Park Yea-In, Park Rackhyun, Lee Siyun, Lee Chunghyeon, Yoo Inkyu, Ka Hakhyun, Huh Yang Hoon, Hong Jongkwang, Park Junsoo
Division of Biological Science and Technology, Yonsei University, Wonju 26493, Republic of Korea.
Department of Life Science, Yong-In University, Yongin 17092, Republic of Korea.
Life (Basel). 2025 Mar 22;15(4):525. doi: 10.3390/life15040525.
AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis, and AMPK contributes to cell growth, apoptosis, and autophagy. Although most cell studies have been performed using two-dimensional (2D) cell culture, recent studies have demonstrated that the three-dimensional (3D) spheroid technique is helpful in various cell research fields, such as tumor biology, due to its resemblance to the 3D tissue structure. However, the role of AMPK in 3D spheroid formation has not been characterized clearly. This study used the AMPK knockout cell line to examine the role of AMPK in 3D spheroid formation and is the first report describing the generation of 3D spheroids using AMPK knockout cells. While control cells produced round spheroids with a similar length-to-width ratio, AMPK knockout produced an oval shape with a more significant length-to-width ratio. We demonstrate that AMPK knockout spheroids contain significantly more prominent lysosomes in each cell, indicating that autophagic flux is impaired in 3D spheroids. Finally, flow cytometry analysis showed that AMPK knockout spheroids contain more apoptotic cells than control cells. These results indicate that AMPK is required for efficient 3D spheroid formation.
腺苷酸活化蛋白激酶（AMPK）是细胞能量稳态的重要调节因子，它对细胞生长、凋亡和自噬都有影响。尽管大多数细胞研究都是使用二维（2D）细胞培养进行的，但最近的研究表明，三维（3D）球体技术由于其与三维组织结构相似，在肿瘤生物学等各种细胞研究领域中很有帮助。然而，AMPK在3D球体形成中的作用尚未得到明确表征。本研究使用AMPK基因敲除细胞系来研究AMPK在3D球体形成中的作用，这是第一篇描述使用AMPK基因敲除细胞生成3D球体的报告。对照细胞产生长宽比相似的圆形球体，而AMPK基因敲除细胞产生的是长宽比更显著的椭圆形球体。我们证明，AMPK基因敲除的球体在每个细胞中含有明显更多、更突出的溶酶体，这表明3D球体中的自噬流受损。最后，流式细胞术分析表明，AMPK基因敲除的球体比对照细胞含有更多的凋亡细胞。这些结果表明，高效的3D球体形成需要AMPK。
