The CD73 immune checkpoint promotes tumor cell metabolic fitness
- Centre de Recherche du Centre Hospitalier l’Université de Montréal, Canada
- Faculté de Pharmacie, Université de Montréal, Canada
- Institut du Cancer de Montréal, Canada
- McGill Goodman Cancer Research Centre, Canada
Figures
Figure 1 with 4 supplements
CD73-deficiency delays tumor growth independently from immune suppression through reduced aspartate biosynthesis.
(A) Sub-cutaneous tumor growth of CD73pos (CD73+) and CD73neg (CD73-) MDA-MB-231 tumors in immune-deficient NRG mice. Results shows combined data of two independent experiments cumulating 15 mice …
-
Figure 1—source data 1
Original raw unedited blots and uncropped blots with the relevant bands labeled that composes Figure 1G.
- https://cdn.elifesciences.org/articles/84508/elife-84508-fig1-data1-v3.zip
-
Figure 1—source data 2
Stable isotope tracing analysis (SITA) raw data for both U-[13C]-glucose and U-[13C]-glutamine tracing in MDA-MB-231 CD73 +and CD73- cells that composes Figure 1E–F and Figure 1—figure supplement 3.
- https://cdn.elifesciences.org/articles/84508/elife-84508-fig1-data2-v3.zip
Figure 1—figure supplement 1
Generation of CD73-knockout MDA-MB-231 cell line.
(A-C) Knockout efficiency of CD73 on MDA-MB-231 cells following CRISPR-Cas9 gene edition. Cell sorting enrichment was confirmed by cell surface (A); FACS. US = unstained control and total (B); …
-
Figure 1—figure supplement 1—source data 1
Original raw unedited blots and uncropped blots with the relevant bands labeled that composes Figure 1—figure supplement 1B, D.
- https://cdn.elifesciences.org/articles/84508/elife-84508-fig1-figsupp1-data1-v3.zip
Figure 1—figure supplement 2
In vitro proliferation and viability analysis of CD73 + and CD73- MDA-MB-231 cells.
(A-B) Examples of in vitro proliferation imaging in presence of excess (2 mM) or limited (0.1 mM) amount of glutamine (Gln) measured using Incucyte technology. Proliferation was quantified by …
Figure 1—figure supplement 3
Glucose and glutamine SITA and transcriptional analysis of TCA cycle enzymes in MDA-231 cells.
(A-D) Mass isotopomer distribution (MID) of U-[13C]-glucose-derived (U-[13C]-GLC; A) or U-[13C]-glutamine-derived (U-[13C]-GLN; B) glycolysis or TCA cycle metabolites compared between CD73 +and …
Figure 1—figure supplement 4
CD73 expression and enzymatic activity in gpASNase1-expressing MDA-MB-231 cells.
(A) CD73 surface protein expression levels on MDA-MB-231 CD73 +and CD73- cells transfected with pLHCX-ev or pLHCX-gpASNase1 plasmids. (B) CD73 enzymatic activity analyzed as in panel (C) compared …
Figure 2 with 3 supplements
CD73 regulates metabolic fitness of cancer cells independently from adenosine signaling.
(A) Schematized metabolic profiling and parameters generated on a Seahorse analyzer. OXPHOS and glycolytic parameters were calculated using Agilent calculator tool. Rot = rotenone, AA = antimycin A, …
Figure 2—figure supplement 1
CD73-deficient MDA-MB-231 cells retain impaired metabolic flexibility in glutamine-limiting conditions.
CD73 + and CD73- MDA-MB-231 cells were analyzed on a seahorse instrument as in Figure 2 but in presence of either excess (2.0 mM) or limiting amount (0.1 mM) of glutamine in seahorse media. (A–B) …
Figure 2—figure supplement 2
Generation of CD73-knockout cancer cell lines.
(A) Knockout efficiency of CD73 on 2 mouse cancer cell lines (4T1.2 and SM1 LWT1) following CRISPR-Cas9 Nt5e gene edition. Cell sorting enrichment was confirmed by FACS. (B) Mouse embryoblastic …
Figure 2—figure supplement 3
Intracellular ATP, ADP and AMP metabolites measurement and transcriptional analysis of metabolic enzymes in MDA-MB-231 CD73 +and CD73- cells.
(A) Absolute intracellular levels of total ATP, ADP, and AMP metabolites measured in CD73 +and CD73- MDA-MB-231 cells by LC-MS (complement to Figure 2I). (B) Transcriptional analysis by microarray …
-
Figure 2—figure supplement 3—source data 1
Raw microarray data that composes Figure 2—figure supplement 3B.
- https://cdn.elifesciences.org/articles/84508/elife-84508-fig2-figsupp3-data1-v3.zip
Figure 3 with 1 supplement
CD73 contributes to metabolic fitness of cancer cells through NAD synthesis.
(A) LC-MS analysis of intracellular nicotinamide adenine levels (NAD+) normalized to protein content of CD73pos (CD73+;+/-1 μg/mL αCD73 mAb or 1 μg/mL rhADA for 48 hr) and CD73neg (CD73-;+/-100 μM …
Figure 3—figure supplement 1
CD73 contributes to metabolic fitness of cancer cells through NAD synthesis.
(A) LC-MS analysis of intracellular nicotinamide adenine levels (NAD+) normalized to protein content of CD73pos (CD73+) and CD73neg (CD73-) 4T1.2 cells (n=1). (B) LC-MS analysis of nicotinamide …
Figure 4 with 1 supplement
CD73 deficiency decreases PARP activity and sensitizes cancer cells to DNA-damaging agents.
(A) Western blot (left panel) showing PARylation levels in CD73pos (CD73+) and CD73neg (CD73-) MDA-MB-231 cells. Densitometry (right panel) is shown relative to CD73 + cells (n=5). (B) Western blot …
-
Figure 4—source data 1
Original raw unedited blots and uncropped blots with the relevant bands labeled that composes Figure 4A–C.
- https://cdn.elifesciences.org/articles/84508/elife-84508-fig4-data1-v3.zip
Figure 4—figure supplement 1
CD73 deficiency sensitizes UWB1.289+BRCA1 cells to DNA-damaging agents.
(A) Knockout efficiency of CD73 expression on UWB1.289+BRCA1 analyzed by FACS. US = unstained control. (B) Western blot showing CD73 protein levels and PARylation levels in CD73+ (CTRL) and CD73- …
-
Figure 4—figure supplement 1—source data 1
Original raw unedited blots and uncropped blots with the relevant bands labeled that composes Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/84508/elife-84508-fig4-figsupp1-data1-v3.zip
Figure 5
Schematic representation of the role of CD73 in NAD + biosynthesis, metabolic function, and genomic stability in cancer cells.
We propose that CD73 regulates to NAD + biosynthesis intracellularly in a NRK1-dependent manner by hydrolyzing extracellular NMN into NR, which in turn favors DNA-damage response, mitochondrial …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-human CD73 (7G2, mouse monoclonal) | Invitrogen | Cat#410200, RRID: AB_2533492 | Inhibition (1 µg/mL) |
| Antibody | Anti-human CD73 BV421 (mouse monoclonal) | BD Bioscience | Cat#562430, RRID: AB_11153119 | FACS (1:200) |
| Antibody | Anti-mouse CD73 PE-Cy7 (Rat monoclonal) | Thermo Fisher Scientific | Cat#25-0731-82, RRID: AB_10853348 | FACS (1:200) |
| Antibody | Anti-β-actin (mouse monoclonal) | Abcam | Cat#ab8226 | WB (1:5000) |
| Antibody | Anti-PLCγ (mouse polyclonal) | Millipore | Cat#05–163 | WB (1:2000) |
| Antibody | Anti-human CD73 (mouse monoclonal) | Abcam | Cat#Ab91086 | WB (1:1000) |
| Antibody | Anti-FLAG (rabbit polyclonal) | Millipore | Cat#F7425 | WB (1:1000) |
| Antibody | Anti-Cas9 S. pyogenes (rabbit monoclonal) | Cell Signaling Technology | Cat#19526 S | WB (1:1000) |
| Antibody | Anti-PAR (mouse monoclonal) | Trevigen | Cat#4335-MC-100 | WB (1:1000) |
| Antibody | HRP-conjugated anti-mouse IgG secondary (goat polyclonal) | Millipore | Cat#AP124P | WB (2.5:10,000) |
| Antibody | IRDye 800CW anti-Mouse IgG secondary (donkey polyclonal) | LI-COR | Cat#926–32212, RRID: AB_621847 | WB (1:10,000) |
| Antibody | IRDye 680RD anti-rabbit IgG secondary (donkey polyclonal) | LI-COR | Cat#926–68073, RRID: AB_10954442 | WB (1:10,000) |
| Antibody | Anti-phospho-Histone H2A.X Ser139 (mouse monoclonal) | Millipore | Cat#05–636 | IF (1:2000) |
| Antibody | Anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Alexa Fluor 488 (donkey polyclonal) | Thermo Fisher Scientific | Cat#A-21202, RRID: AB_141607 | IF (1:800) |
| Chemical compound, drug | Adenosine 5'-(α,β-methylene) diphosphate (APCP) | Sigma-Aldrich | Cat#M3763, CAS: 3768-14-7 | |
| Chemical compound, drug | Nicotinamide riboside (NR) | Cayman Chemical | Cat#23132, CAS: 1341-23-7 | |
| Chemical compound, drug | Nicotinamide mononucleotide (NMN) | Sigma-Aldrich | Cat#N3501, CAS: 1094-61-7 | |
| Chemical compound, drug | 5'-N-Ethylcarboxamidoadenosine (NECA) | Tocris | Cat#1691, CAS: 35920-39-9 | |
| Chemical compound, drug | SCH58261 | Tocris | Cat#2270, CAS: 160098-96-4 | |
| Chemical compound, drug | PSB1115 | Tocris | Cat#2009, CAS: 152529-79-8 | |
| Chemical compound, drug | Olaparib (AZD2281) | Selleckchem | Cat#S1060, CAS: 763113-22-0 | |
| Chemical compound, drug | Doxorubicin | CRCHUM pharmacy | CAS: 23214-92-8 | |
| Chemical compound, drug | Carboplatin | CRCHUM pharmacy | CAS: 41575-94-4 | |
| Chemical compound, drug | 2-deoxy-2-[(7-nitro-2,1,3- benzoxadiazol-4-yl)amino]- D-glucose (2-NBDG) | Cayman Chemical | Cat#11046, CAS: 186689-07-6 | |
| Chemical compound, drug | U-[13C]-glucose | Cambridge Isotope Laboratories | Cat#CLM-1396, CAS: 110187-42-3 | |
| Chemical compound, drug | U-[13C]-glutamine | Cambridge Isotope Laboratories | Cat#CLM-1822, CAS: 184161-19-1 | |
| Chemical compound, drug | Oligomycin | Sigma-Aldrich | Cat#O4876, CAS: 1404-19-9 | |
| Chemical compound, drug | Carbonyl cyanide p- trifluoromethoxyphenylhydrazone (FCCP) | Sigma-Aldrich | Cat#C2920, CAS: 370-86-5 | |
| Chemical compound, drug | Rotenone | Sigma-Aldrich | Cat#R8875, CAS: 83-79-4 | |
| Chemical compound, drug | Antimycin A from Streptomyces sp. | Sigma-Aldrich | Cat#A8674, CAS: 1397-94-0 | |
| Chemical compound, drug | CelLytic M buffer | Sigma-Aldrich | Cat#C2978 | |
| Chemical compound, drug | Halt protease and phosphatase cocktail inhibitors | Sigma-Aldrich | Cat#78440 | |
| Chemical compound, drug | Bradford protein assay dye reagent | Bio-Rad | Cat#500–0006 | |
| Chemical compound, drug | Protein Block DAKO solution | Agilent | Cat#X0909 | |
| Chemical compound, drug | Fluoromount Aqueous Mounting Medium | Sigma-Aldrich | Cat#F4680 | |
| Chemical compound, drug | Formalin solution, neutral buffered, 10% | Sigma-Aldrich | Cat#HT5011 | |
| Chemical compound, drug | Triton X-100 reduced | Sigma-Aldrich | Cat#X100RS; CAS: 92046-34-9 | |
| Chemical compound, drug | 7-amino-actinomycin D (7-AAD) | eBioscience | Cat#00-6993-50 | |
| Peptide, recombinant protein | Recombinant human adenosine deaminase (rhADA) | R&D systems | Cat#7048-AD, Acc#P00813 | |
| Peptide, recombinant protein | Recombinant human CD73 (rhCD73) | R&D systems | Cat#5795-EN, Acc#AAH659937 | |
| Commercial assay, kit | Malachite green phosphate detection kit | R&D systems | Cat#DY996 | |
| Commercial assay, kit | SuperScript VILO cDNA Synthesis Kit | Thermo Fisher Scientific | Cat#11754050 | |
| Commercial assay, kit | RNeasy mini kit | QIAGEN | Cat#74104 | |
| Commercial assay, kit | ALDEFLUOR kit | Stemcell | Cat#01700 | |
| Commercial assay, kit | Amersham ECL prime detection reagent | GE healthcare lifescience | Cat#RPN2232 | |
| Commercial assay, kit | Glutamine/Glutamate-Glo Assay kit | Promega | Cat#J8021 | |
| Cell line (homo-sapiens) | MDA-MB-231 (TNBC) | ATCC | HTB-26 | |
| Cell line (homo-sapiens) | PANC-1 (pancreatic cancer) | ATCC | CRL-1469 | |
| Cell line (homo-sapiens) | SK23MEL (metastatic melanoma) | MSK Cancer Center | RRID: CVCL_6027 | |
| Cell line (homo-sapiens) | HCC1954 (breast cancer) | ATCC | CRL-2338 | |
| Cell line (homo-sapiens) | OV1369 (ovarian cancer) | Dr. Anne-Marie Mes-Masson | Fleury et al., 2019 | |
| Cell line (homo-sapiens) | SKOV3 (ovarian cancer) | ATCC | HTB-77 | |
| Cell line (homo-sapiens) | HEK293FT (human embryonic kidney) | Dr. Francis Rodier | RRID: CVCL_6911 | |
| Cell line (homo-sapiens) | UWB1.289+BRCA1 | Dr. Madhuri Koti | RRID: CVCL_B078 | |
| Cell line (Mus musculus) | 4T1.2 (mammary cancer) | ATCC | CRL-2539 | |
| Cell line (Mus musculus) | SM1 LWT1 (metastatic melanoma) | Dr. Mark Smyth | ||
| Cell line (Mus musculus) | MEF from C57BL/6 J | This paper | Generated in Dr. Stagg’s laboratory | |
| Cell line (Mus musculus) | MEF from Nt5e-/- C57BL/6 J | This paper | Generated in Dr. Stagg’s laboratory | |
| Strain, strain background (mouse) | NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) | Jackson Laboratory | JAX: 007799 | |
| Strain, strain background (mouse) | C57BL/6 J | Jackson Laboratory | JAX: 000664 | |
| Strain, strain background (mouse) | B6.129S1-Nt5etm1Lft/J (CD73-/- C57BL/6 J) | Dr. Linda Thompson | JAX: 018986 | |
| Sequence-based reagent | Nt5e | Thermo Fisher Scientific | Cat#4331182, Mm00501910_m1 | |
| Sequence-based reagent | Actb | Thermo Fisher Scientific | Cat#4331182, Mm00607939_s1 | |
| Sequence-based reagent | NMRK1 | Thermo Fisher Scientific | Cat#4331182, Hs00944470_m1 | |
| Sequence-based reagent | ACTB | Thermo Fisher Scientific | Cat#4331182, Hs99999903_m1 | |
| Sequence-based reagent | ADORA2A | Thermo Fisher Scientific | Cat#4331182, Hs00169123_m1 | |
| Sequence-based reagent | ADORA2B | Thermo Fisher Scientific | Cat#4331182, Hs00386497_m1 | |
| Recombinant DNA reagent | pLenti-PGK-ER- KRAS G12V (plasmid) | Dr. Francis Rodier | Rodier et al., 2009; Addgene#35635 | |
| Recombinant DNA reagent | pLHCX (plasmid) | Dr. Lucas Sullivan | Sullivan et al., 2018 | |
| Recombinant DNA reagent | pLHCX-gpASNase1 | Dr. Lucas Sullivan | Sullivan et al., 2018; Addgene#121526 | |
| Recombinant DNA reagent | shGFP | Sigma-Aldrich | TRCN0000075219 | |
| Recombinant DNA reagent | shNRK1 | Sigma-Aldrich | TRCN0000160469 | |
| Other | DMEM | Wisent | Cell culture reagent | |
| Other | Glucose/glutamine/sodium pyruvate-free DMEM | Wisent | Cell culture reagent | |
| Other | RPMI 1640 | Wisent | Cell culture reagent | |
| Other | OSE | Wisent | Cell culture reagent | |
| Other | Dialyzed FBS | Wisent | Cell culture reagent | |
| Other | Seahorse base media | Agilent | Cell culture reagent | |
| Other | Glutamax | Gibco | Cat#35050–061 | Cell culture reagent |
| Other | D-(+)-glucose (cell culture grade) | Sigma-Aldrich | Cat#G7021, CAS: 50-99-7 | Cell culture reagent |
| Other | L-glutamine (cell culture grade) | Sigma-Aldrich | Cat#G8540, CAS: 56-85-9 | Cell culture reagent |
| Software, algorithm | FlowJo (10.8.0) | FlowJo | RRID:SCR_008520 | |
| Software, algorithm | GraphPad Prism 9 (9.2.0) | GraphPad | RRID:SCR_002798 | |
| Software, algorithm | StepOne (2.3) | Thermo Fisher Scientific | RRID:SCR_014281 | |
| Software, algorithm | Incucyte base analysis software | Sartorius | RRID:SCR_019874 | |
| Software, algorithm | Seahorse Wave software | Agilent | RRID:SCR_014526 | |
| Software, algorithm | Image Lab Software (6.0.1) | Bio-Rad | RRID:SCR_014210 | |
| Software, algorithm | Visiomorph software | B&B microscopes | http://www.bbmicro.com/products/product.php?pid=204 |
