AMPK Knockout Impairs the Formation of Three-Dimensional Spheroids

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.

Keywords: 3D culture; AMPK; knockout; lysosome; spheroid.

Figure 1

Formation of HEK293T spheroid using ultra-low attachment plate. (A) HEK293T cells were seeded at 1000, 10,000, and 100,000 cells/well and incubated for the indicated days. Bars, 100 μm. (B) The diameters of the spheroids were measured and are shown in the graph.

Figure 2

AMPK knockout cells produced oval-shaped spheroids. (A) Control HEK293T and AMPK knockout cells were seeded at 10,000 cells/well, and spheroid shapes were captured at the indicated days. Bars, 100 μm. (B) The spheroids of AMPK knockout cells were smaller than the spheroids of control cells. The surface area was calculated in each sample and is shown in the graph. N = 5, control vs. AMPK knockout, * p < 0.05, ** p < 0.01. (C) The spheroids of AMPK knockout cells were oval-shaped rather than round-shaped. (D) The length-to-width ratio of each spheroid was calculated. N = 5, control vs. AMPK knockout, ** p < 0.01.

Figure 3

The expressions of autophagy markers are induced in the core of spheroids. (A) Control and AMPK knockout spheroid were used to make a paraffin-embedded tissue block before sectioning. Each slide was stained with an anti-LC3 antibody. (B) The control cell and AMPK knockout cell spheroid were stained with an anti-LC3 antibody, and the stained cells were observed using a confocal microscope. Bars, 100 μm. (C) The expression of LC3 proteins is upregulated in AMPK knockout spheroids. GAPDH was used as a loading control. (D) The reported activity of pGL2-CLEAR Luc was reduced in AMPK knockout cells. Control vs. AMPK knockout (N = 3), * p < 0.05, ** p < 0.01.

Figure 4

AMPK knockout cells produced larger lysosomes in 3D spheroids. (A) Control cells and AMPK knockout cells were analyzed using a transmission electron microscope. (B) The size of the lysosome was measured and is shown in the graph. Control vs. AMPK knockout, *** p < 0.001.

Figure 5

AMPK knockout cells spheroids contain more apoptotic cells than control cells. (A) Control HEK293T cells and AMPK knockout cells were used to form the spheroids, and the spheroids were disassembled for flow cytometry analysis. (B) The sub-G1 population of each cell was analyzed and is shown in the graph. Control vs. AMPK knockout (N = 3), * p < 0.05, ** p < 0.01. (C) Control and AMPK knockout spheroids were used to make a paraffin-embedded tissue block before sectioning. Each slide was stained with an anti-cleaved PARP-1 antibody. Arrowheads indicate the positively stained cells.