Data from: The CD73 immune checkpoint promotes tumor cell metabolic fitness
Data files
Jun 06, 2023 version files 11.44 MB
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Blots-original-files.zip
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Microarray_MDA.zip
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README.md
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SITA-analyses.zip
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Uncropped-blots.pdf
Abstract
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.
Methods
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 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, 1x106 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 H2O2 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 Génome Québec (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 NT5E mRNA expression and gene expression using Spearman correlation.