The activation of Wnt signaling has been reported in many types of squamous cell carcinoma. In this study, using human oral squamous cell carcinoma (OSCC) cells with different metastatic potential, we investigated the involvement of Wnt signaling in metastasis. Further, we aimed to elucidate the characteristic biological features related to high metastatic potential and to identify new target molecules for the suppression of OSCC lymph node metastasis. We compared SAS-Venus (SAS OSCC cells expressing green fluorescent protein) and SAS-LM8, which is a highly metastatic cell line derived from SAS-Venus by in vivo selection. The SAS-LM8 cell line had greater ability of migration and invasion compared to SAS-Venus. Furthermore, a higher number of filopodia-like protrusive structures were produced in SAS-LM8 cells compared to SAS-Venus cells, and the levels of active Cdc42 and active RhoA protein were higher in SAS-LM8 cells compared to SAS-Venus cells. We did not observe any differences in the expression of Wnt/β-catenin target genes between the two cell lines; however, the mRNA levels of Wnt5b were higher in SAS-LM8 cells compared to SAS-Venus cells. To confirm the involvement of Wnt5b in migration in OSCC cells, we examined the effects of the siRNA-mediated knockdown of Wnt5b in SAS-Venus cells and SAS-LM8 cells. The siRNA treatment significantly inhibited migration and the formation of filopodia-like protrusive structures. Conversely, when stimulated with Wnt5b, the migration and formation of filopodia-like protrusions were significantly enhanced and the levels of active Cdc42 and active RhoA proteins were also increased. These results indicate that Wnt5b is involved in the migration ability of OSCC cells through active Cdc42 and RhoA.

: The activation of Wnt signaling has been reported in many types of squamous cell carcinoma. In this study, we used human oral squamous cell carcinoma (OSCC) cells with different metastatic potentials to investigate the involvement of Wnt signaling in metastasis. In addition, we aimed to elucidate the characteristic biological features related to high metastatic potential and to identify new target molecules for the suppression of OSCC lymph node metastasis. We compared SAS-Venus (SAS OSCC cells expressing green fluorescent protein) and SAS-LM8, which is a highly metastatic cell line derived from SAS-Venus by in vivo selection. The SAS-LM8 cell line had greater ability of migration and invasion than did SAS-Venus. Furthermore, a higher number of filopodia-like protrusive structures were produced in SAS-LM8 cells than in SAS-Venus cells, and the levels of active Cdc42 and active RhoA protein were higher in SAS-LM8 cells than in SAS-Venus cells. We did not observe any differences in the expression of Wnt/beta-catenin target genes between the two cell lines; however, the mRNA levels of Wnt5b were higher in SAS-LM8 cells than in SAS

Preventing cancer metastasis requires a thorough understanding of cancer cell invasion. These phenomena occur in human 3-D living tissues. To this end, we developed a human cell-based three-dimensional (3-D) cultured tissue constructs that imitate in vivo human tissue organization. We investigated whether our 3-D cell culture system can be used to analyze the invasion of human oral squamous cell carcinoma (OSCC) cells. The 3-D tissue structure consisted of five layers of normal human dermal fibroblasts along with human dermal lymphatic endothelial cell tubes and was generated by the cell accumulation technique and layer-by-layer assembly using fibronectin and gelatin. OSCC cells with different lymph metastatic capacity were inoculated on the 3-D tissues and their invasion through the 3-D tissue structure was observed. Conventional methods of analyzing cell migration and invasion, that is, 2-D culture-based transwell and Matrigel assays were also used for comparison. The results using the 3-D cultured tissue constructs were comparable to those obtained using conventional assays; moreover, use of the 3-D system enabled visualization of differential invasion capacities of cancer cells. These results indicate that our 3-D cultured tissue constructs can be a useful tool for analysis of cancer cell invasion in a setting that reflects the in vivo tissue organization. © 2018 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J. Biomed. Mater. Res. Part A: 00A: 000-000, 2018.

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