# AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids

## TL;DR

This study shows that knocking out AMPK impairs the formation of 3D cell spheroids, affecting their shape and increasing cell death.

## Contribution

This is the first report on 3D spheroid formation using AMPK knockout cells, revealing AMPK's role in spheroid development.

## Key findings

- AMPK knockout spheroids are oval-shaped with a higher length-to-width ratio compared to control cells.
- AMPK knockout spheroids show increased lysosomes, indicating impaired autophagic flux.
- AMPK knockout spheroids have more apoptotic cells than control cells.

## Abstract

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.