Measurement of radiation-induced autophagic flux in human brain cancer cells

Abstract

Radiation-induced autophagy mediates susceptibility to radiation [1]. However, autophagy is a multifaceted process. Herein, 2 aspects of autophagy, induction of autophagy or changes in autophagic flux, in radiation sensitivity were investigated. Relying on a systems-based approach and adding to a large, data dense morphome database [2, 3], we mined for complex cellular information on autophagy due to irradiation of human brain cancer (glioblastoma or GBM) cells. A quantitative stereology method is described here to chronicle these two important aspects of autophagy. These results add to our understanding of the role of autophagy in the efficacy of radiation therapy by providing a basis for the adaptive role for radiation-induced autophagy in GBM cells. Further, because GBM tumors are so resistant to radiotherapy, a novel strategy to radiosensitize GBM cells is urgently needed.Autophagy is a normal, basal housekeeping process in a cell. The main function of autophagy is to recycle damaged or old cellular constituents by encapsulating cargo within autophagosomes that are then trafficked to fuse with lysosomes, allowing lysosomal hydrolytic enzymes to catabolize the cellular material into simple macromolecular subunits. Induction of autophagy above basal levels provided some of the first insights into the complex role of this dynamic process in cell death and its role in radiation sensitization [4,5].To provide a proof-of-principle, morphomics data was collected and analyzed for human glioblastoma (U87) monolayer cultured cells irradiated with 0 or 10 Gy  rays [2] and 3 days after treatment were collected and fixed for TEM (the most sensitive method to monitor autophagy [6]). Biased sampling methods were used to selectively acquire at least 30 cell profiles for each treatment. The number of autophagosomes was scored for each cell to reflect changes in induction of autophagy in the cell population and within each cell. Autophagic flux was estimated by measuring the area of the cytoplasm occupied by autophagosomes using ImageJ. The area measurement integrates both the number of autophagosomes and size of autophagosomes in the cell. Increased flux, a possible outcome of the area analysis, is revealed when a decreased number and area of autophagosomes is found because hydrolytic digestion of autophagosomes indicates completion of the autophagic process. In contrast, decreased autophagic flux is revealed by increased number or area of autophagosomes resulting from a blockade in autophagy before the final hydrolysis of autophagosomes. Finally, a third outcome of the analysis is the identification of cells that have cytoplasmic areas occupied by autophagosomes that are more than 1 standard deviation of the average area. These outliers focus our attention on those cells having the most dramatic change in radiation-induced autophagy where flux is decreased AND continued autophagosome formation is abnormal. Indeed, previous experiments identified a significant proportion of an irradiated population as outliers, having extre...