Response of Human and Canine Tumor Cell Lines to Pharmacologic and Genetic Autophagy Inhibition

Background: The intrinsic autophagy-dependence of human tumor cell lines has been demonstrated using CRISPR screens showing that some respond in similar ways to loss core autophagy genes as they do an established essential gene such proliferating nuclear antigen (PCNA). Pharmacologic inhibition can be achieved lysosomotropic agents hydroxychloroquine (HCQ) interfere with fusion the autophagosome lysosome thus preventing completion recycling process. sensitivity cells antiproliferative and cytotoxic activity HCQ is complicated by pH non-selective mechanism action determining based on not explicitly established. goal present study determine canine osteosarcoma (cOSA) (hTC) effects lysosomal inhibitors (HCQ LysoV), compare these results measured a CRISPR/Cas9 live-cell imaging assay. Materials Methods: RFP labelled line response LysoV was determined live YOYO-1 staining buffered media INCUCYTE ZOOMÒ. screen done species specific guide RNA’s transfection reagents into were dual GFP labelled. Guide RNA for included CRISPR/ Cas9 reagent mix selection only indicating uptake targeted (ATG7, FIP200, LAMP2, ULK2, ATG12, ATG13, STX17 or VPS34). Response compared (PCNA) non-essential (PTEN) gene. Results: cOSA hTC showed responses median lethal dose (Dm) values ranging from 7.1–14.6 μM 1.1–5.2 measures anti-proliferative response, respectively. Dm death ranged 11.4–36.4 3.4–7.9 LysoV. Analysis key varied within lines. A clear relationship observed between VPS34 score correlations (r2 = 0.9463 0.6395) independent manner. also although scores other FIP200) enhanced predictive ability. Conclusions: shows strong correlation knockout. This lineage suggests variable cellular knockout select genes. No conflict interest.