1. Cancer Biology

Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

  1. Caroline Contat
  2. Pierre-Benoit Ancey
  3. Nadine Zangger
  4. Silvia Sabatino
  5. Justine Pascual
  6. Stéphane Escrig
  7. Louise Jensen
  8. Christine Goepfert
  9. Bernard Lanz
  10. Mario Lepore
  11. Rolf Gruetter
  12. Anouk Rossier
  13. Sabina Berezowska
  14. Christina Neppl
  15. Inti Zlobec
  16. Stéphanie Clerc-Rosset
  17. Graham William Knott
  18. Jeffrey C Rathmell
  19. E Dale Abel
  20. Anders Meibom
  21. Etienne Meylan  Is a corresponding author
  1. Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Switzerland
  2. Swiss Cancer Center Léman, Switzerland
  3. Bioinformatics Core Facility, Swiss Institute of Bioinformatics, Switzerland
  4. Laboratory for Biological Geochemistry, School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne, Switzerland
  5. Institute of Animal Pathology (COMPATH), University of Bern, CH-3012 Bern, and Histology Core Facility, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Switzerland
  6. Laboratory for Functional and Metabolic Imaging (LIFMET), Ecole Polytechnique Fédérale de Lausanne, Switzerland
  7. Center for Biomedical Imaging (CIBM), Ecole Polytechnique Fédérale de Lausanne, Switzerland
  8. Institute of Pathology, University of Bern, Switzerland
  9. BioEM Facility, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Switzerland
  10. Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, United States
  11. Fraternal Order of Eagles Diabetes Research Center and Division of Endocrinology and Metabolism, Carver College of Medicine, University of Iowa, United States
  12. Center for Advanced Surface Analysis, Faculty of Geosciences and Environment, University of Lausanne, Switzerland
https://doi.org/10.7554/eLife.53618
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Cite this article
  1. Caroline Contat
  2. Pierre-Benoit Ancey
  3. Nadine Zangger
  4. Silvia Sabatino
  5. Justine Pascual
  6. Stéphane Escrig
  7. Louise Jensen
  8. Christine Goepfert
  9. Bernard Lanz
  10. Mario Lepore
  11. Rolf Gruetter
  12. Anouk Rossier
  13. Sabina Berezowska
  14. Christina Neppl
  15. Inti Zlobec
  16. Stéphanie Clerc-Rosset
  17. Graham William Knott
  18. Jeffrey C Rathmell
  19. E Dale Abel
  20. Anders Meibom
  21. Etienne Meylan
(2020)
Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth
eLife 9:e53618.
https://doi.org/10.7554/eLife.53618
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11 figures, 4 tables and 1 additional file

Figures

Figure 1 with 2 supplements
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Glut1 deletion does not impact disease progression in KrasLSL-G12D/WT; Trp53Flox/Flox mice.

(a) Gene expression level (RSEM) of glucose transporters in TCGA-LUAD samples (n = 511). (b) Immunohistochemistry (IHC) from a next-generation tissue microarray of human LUAD and LUSC showing score …

Figure 1—source data 1

Source files for tumor growth, grades and survival of KP and KPG1 mice.

https://cdn.elifesciences.org/articles/53618/elife-53618-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
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Non-negative matrix factorization generates four LUAD subtypes, with NMF4 being the closest to KP tumors.

(a) Kaplan-Meier survival analyses of glucose transporters in TCGA-LUAD (n = 448). Samples are split by median expression into high and low groups. p-values were computed with Wald test. (b) Heatmap …

Figure 1—figure supplement 2
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Glut1 is detectable in tumors and the bronchiolar epithelium but not in the alveolar compartment.

(a) (upper panels) Representative examples of Glut1 staining by immunohistochemistry (IHC) in KP tumors showing weak, intermediate or strong expression. Scale bars: 100 μm. (lower panel) IHC …

Figure 1—figure supplement 2—source data 1

Source files for Glut1 protein expression analysis by KP tumor grade.

https://cdn.elifesciences.org/articles/53618/elife-53618-fig1-figsupp2-data1-v1.xlsx
Figure 2
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Lamellar body-like organelles are produced by tumor cells and accumulate most glucose-derived biomass.

(a) Experimental setting. (b) Representative examples of a scanning electron microscopy (SEM) images (left), NanoSIMS isotopic image (middle) and their superposition (right) obtained from a KP …

Figure 3 with 3 supplements
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Glut1-deficient tumor analyses suggest Glut3- or PPARα-dependent compensatory mechanisms.

(a) (left) Tumor-derived single cell preparations were placed in a Seahorse XF analyzer and subjected to longitudinal extracellular acidification rate (ECAR) measurements. Glucose or 2-deoxyglucose …

Figure 3—source data 1

Source files for Seahorse analysis of KP and KPG1 tumors.

https://cdn.elifesciences.org/articles/53618/elife-53618-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
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Glut1 deletion compromises ex vivo glycolytic metabolism.

(a) Freshly-isolated tumor-derived cells were placed in a Seahorse XF analyzer and subjected to longitudinal extracellular acidification rate (ECAR) measurements. Glucose or 2-deoxyglucose (2-DG) …

Figure 3—figure supplement 1—source data 1

Source files for Seahorse analysis of KP and KPG1 tumors.

https://cdn.elifesciences.org/articles/53618/elife-53618-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
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Hallmark ‘bile acid metabolism’ is upregulated in Glut1-deficient tumors.

(a) (left panels) Top 20 Hallmark pathways enriched in genes induced in KPG1 versus KP in SPC-Cre (top left), CC10-Cre (middle left) and PGK-Cre (bottom left). Bar length shows the normalized …

Figure 3—figure supplement 3
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PPARαΔ13 does not interfere with tumor development of KP and KPG1 mice.

(a) Mouse models and viral vector used for in vivo PPARαΔ13 induction in tumor cells. (b) Western blot from transfected 293T cells’ extracts validating the doxycycline-mediated PPARαΔ13 induction in …

Figure 3—figure supplement 3—source data 1

Source files for tumor growth of KP and KPG1 mice, with or without PPARαΔ13 induction.

https://cdn.elifesciences.org/articles/53618/elife-53618-fig3-figsupp3-data1-v1.xlsx
Figure 4 with 4 supplements
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Combined Glut1 and Glut3 deletion impairs lung tumor growth.

(a) Representative examples of H&E, Glut1 or Glut3 staining by IHC from serial sections of a KP tumor showing Glut1-3 co-localization. Scale bars: 200 μm. (b) Histogram with mean ± s.d. shows the …

Figure 4—source data 1

Source files for tumor growth, grades and survival of KP and KPG1G3 mice.

https://cdn.elifesciences.org/articles/53618/elife-53618-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
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Glut1 and Glut3 co-localize frequently within tumors.

(a) Real time PCR for Slc2a3 on KP (n = 19), KPG1 (n = 32), KL (n = 12) and KLG1 (n = 11) tumors. ns: not significant by Mann-Whitney test; ****: p < 0.0001 by Mann-Whitney test. (b) Representative …

Figure 4—figure supplement 2
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Glut3 deletion alone does not affect lung tumor development.

(a) Graph with mean ± s.e.m. shows the fold changes of KP and KPG3 tumor volumes (n = 7, respectively) monitored during 56 days by μCT, starting at 16 weeks post-tumor initiation with tumor volumes …

Figure 4—figure supplement 2—source data 1

Source files for tumor growth, Seahorse analysis, survival and grades of KP and KPG3.

https://cdn.elifesciences.org/articles/53618/elife-53618-fig4-figsupp2-data1-v1.xlsx
Figure 4—figure supplement 3
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Dual knockdown of GLUT1 and GLUT3 most strongly reduces human non-small cell lung cancer cell viability in vitro, corroborating the in vivo data.

(a) Graph with mean ± s.e.m. shows the fold changes of KP and KPG1G3 tumor volumes (n = 32 and 20, respectively) monitored during 28 days by X-rays micro-computed tomography (μCT), starting at 16 …

Figure 4—figure supplement 3—source data 1

Source files for tumor growth of KP and KPG1G3 mice.

https://cdn.elifesciences.org/articles/53618/elife-53618-fig4-figsupp3-data1-v1.xlsx
Figure 4—figure supplement 4
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KP tumors do not express differently another glucose transporter upon dual Glut1 and Glut3 deletion.

(a) Dot plots with mean ± s.d. show the relative glucose transporter mRNA expression in CD45 negative fraction from KP and KPG1G3 tumors (Slc2a1 and Slc2a3: n = 8 and 10 tumors, respectively; Slc2a2,…

Author response image 1
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All human NSCLC cell lines were mycoplasma negative.

(a) PCR showing that Calu-6, H460, A549, and SW 1573 were tested negative for mycoplasma. Black arrow indicates the positive band for mycoplasma detection.

Author response image 2
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Acute Glut1 deletion in established KPfrt tumors may decrease tumor growth and may induce Glut3 expression.

(a) Mouse model of acute Glut1 deletion. Flp-frt recombinations upon Flp-recombinase induce KrasG12D activation and Tp53 deletion, driving tumor development. Intraperitoneal injection of tamoxifen …

Author response image 3
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Glucose transporter expression in human lung squamous cell carcinomas (LUSC) and in human lung adenocarcinomas (LUAD).

Gene expression level (RSEM) of glucose transporters in TCGA-LUSC (n = 501) and in TCGA-LUAD samples (n = 515).

Author response image 4
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Survival analysis of 448 lung adenocarcinoma patients (TCGA-LUAD) and 475 lung squamous cell carcinoma patients (TCGA-LUSC).

Median glucose transporter GLUT1 expression value splits down the patient tumors into low and high GLUT1 expressers. Kaplan-Meier plots show that GLUT1 expression is associated with a poor overall …

Author response image 5
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KPG1G3 tumors proliferate significantly more than KP lesions.

(a) Dot plot with mean ± s.d shows percent of pHH3 positive cells per KP or KPG1G3 tumors (n = 4, 6 mice and n = 115, 101 tumors, respectively) at sacrifice. ****: p < 0.0001 by Mann-Whitney test.

Author response image 6
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Glut3 and Pparα expressions correlate neither in KP nor in KPG1 tumors.

(a) Representative serial KPG1 tumor-bearing lung sections stained by immunohistochemistry (IHC) for (upper panel) Glut3 and (lower panel) Pparα. Tumors analyzed for this KPG1 mouse are encircled in …

Author response image 7
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The increased KLG1 tumor growth does not impact the overall mouse survival.

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (M. musculus, male, female)KThe Jackson LaboratoryRRID:IMSR_JAX:008179KrasLSL-G12D/WT
Trp53Flox/Flox
K and P interbred at EPFL
Strain, strain background (M. musculus, male, female)PThe Jackson LaboratoryRRID:IMSR_JAX:008462KrasLSL-G12D/WT
Trp53Flox/Flox
K and P interbred at EPFL
Strain, strain background (M. musculus, male, female)KPG1Interbred at EPFLKrasLSL-G12D/WT
Trp53Flox/Flox
Slc2a1Flox/Flox
Strain, strain background (M. musculus, male, female)KPG3Interbred at EPFLKrasLSL-G12D/WT
Trp53Flox/Flox
Slc2a3Flox/Flox
Strain, strain
background (M. musculus, male, female)		KPG1G3Interbred at EPFLKrasLSL-G12D/WT
Trp53Flox/Flox
Slc2a1Flox/Flox
Slc2a3Flox/Flox
Strain, strain background (M. musculus, male, female)KPRInterbred at EPFLKrasLSL-G12D/WT
Trp53Flox/Flox
CCSP-rtTA
Strain, strain background (M. musculus, male, female)KPRG1Interbred at EPFLKrasLSL-G12D/WT
Trp53Flox/Flox
Slc2a1Flox/Flox
CCSP-rtTA
Strain, strain background (M. musculus, male, female)KLInterbred at EPFLKrasLSL-G12D/WT
Stk11Flox/Flox
Strain, strain background (M. musculus, male, female)KLG1Interbred at EPFLKrasLSL-G12D/WT
Stk11Flox/Flox
Slc2a1Flox/Flox
Cell line (Homo sapiens)A549ATCCRRID:CVCL_0023Authentication: 100%
Cell line (Homo sapiens)Calu-6ATCCRRID:CVCL_0236Authentication: 100%
Cell line (Homo sapiens)H460ATCCRRID:CVCL_0459Authentication: 100%
Cell line (Homo sapiens)SW 1573ATCCRRID:CVCL_1720Authentication: 93.3%
Transfected construct (Homo sapiens and M. musculus)siCTRInvitrogenCat# 4390843Transfected construct
Transfected construct (Homo sapiens)siSLC2A1#1ThermoFisher Scientifics12926
Cat# 4390824		Transfected construct
Transfected construct (Homo sapiens)siSLC2A1#2ThermoFisher Scientifics12927
Cat# 4390824		Transfected construct
Transfected construct (Homo sapiens)siSLC2A3#1ThermoFisher Scientifics12931
Cat# 4390824		Transfected construct
Transfected construct (Homo sapiens)siSLC2A3#2ThermoFisher Scientifics12932
Cat# 4390824		Transfected construct
AntibodyGLUT1 (Rabbit monoclonal)AbcamRRID:AB_10903230IHC (1:650)
AntibodyGLUT1 (Rabbit polyclonal)MilliporeRRID:AB_1587074IHC (ngTMA) (1:5000);
WB (1:1000)
AntibodyGLUT3 (Rabbit polyclonal)AbcamRRID:AB_732609IHC (1:200);
WB (1:1000)
AntibodyPPARα (Rabbit polyclonal)ThermoFisher ScientificRRID:AB_2165595WB (1:1000)
Antibodyβ-tubulin (Rabbit polyclonal)Santa Cruz BiotechnologyRRID:AB_2241191WB (1:1000)
Antibodyγ-tubulin (Mouse monoclonal)Sigma-AldrichRRID:AB_477584WB (1:10000)
Recombinant DNA reagentrtTA (plasmid)Produced at EPFL
Recombinant DNA reagentLenti.PGK-Cre (Viral construct)Produced at EPFL
Recombinant DNA reagentLenti.TRE-PPARαΔ13_
PGK-Cre (Viral construct)		Produced at EPFLCloned from
PPARαΔ13 plasmid (L. Michalik, UNIL, Lausanne)
Recombinant DNA reagentAde5.SPC-Cre (Viral construct)University of Iowa Viral Vector Core FacilityAde5.
SPC-Cre
Recombinant DNA reagentAde5.CC10-Cre (Viral construct)University of Iowa Viral Vector Core FacilityAde5.
CC10-Cre
Sequence-based reagentRpl30 (M. musculus)ThermoFisher ScientificMm01611464_g1
Cat# 4331182		Taqman probe
(Housekeeping gene)
Sequence-based reagentSlc2a1 (M. musculus)ThermoFisher ScientificMm00441480_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a2 (M. musculus)ThermoFisher ScientificMm00446229_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a3 (M. musculus)ThermoFisher ScientificMm00441483_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a4 (M. musculus)ThermoFisher ScientificMm00436615_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a5 (M. musculus)ThermoFisher ScientificMm00600311_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a6 (M. musculus)ThermoFisher ScientificMm00554217_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a7 (M. musculus)ThermoFisher ScientificMm01260610_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a8 (M. musculus)ThermoFisher ScientificMm00444634_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a9 (M. musculus)ThermoFisher ScientificMm00455122_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a10 (M. musculus)ThermoFisher ScientificMm01249519_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a12 (M. musculus)ThermoFisher ScientificMm02375931_s1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc2a13 (M. musculus)ThermoFisher ScientificMm01306489_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc5a2 (Sglt2) (M. musculus)ThermoFisher ScientificMm00453831_m1
Cat# 4331182		Taqman probe
Sequence-based reagentSlc50a1 (Sweet) (M. musculus)ThermoFisher ScientificMm00485707_m1
Cat# 4331182		Taqman probe
Commercial assay or kitCD45 microbeadsMiltenyi Biotec130-052-301CD45 magnetic cell sorting
Chemical compound, drugDoxycycline diet (dox)Kliba Nafag3242 diet + DOX 0.625 g kg−1625 mg kg−1
Chemical compound, drugDoxycycline (dox)Sigma-AldrichD98912 μg ml−1
Software, algorithmAnalyze 12.0PerkinElmerAnalyze 12.0Tumor volume analysis
Software, algorithmOsiriX MDPixmeoRRID:SCR_013618Tumor volume analysis
Software, algorithmQuPathQuPathRRID:SCR_018257
DOI: https://doi.org/10.1038/s41598-017-17204-5
Table 1
References of the siRNAs used for in vitro experiments.
siRNAMixes ofSpeciesSupplierReferences
siCTRsiCTRHuman and MouseInvitrogenCat# 4390843
siSLC2A1siSLC2A1#1HumanThermoFisher Scientifics12926
Cat# 4390824
siSLC2A1#2HumanThermoFisher Scientifics12927
Cat# 4390824
siCTRHuman and MouseInvitrogen4390843
siSLC2A3siSLC2A3#1HumanThermoFisher Scientifics12931
Cat# 4390824
siSLC2A3#2HumanThermoFisher Scientifics12932
Cat# 4390824
siCTRHuman and MouseInvitrogenCat# 4390843
siSLC2A1
+ siSLC2A3		siSLC2A1#1HumanThermoFisher Scientifics12926
Cat# 4390824
siSLC2A1#2HumanThermoFisher Scientifics12927
Cat# 4390824
siSLC2A3#1HumanThermoFisher Scientifics12931
Cat# 4390824
siSLC2A3#2HumanThermoFisher Scientifics12932
Cat# 4390824
Author response table 1
Detailed conclusions of the cell line authentication.
Sample nameOrganismConclusion
A549HumanAccording to our analysis of the submitted sample there is no detectable contamination with human origin.
The analyzed data of the submitted sample match 100% to the DNA profile of the cell line A549 (ATCC CRM-CCL-185TM) and 100% over all 15 autosomal STRs to Microsynth’s reference DNA profile of A549 (Mic_150733).
Calu-6HumanAccording to our analysis of the submitted sample there is no detectable contamination with human origin.
The analyzed data of the submitted sample match 100% to the DNA profile of the cell line Calu-6 (ATCC HTB-56TM) and 100% over all 15 autosomal STRs to the DNA profile of Calu-6 (Cellosaurus, RRID:CVCL_0236).
H460HumanAccording to our analysis of the submitted sample there is no detectable contamination with human origin.
The analyzed data of the submitted sample match 100% to the DNA profile of the cell line NCI-H460 [H460] (ATCC HTB-177TM) and 100% over all 15 autosomal STRs to the DNA profile of NCI-H460 (Cellosaurus, RRID:CVCL_0459).
SW 1573HumanAccording to our analysis of the submitted sample there is no detectable contamination with human origin.
The analyzed data of the submitted sample match 93.3% to the DNA profile of the cell line SW 1573 [SW-1573, SW1573] (ATCC CRL-2170TM) and 93.1% over all 15 autosomal STRs to the DNA profile of SW 1573 (Cellosaurus, RRID:CVCL_1720).
Cell line samples matching at ≥ 80% of alleles across the eight core reference loci are said to be related.
Author response table 2
References of the siRNAs used for in vitro experiments.
siRNAMixes ofSpeciesSupplierReferences
siCTRsiCTRHuman and MouseInvitrogen4390843
siGLUT1siGLUT1#1HumanThermoFisher Scientifics12926
siGLUT1#2HumanThermoFisher Scientifics12927
siCTRHuman and MouseInvitrogen4390843
siGLUT3siGLUT3#1HumanThermoFisher Scientifics12931
siGLUT3#2HumanThermoFisher Scientifics12932
siCTRHuman and MouseInvitrogen4390843
siGLUT1 + siGLUT3siGLUT1#1HumanThermoFisher Scientifics12926
siGLUT1#2HumanThermoFisher Scientifics12927
siGLUT3#1HumanThermoFisher Scientifics12931
siGLUT3#2HumanThermoFisher Scientifics12932

Additional files

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https://cdn.elifesciences.org/articles/53618/elife-53618-transrepform-v1.docx

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