Visualization of Apoptosis in Three-Dimensional Cell Aggregates Based on Molecular Beacon Imaging

Abstract

The objective of this study is to visualize cell apoptosis in three-dimensional (3D) cell aggregates based on molecular beacons (MB). Two types of MB for messenger RNA were used: caspase-3 MB as a target for apoptosis and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) MB as a control of stable fluorescence in cells. To enhance the MB internalization into cells, caspase-3 and GAPDH MB were incorporated in cationized gelatin nanospheres (cGNS), respectively (cGNS_{casp3 MB} and cGNS_{GAP MB}). In addition, cGNS co-incorporating caspase-3 and GAPDH MB (cGNS_{dual MB}) were prepared to perform the dual-color imaging for the same cell aggregate. The cGNS_MB were incubated with mouse mesenchymal stem cells to label with MB in the two-dimensional culture. The cell apoptosis mediated by the addition of antibody for a death receptor Fas was ratiometrically detected by the cGNS_{dual MB} to the same extent as single MB. The cell aggregates were prepared from MB-labeled cells, and the MB fluorescence was detected from almost all the cells even in the 3D aggregates to show the homogenous distribution. In addition to the Fas-mediated apoptosis, the aggregates were treated with camptothecin of a low-molecular weight apoptosis inducer. The fluorescence of caspase-3 MB was mainly distributed at the surface surrounding site of Fas-mediated apoptotic aggregates rather than the center site, while that of GAPDH MB was detected even in the interior site. On the other hand, in the camptothecin-induced apoptotic aggregates, both caspae-3 and GAPDH MB fluorescence were detected from the interior site of aggregates as well as the surrounding site. It is likely that the MB fluorescence reflected the localization of apoptotic position caused by the different molecular sizes of apoptosis inducer and the consequent penetration into the aggregates. It is concluded that the cGMS_MB are a promising system to visualize cell apoptosis in 3D cell aggregates without the destruction of aggregates.

Impact statement

This study demonstrates the methodological feasibility of cationized gelatin nanospheres incorporating molecular beacons (cGNS_MB) to visualize cell apoptosis in three-dimensional (3D) cell aggregates without the destruction of aggregates. When cell apoptosis was induced by the addition of anti-Fas antibody and camptothecin of a low-molecular weight drug, the cGNS_MB imaging system achieved the discrimination of apoptotic position in aggregates caused by the different penetrations of apoptosis inducers. The imaging technology developed in this study would be useful and informative for wide research fields, including 3D cell culture, drug screening, and transplantation therapy.