Departments of Ophthalmology, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan.
Departments of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan.
Cells. 2023 Feb 27;12(5):759. doi: 10.3390/cells12050759.
To study the molecular mechanisms responsible for inducing the spatial proliferation of malignant melanomas (MM), three-dimension (3D) spheroids were produced from several MM cell lines including SK-mel-24, MM418, A375, WM266-4, and SM2-1, and their 3D architectures and cellular metabolisms were evaluated by phase-contrast microscopy and Seahorse bio-analyzer, respectively. Several transformed horizontal configurations were observed within most of these 3D spheroids, and the degree of their deformity was increased in the order: WM266-4, SM2-1, A375, MM418, and SK-mel-24. An increased maximal respiration and a decreased glycolytic capacity were observed within the lesser deformed two MM cell lines, WM266-4 and SM2-1, as compared with the most deformed ones. Among these MM cell lines, two distinct cell lines, WM266-4 and SK-mel-24, whose 3D appearances were the closest and farthest, respectively, from being horizontally circular-shaped, were subjected to RNA sequence analyses. Bioinformatic analyses of the differentially expressed genes (DEGs) identified KRAS and SOX2 as potential master regulatory genes for inducing these diverse 3D configurations between WM266-4 and SK-mel-24. The knockdown of both factors altered the morphological and functional characteristics of the SK-mel-24 cells, and in fact, their horizontal deformity was significantly reduced. A qPCR analysis indicated that the levels of several oncogenic signaling related factors, including KRAS and SOX2, PCG1α, extracellular matrixes (ECMs), and ZO1 had fluctuated among the five MM cell lines. In addition, and quite interestingly, the dabrafenib and trametinib resistant A375 (A375DT) cells formed globe shaped 3D spheroids and showed different profiles in cellular metabolism while the mRNA expression of these molecules that were tested as above were different compared with A375 cells. These current findings suggest that 3D spheroid configuration has the potential for serving as an indicator of the pathophysiological activities associated with MM.
为了研究导致恶性黑色素瘤（MM）空间增殖的分子机制，我们从包括 SK-mel-24、MM418、A375、WM266-4 和 SM2-1 在内的几种 MM 细胞系中产生了三维（3D）球体，并分别通过相差显微镜和 Seahorse 生物分析仪评估了它们的 3D 结构和细胞代谢。在大多数 3D 球体中观察到了几种变形的水平结构，其变形程度的顺序为：WM266-4、SM2-1、A375、MM418 和 SK-mel-24。与变形最大的细胞系相比，在变形较小的两个 MM 细胞系 WM266-4 和 SM2-1 中，观察到最大呼吸增加和糖酵解能力降低。在这些 MM 细胞系中，两个明显的细胞系 WM266-4 和 SK-mel-24，其 3D 外观分别最接近和最远离水平圆形，进行了 RNA 序列分析。差异表达基因（DEG）的生物信息学分析鉴定出 KRAS 和 SOX2 作为诱导 WM266-4 和 SK-mel-24 之间这些不同 3D 构型的潜在主调控基因。这两个因素的敲低改变了 SK-mel-24 细胞的形态和功能特征，实际上，它们的水平变形显著减少。qPCR 分析表明，在这五种 MM 细胞系中，包括 KRAS 和 SOX2、PCG1α、细胞外基质（ECM）和 ZO1 在内的几种致癌信号相关因子的水平发生了波动。此外，相当有趣的是，达拉非尼和曲美替尼耐药的 A375（A375DT）细胞形成了球状 3D 球体，并在细胞代谢方面表现出不同的特征，而上述测试的这些分子的 mRNA 表达与 A375 细胞不同。这些发现表明，3D 球体结构有可能作为与 MM 相关的病理生理活动的指标。
