fibroblasts. (EPS 1824 KB)
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Supplementary file2 Fig. S2. Effects of fluid flow and human fibroblasts on the cellular kinetics of tongue SCC. A, Representative histopathological images of SAS cells at day 10 determined by hematoxylin-eosin staining. B, Quantitative analysis of SAS cell layer thickness under each condition. On day 10, fluid flow and fibroblasts (TIG-121 cells) synergistically promoted cellular hypertrophy and thickened the SAS cell layer. C, Representative HSC-3 images determined by hematoxylin-eosin staining under each condition. On day 10, monocultured HSC-3 cells under static or fluid stimulation showed no difference in the cell layer thickness. HSC-3 cells co-cultured with TIG-121 cells exhibited an increased cell layer thickness and cell enlargement, and fluid stimulation further increased the cell layer thickness and also cell enlargement. D, Thickness of HSC-3 cell layers. All data represent means ± SD of 3 determinations. * P < 0.05. Mono, monoculture; S, static conditions; F, fluid flow stimulation (EPS 1353 KB)
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Supplementary file3 Fig. S3. Effect of fibroblasts and fluid flow on the JNK signaling pathway in tongue SCC. JNK expression levels in A, SAS or B, HSC-3 cells were evaluated by western blotting. Relative expression is depicted as the ratio of target protein expression to α/β-tubulin expression. All data represent means ± SD of 4–5 determinations. * P < 0.05. Mono, monoculture; PF, primary fibroblasts; S, static conditions; F, fluid flow stimulation. (EPS 1932 KB)
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Supplementary file4 Fig. S4. Uncropped western blot images of Fig. 4. Mono, monoculture; PF, primary fibroblasts; S, static conditions; F, fluid flow stimulation. (EPS 838 KB)
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Supplementary file5 Fig. S5. Uncropped western blot images of Fig. 7. Mono, monoculture; PFs, primary fibroblasts; S, static conditions; F, fluid flow stimulation; CDDP, cis-diamminedichloroplatinum. (EPS 2557 KB)
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Iwamoto, S., Nishiyama, M., Kawasaki, M. et al. Oral-specific microenvironments regulate cell behavior and anticancer drug sensitivity of tongue squamous cell carcinoma. Human Cell 36, 643–656 (2023). https://doi.org/10.1007/s13577-023-00866-x
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DOI: https://doi.org/10.1007/s13577-023-00866-x