The University of Texas MD Anderson Cancer Center Houston United States
Purpose We are investigating if sequestration of metabolically dysfunctional mitochondria by the autophagosomes mitophagy imparts anti-androgen resistance and if this phenomenon can be applied in circulating tumor cells in patient blood samples as a biomarker for development of drug resistance. Method Effects of the anti-androgen enzalutamide on the autophagy and mitophagy of androgen-dependent LNCaP and -independent C4-2 and CWR22v1 cells are studied first. Autophagy is monitored by fluorescence of cells with anti-LC3B antibody. Cellular fluorescence due to Mitosox dye oxidation is used to identify mitochondria producing high superoxide O2-. Mitophagy is monitored using fluorescence resonance energy transfer FRET by visualization of FRET images and quantitation of FRET image intensities using a Leica Sp8 fluorescence STED confocal microscope and Image J software. Results and Discussion The degree of mitophagy is more in the surviving androgen-dependent LNCaP cells than in the -independent C4-2 cells, when grown in androgen-depleted media. Enzalutamide treatment induces mitophagy in both cell lines. However, the increase in mitophagy is significantly more in the enzalutamide resistant C4-2 than in the sensitive LNCaP cells. Mitosox fluorescence and mitophagy in circulating tumor cells CTCs isolated from patient blood samples are being quantitated to identify drug resistance.