The CD73 immune checkpoint promotes tumor cell metabolic fitness

CD73 is an ectonucleotidase overexpressed on tumor cells that suppresses anti-tumor immunity. Accordingly, several CD73 inhibitors are currently being evaluated in the clinic, including in large randomized clinical trials. Yet, the tumor cell-intrinsic impact of CD73 remains largely uncharacterized. Using metabolomics, we discovered that CD73 significantly enhances tumor cell mitochondrial respiration and aspartate biosynthesis. Importantly, rescuing aspartate biosynthesis was sufficient to restore proliferation of CD73-deficient tumors in immune-deficient mice. Seahorse analysis of a large panel of mouse and human tumor cells demonstrated that CD73 enhanced oxidative phosphorylation (OXPHOS) and glycolytic reserve. Targeting CD73 decreased tumor cell metabolic fitness, increased genomic instability and suppressed poly ADP ribose polymerase (PARP) activity. Our study thus uncovered an important immune-independent function for CD73 in promoting tumor cell metabolism and provides the rati..., For SITA, 3x106 cells/well were seeded in a 6-well plate and cultured in 2 mL assay media supplemented with 10% dialyzed FBS (Wisent) and either 25 mM U-[13C]-glucose (Cambridge Isotope Laboratories) and 4 mM glutamine (Sigma) or 4 mM U-[13C]-glutamine (Cambridge Isotope Laboratories) and 25 mM glucose (Sigma) for 3h at 37°C. Metabolites were extracted from cells using dry ice-cold 80% methanol, followed by sonication and removal of cellular debris by centrifugation at 4°C. Metabolite extracts were dried, derivatized as tert-butyldimethylsilyl esters, and analyzed via GC-MS by the metabolomic core from the Goodman cancer research centre (McGill University). Labeled metabolite abundance was expressed relative to the internal standard D27 (D-myristic acid) and normalized to protein content.
For Western Blots. Adherent cells were washed with ice-cold PBS and lysed and scrapped in CelLytic™ M buffer (Sigma) with 1X Halt™ protease and phosphatase cocktail inhibitors (Thermo Fisher Scientifi...,