DOI: 10.5061/dryad.t1g1jwt62
title: Data from: The CD73 immune checkpoint promotes tumor cell metabolic fitness

description: 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 rationale for previously
 unforeseen combination therapies incorporating CD73 inhibition.
description: For SITA, 3x10<sup>6
 </sup>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-[<sup>13</sup>C]-glucose (Cambridge Isotope Laboratories)
 and 4 mM glutamine (Sigma) or 4 mM
 U-[<sup>13</sup>C]-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 <em>Goodman cancer research
 centre </em>(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 Scientific) before being centrifuged for 15 min at 20,000g
 at 4°C. Proteins were harvested from supernatant and quantified by using
 Bradford protein assay dye reagent (Bio-Rad). 25 µg of protein from whole
 cell extract were loaded in 4–10% acrylamide gels and transferred on
 nitrocellulose membranes. Membranes were stained overnight in 5%
 BSA-containing PBS Tween 0.1% with following antibodies: mouse
 anti-β-actin (1:5000),
 mouse anti-PLCγ (1:2000),
 mouse anti-CD73 (human; 1:1000), mouse anti-FLAG (1:1000) and rabbit
 anti-Cas9 (S. pyogenes; 1:1000). For PARylation assay,
 1x10<sup>6</sup> cells were seeded in 6-well plates and drugs
 was added in media for 48 hours. Before proceeding to protein extraction,
 cells were treated with 2 mM
 H<sub>2</sub>O<sub>2</sub> for 20 minutes at room
 temperature. 50µg proteins were loaded in a 4-10% acrylamide gels,
 transferred on nitrocellulose membrane and stained overnight in 5%
 milk-containing PBS Tween 0.1% with mouse anti-PAR (1:1000). Proteins were
 revealed with fluorescent secondary anti-rabbit or anti-mouse antibodies
 (1:10 000; LI-COR) using the LI-COR fluorescent scanner or by
 chemiluminescence (Bio-Rad) using HRP-conjugated secondary anti-mouse
 antibody (2.5:10 000) and Amersham ECL prime detection reagent (GE
 Healthcare Lifescience). For Genome-wide transcriptional
 analysis. Total RNA was isolated from MDA-MB-231 cells using the RNeasy
 Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA
 was quantified using a DS-11 spectrophotometer (DeNovix). Samples were
 sent to <em>Génome Québec</em> (Montréal, QC, CA) for gene
 expression analysis using an Affymetrix microarray chip. Expression values
 were computed using the robust multi-array analysis (RMA) normalization
 method (‘affy’ package in Bioconductor). When multiple probe sets mapped
 to the same official gene symbol, we computed their average value.
 Differential expression (DE) analysis was performed using ‘limma’ standard
 analysis pipeline. Significant DE genes were defined as genes with
 absolute fold-change ≥ 1.5 and adjusted P-value ≤ 0.05. Statistical
 analyses were performed between <em>NT5E</em> mRNA expression
 and gene expression using Spearman correlation.