AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids

Area of Science:

• Cell Biology
• Biochemistry
• Cancer Research

Background:

• AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis, impacting cell growth, apoptosis, and autophagy.
• Three-dimensional (3D) spheroid culture mimics in vivo tissue structure, offering advantages over 2D cultures for various research fields, including tumor biology.

Purpose of the Study:

• To investigate the role of AMPK in 3D spheroid formation.
• To characterize the morphological and cellular differences in 3D spheroids derived from AMPK knockout cells.

Main Methods:

• Generation of 3D spheroids using AMPK knockout and control cell lines.
• Microscopic analysis of spheroid morphology (shape, length-to-width ratio).
• Assessment of lysosome prominence and autophagic flux.
• Flow cytometry analysis to quantify apoptosis.

Main Results:

• AMPK knockout cells formed oval-shaped spheroids with an increased length-to-width ratio compared to round control spheroids.
• AMPK knockout spheroids exhibited significantly more prominent lysosomes, indicating impaired autophagic flux.
• Flow cytometry revealed a higher percentage of apoptotic cells in AMPK knockout spheroids.

Conclusions:

• AMPK is essential for the proper formation of 3D spheroids.
• Disruption of AMPK signaling affects spheroid morphology, lysosomal function, and cell viability.
• These findings underscore the importance of AMPK in maintaining cellular homeostasis within 3D tissue models.