Abstract Date: 5/2/2015
Christopher Paul Deibert, MD
Wi Jin Kim, BA
Michael Brancho, BS
Michael Lotze, MD
Nduka Amankulor, MD (Pittsburgh, PA)
Our studies have shown that IDH mutant gliomas avoid natural killer cell-mediated death, unlike IDH wild-type counterparts. The basis for NK cell resistance in IDH mutants remains unknown. Autophagy has been described as a mechanism for NK cell resistance in various cancers. Therefore, we explored whether IDH mutations induce autophagy as survival mechanism following NK cell-induced stress.
To characterize autophagy in astrocytic cells, we utilized immunocytochemistry to identify LC3 puncta, a marker for autophagosome formation. We cultured IDH mutant and wild-type astrocytes with NK cells for 48 hours at a 20:1 effector:target ratio. Astrocytes were fixed on microscope slides and stained using anti-LC3 antibodies. Slides were finalized and imaged at 1000x using an oil-emersion fluorescent microscope to determine the number, average area and fluorescence intensities of LC3 puncta.
Co-culturing IDHmut with NK cells showed 97%, 113% and 104% increase in puncta area in early, middle and late passage astrocytes respectively; 138% and 40% increase in puncta intensity in early and middle passages respectively; and 104% increase the number of puncta/cell in late passages compared to controls. All values were statistically significant compared to controls. In contrast, co-culturing IDHwt with NK cells showed a 14% decrease and 53% increase in puncta intensity in middle and late passages respectively when compared to controls.
These results indicate significant increases autophagy in IDH mutant cells compared to IDH wild-type cells when stressed by NK cells. These findings suggest autophagy inhibitors maybe an effective means of treatment in various IDH mutant cancers including astrocytomas.
Article ID: AA-32157