3D Spheroid Configurations Are Possible Indictors for Evaluating the Pathophysiology of Melanoma Cell Lines

Hiroshi Ohguro; Megumi Watanabe; Tatsuya Sato; Fumihito Hikage; Masato Furuhashi; Masae Okura; Tokimasa Hida; Hisashi Uhara

doi:10.3390/cells12050759

3D Spheroid Configurations Are Possible Indictors for Evaluating the Pathophysiology of Melanoma Cell Lines

Cells. 2023 Feb 27;12(5):759. doi: 10.3390/cells12050759.

Authors

Hiroshi Ohguro¹, Megumi Watanabe¹, Tatsuya Sato^{2

3}, Fumihito Hikage¹, Masato Furuhashi², Masae Okura⁴, Tokimasa Hida⁴, Hisashi Uhara⁴

Affiliations

¹ Departments of Ophthalmology, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan.
² Departments of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan.
³ Departments of Cellular Physiology and Signal Transduction, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan.
⁴ Departments of Dermatology, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan.

Abstract

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.

Keywords: 3D spheroid culture; KRAS; RNA sequencing; SOX2; melanoma.

MeSH terms

Cell Line, Tumor
Humans
Melanoma* / pathology
Proto-Oncogene Proteins p21(ras)* / metabolism

Substances

Proto-Oncogene Proteins p21(ras)

Grants and funding

This research received no external funding.