GPIHBP1 expression in gliomas promotes utilization of lipoprotein-derived nutrients

  1. Xuchen Hu
  2. Ken Matsumoto
  3. Rachel S Jung
  4. Thomas A Weston
  5. Patrick J Heizer
  6. Cuiwen He
  7. Norma P Sandoval
  8. Christopher M Allan
  9. Yiping Tu
  10. Harry V Vinters
  11. Linda M Liau
  12. Rochelle M Ellison
  13. Jazmin E Morales
  14. Lynn J Baufeld
  15. Nicholas A Bayley
  16. Liqun He
  17. Christer Betsholtz
  18. Anne P Beigneux
  19. David A Nathanson
  20. Holger Gerhardt
  21. Stephen G Young  Is a corresponding author
  22. Loren G Fong  Is a corresponding author
  23. Haibo Jiang  Is a corresponding author
  1. University of California, Los Angeles, United States
  2. VIB-KU Leuven Center for Cancer Biology (CCB), Belgium
  3. Uppsala University, Sweden
  4. Karolinska Institutet, Sweden
  5. Max Delbrück Center for Molecular Medicine, Germany
  6. University of Western Australia, Australia
9 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
GPIHBP1 expression in the endothelial cells of several human gliomas.

Immunohistochemical studies on surgically resected gliomas (Gliomas 1, 5, 9; Table 1) and non-diseased human frontal lobe (n = 3), revealing GPIHBP1 expression in capillaries of gliomas but not in …

https://doi.org/10.7554/eLife.47178.002
Figure 1—figure supplement 1
Detecting GPIHBP1 in glioma capillaries with immunoperoxidase staining.

Immunohistochemical studies on glioma sample 5 revealed GPIHBP1 in capillaries (detected by mAb RF4; left). In the control panel (right), the section was handled in the same fashion, except that the …

https://doi.org/10.7554/eLife.47178.003
Detecting GPIHBP1 in capillaries of human glioma specimens with three different monoclonal antibodies (mAbs) against GPIHBP1.

(A) Confocal fluorescence microscopy studies on sections from glioma sample 1 (Table 1), demonstrating the detection of GPIHBP1 with three different human GPIHBP1–specific monoclonal antibodies …

https://doi.org/10.7554/eLife.47178.004
Figure 3 with 3 supplements
GPIHBP1 is expressed by capillary endothelial cells in mouse gliomas.

Confocal microscopy images of a BFP-tagged CT-2A glioma implanted in a ROSAmT/mG::Pdgfb-iCreERT2 mouse, revealing the expression of GPIHBP1 in capillary endothelial cells of the glioma but not those …

https://doi.org/10.7554/eLife.47178.006
Figure 3—figure supplement 1
GPIHBP1 is expressed in the capillaries of mouse glioma but not normal brain.

Confocal immunofluorescence imaging of a BFP-tagged CT-2A glioma and adjacent normal brain in a ROSAmT/mG::Pdgfb-iCreERT2 mouse, revealing GPIHBP1 in the capillary endothelial cells of the glioma …

https://doi.org/10.7554/eLife.47178.007
Figure 3—figure supplement 2
The morphology of glioma capillaries differs from that of capillaries in normal brain, as revealed by transmission electron microscopy (TEM).

A four-month-old C57BL/6 mouse harboring a CT-2A glioma was euthanized and perfusion-fixed with carbodiimide/glutaraldehyde before sections of glioma and brain were processed for TEM. Electron …

https://doi.org/10.7554/eLife.47178.008
Figure 3—figure supplement 3
Vascular endothelial growth factor (VEGF) increases Gpihbp1 transcript levels in the mouse brain microvascular endothelial cell line bEnd.3.

The endothelial cells were cultured in medium in the presence or absence of mouse VEGF (100 ng/ml) for 24 hr. (A) Transcript levels for Gpihbp1, Cd31 (an endothelial cell gene), and three genes …

https://doi.org/10.7554/eLife.47178.009
Figure 4 with 4 supplements
Expression of GPIHBP1 and GLUT1 in the endothelial cells of mouse gliomas.

Immunohistochemical studies of a BFP-expressing CT-2A glioma (after three weeks of growth). Mice were injected via the tail vein with an Alexa Fluor 647–labeled antibody against mouse GPIHBP1 (11A12;…

https://doi.org/10.7554/eLife.47178.010
Figure 4—figure supplement 1
GPIHBP1 and GLUT1 expression in glioma capillaries.

Immunohistochemical studies on a CT-2A glioma collected from a mouse that had been given an intravenous injection of a GPIHBP1-specific antibody. Tissue sections (200-μm-thick) were fixed with 2% …

https://doi.org/10.7554/eLife.47178.011
Figure 4—figure supplement 2
GPIHBP1 and GLUT1 in glioma capillaries.

Immunohistochemical studies on mouse glioma and normal brain, revealing GPIHBP1 in gliomas but not normal brain. Tissue sections (10-μm-thick) were fixed with 3% PFA in PBS and stained with …

https://doi.org/10.7554/eLife.47178.012
Figure 4—figure supplement 3
Single-cell RNA-seq observations on normal mouse brain and mouse gliomas.

RNA expression data for normal mouse brain vascular cells and mouse glioma vascular cells reveal high levels of Gpihbp1 expression in the endothelial cells of gliomas but not those of normal brain. G…

https://doi.org/10.7554/eLife.47178.013
Figure 4—figure supplement 4
GLUT1 is detectable in the endothelial cells of gliomas and normal brain in wild-type (Gpihbp1+/+) and Gpihbp1–/– mice.

Immunohistochemical studies of BFP-expressing CT-2A gliomas (after three weeks of growth) in Gpihbp1+/+ and Gpihbp1–/– mice. Mice were perfused with PBS and perfusion-fixed with 2% PFA. Glioma and …

https://doi.org/10.7554/eLife.47178.014
Figure 5 with 8 supplements
Lipoprotein lipase (LPL) colocalizes with GPIHBP1 in glioma capillaries.

Confocal immunofluorescence microscopy studies on glioma and normal brain from wild-type and Gpihbp1–/– mice, along with the brain from an Lpl–/– mouse carrying a skeletal muscle–specific human LPL …

https://doi.org/10.7554/eLife.47178.015
Figure 5—figure supplement 1
Large numbers of macrophages in mouse gliomas.

Confocal immunofluorescence microscopy studies on a mouse glioma (10-μm section), revealing large numbers of macrophages (detected with an antibody against F4/80 [red]) in the glioma. Endothelial …

https://doi.org/10.7554/eLife.47178.016
Figure 5—figure supplement 2
LPL is expressed in peritoneal macrophages from wild-type mice but not in macrophages from Lpl–/–MCK-hLPL mice, as revealed by confocal immunofluorescence microscopy.

Peritoneal macrophages were harvested and plated on poly-D-lysine–coated glass coverslips and then fixed with 3% PFA. Using a goat antibody against mouse LPL, LPL (red) was detected in macrophages …

https://doi.org/10.7554/eLife.47178.017
Figure 5—figure supplement 3
LPL is present in the macrophages of brain and gliomas, as revealed by confocal immunofluorescence microscopy.

Brain and glioma from wild-type mice and brain from Lpl–/–MCK-hLPL (MCK) mice were harvested, sectioned (10-μm), and fixed with 3% PFA. LPL (detected with a goat antibody against mouse LPL [red]) …

https://doi.org/10.7554/eLife.47178.018
Figure 5—figure supplement 4
Heat map showing genes related to fatty acid metabolism that are upregulated in human gliomas, compared to normal brain.

Rows in the heat map correspond to individual genes; columns correspond to individual specimens, normal brain specimens on the left (GTEx; green) and tumors on the right (TCGA; purple). LPL was the …

https://doi.org/10.7554/eLife.47178.019
Figure 5—figure supplement 5
LPL colocalizes with GPIHBP1 in glioma capillaries, as revealed by confocal immunofluorescence microscopy.

Sections (10-μm) of brain and glioma tissue from wild-type and Gpihbp1–/– mice and brain tissue from Lpl–/–MCK-hLPL mice (MCK) were fixed with 3% PFA and stained with a goat antibody against mouse …

https://doi.org/10.7554/eLife.47178.020
Figure 5—figure supplement 6
Mouse LPL is absent from tissues of an Lpl–/–MCK-hLPL mouse, as revealed by confocal immunofluorescence microscopy.

Sections (10-μm) of heart and brain from Lpl–/–MCK-hLPL mice and of heart tissue from wild-type mice were fixed with 3% PFA and stained with a goat antibody against mouse LPL, a mAb against GPIHBP1 …

https://doi.org/10.7554/eLife.47178.021
Figure 5—figure supplement 7
LPL is present in the hippocampal neurons of wild-type mice, as revealed by confocal immunofluorescence microscopy.

Sections (10-μm) of brain specimens from wild-type and Lpl–/–MCK-hLPL mice were fixed with 3% PFA and stained with antibodies against LPL (red) and CD31 (green). The left panel shows a tiled …

https://doi.org/10.7554/eLife.47178.022
Figure 5—figure supplement 8
Mouse Gpihbp1, mouse Lpl, and human LPL transcript levels in 25-week-old wild-type and Lpl–/–MCK-hLPL mice (MCK-hLPL).

Mouse Gpihbp1 (A), mouse Lpl (B), and human LPL (C) transcript levels, as measured by qRT-PCR (n = 3/group). Expression levels were normalized to the expression of cyclophilin A. BAT, brown adipose …

https://doi.org/10.7554/eLife.47178.023
NanoSIMS imaging reveals margination of [2H]TRLs along glioma capillaries and 2H enrichment in adjacent glioma cells.

Four-month-old C57BL/6 mice harboring CT-2A gliomas were fasted for 4 hr and then injected intravenously with 200 μl of [2H]TRLs. After 1 min, mice were euthanized and perfusion-fixed with …

https://doi.org/10.7554/eLife.47178.024
Figure 7 with 2 supplements
NanoSIMS imaging showing 2H enrichment in gliomas 30 min after an intravenous injection of [2H]TRLs.

Four-month-old C57BL/6 mice harboring CT-2A gliomas were fasted for 4 hr and then injected intravenously with 200 μl of [2H]TRLs. After 30 min, mice were euthanized and perfusion-fixed with …

https://doi.org/10.7554/eLife.47178.025
Figure 7—figure supplement 1
Absence of 2H enrichment in glioma and brain of a mouse that had been given an injection of PBS alone.

Four-month-old C57BL/6 mice harboring CT-2A gliomas were fasted for 4 hr and then injected intravenously with 200 μl of PBS. After 1 min, the mouse was euthanized and perfusion-fixed with …

https://doi.org/10.7554/eLife.47178.026
Figure 7—figure supplement 2
Uptake of [2H]TRLs in heart and brain of Gpihbp1–/– mice.

A C57BL/6 wild-type mouse (Gpihbp1+/+) and a Gpihbp1-deficient mouse (Gpihbp1–/–) were injected intravenously with 80 μl of [2H]TRLs. After 15 min, the mouse was euthanized; perfusion-fixed with …

https://doi.org/10.7554/eLife.47178.027
Figure 8 with 4 supplements
Tissue uptake of fatty acid and glucose-derived nutrients by mice harboring CT-2A gliomas.

(A) NanoSIMS images showing 13C enrichment in mouse tissues (brain, glioma, and heart) after oral administration of 13C-labeled mixed fatty acids to mice (three 80-mg doses administered 12 h apart). …

https://doi.org/10.7554/eLife.47178.028
Figure 8—figure supplement 1
13C-enriched lipid droplets in mouse glioma cells.

13C-labeled mixed fatty acids were administered by gastric gavage to C57BL/6 mice harboring CT-2A gliomas (three doses of 80 mg administered 12 hr apart). Four hours after the last dose, the mice …

https://doi.org/10.7554/eLife.47178.029
Figure 8—figure supplement 2
NanoSIMS imaging showing 13C enrichment in capillary endothelial cells of normal brain after administering 13C-labeled mixed fatty acids by oral gavage (three doses of 80 mg administered 12 hr apart).

Four hours after the last dose, mice were euthanized; perfusion-fixed with carbodiimide/glutaraldehyde; and sections of normal brain were prepared for NanoSIMS imaging. 12C14N images were generated …

https://doi.org/10.7554/eLife.47178.030
Figure 8—figure supplement 3
Absence of 13C enrichment in the glioma and brain of a mouse that had been given an injection of PBS alone.

Four-month-old C57BL/6 mice harboring CT-2A gliomas were fasted for 4 hr and then injected intravenously with 200 μl of PBS. After 1 min, the mouse was euthanized; perfusion-fixed with …

https://doi.org/10.7554/eLife.47178.031
Figure 8—figure supplement 4
Glioma studies in Gpihbp1+/+ and Gpihbp1–/– mice.

CT-2A glioma cells that were stably transfected with a Gaussia luciferase reporter were injected intracranially into Gpihbp1+/+ (blue) and Gpihbp1–/– (red) mice (n = 11/group). Tumor growth in live …

https://doi.org/10.7554/eLife.47178.032
Intravascular lipolysis as a source of lipid nutrients for gliomas.

In normal brain (left panel), LPL is produced by astrocytes, neurons, oligodendrocytes, and fibroblasts. Because GPIHBP1 is not expressed in the capillaries of the brain parenchyma, we have proposed …

https://doi.org/10.7554/eLife.47178.033

Tables

Table 1
Human glioma tumor specimens.

Expression of GPIHBP1 was assessed by immunohistochemistry with mAbs against human GPIHBP1 (RF4, RE3, RG3). Those conducting the studies were blinded to diagnoses. This table details the tumor …

https://doi.org/10.7554/eLife.47178.005
Sample IDTissue diagnosisLocation1p/19q co-deletionIDH1 mutationGPIHBP1
1Glioblastoma (GBM)Right frontal, parietalNoNegativeYes
2GBMLeft temporalNoNegativeYes
3GBMRight occipitalNoNegativeYes
4GBMLeft frontalNoNegativeYes
5Oligodendroglioma Grade IILeft anterior temporal, left posterior temporalYesNegativeYes
6Oligoastrocytoma Grade IIIRight temporalNoNegativeYes
7GBM + oligodendroglial componentLeft frontalYesNegativeYes
8GBM + extensive oligodendroglial componentRight frontalNoNegativeYes
9Oligodendroglioma Grade IIILeft frontalYes+R132HYes
10Oligodendroglioma Grade IIILeft frontalYes+R132HYes
11OligoastrocytomaRight parietalNoNegativeYes
12Oligodendroglioma Grade IIIRight parietalYes+R132HYes
13Oligodendroglioma Grade IIIRight parietalYesNegativeYes
14Oligoastrocytoma Grade IIILeft temporalNo+R132HYes
15Oligoastrocytoma Grade IIIRight temporalNo+R132GNo
16Oligoastrocytoma Grade IIIRight frontalNo+R132HNo
17Oligodendroglioma Grade IIILeft frontalYesNegativeNo
18Oligodendroglioma Grade IIILeft frontalYes+R132HNo
19Oligodendroglioma Grade IIILeft temporalYesNegativeNo
20Oligodendroglioma Grade IIIRight temporalYes+R132HNo
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (M. musculus)Gpihbp1–/–Beigneux et al., 2007RRID: MGI:3771172Dr. Stephen G Young (UCLA)
Genetic reagent (M. musculus)Lpl–/–MCK-hLPLLevak-Frank et al., 1995RRID: MGI:3624988Dr. Rudolph Zechner (Graz University)
Genetic reagent (M. musculus)ROSAmT/mGPdgfb-iCreT2Mathivet et al., 2017Dr. Holger Gerhardt (VIB KU-Leuven)
Cell line (M. musculus)CT-2ASeyfried et al., 1992Dr. Thomas Seyfried (Boston College)
Cell line (M. musculus)CT-2A–BFPPMID: 24658686Dr. Holger Gerhardt (VIB KU-Leuven)
Cell line (M. musculus)bEnd.3ATCCCatalog No. CRL-2299
RRID: CVCL_0170
Transfected construct (lentiviral plasmid)plenti-GLuc-IRES-EGFPTargeting SystemsCatalog No. GL-GFP
AntibodyRat monoclonal anti-mouse GPIHBP1 (11A12)Beigneux et al., 2009Dr. Stephen G Young (UCLA); IHC (10 μg/ml)
AntibodyMouse monoclonal anti-human
GPIHBP1 (RE3)
Hu et al., 2017Dr. Stephen G Young (UCLA); IHC (10 μg/ml)
AntibodyMouse monoclonal anti-human GPIHBP1 (RF4)Hu et al., 2017Dr. Stephen G Young (UCLA); IHC (10 μg/ml)
AntibodyMouse monoclonal anti-human GPIHBP1 (RG3)Hu et al., 2017Dr. Stephen G Young (UCLA); IHC (10 μg/ml)
AntibodyRabbit polyclonal anti-vWFDakoCatalog No. A0082
RRID: AB_2315602
IHC (1:200)
AntibodyGoat polyclonal anti-GFAPAbcamCatalog No. ab53554
RRID: AB_880202
IHC (1:200)
AntibodyRabbit polyclonal anti-GLUT1Millipore-SigmaCatalog No. 07–1401
RRID: AB_1587074
IHC (1:200)
AntibodyRabbit polyclonal anti-CD31AbcamCatalog No. ab28364
RRID: AB_726362
IHC (1:50)
AntibodyRat monoclonal anti-F4/80AbcamCatalog No. ab6640
RRID: AB_1140040
IHC (10 μg/ml)
AntibodyGoat polyclonal anti-mouse LPLPage et al., 2006Dr. André Bensadoun (Cornell); IHC (12 μg/ml)
AntibodyAlexa Fluor 488, 568, 647 secondariesThermoFisher ScientificIHC (1:500)
Commercial assay or kitImmPRESS Excel Staining KitVector LaboratoryCatalog No. MP-7602
Sequence-based reagentMouse Gpihbp1 primers5′-AGCAGGGACAGAGCACCTCT-3′ and 5′-AGACGAGCGTGATGCAGAAG-3′
Sequence-based reagentMouse Cd31 primers5′-AACCGTATCTCCAAAGCCAGT-3′ and 5′-CCAGACGACTGGAGGAGAACT-3′
Sequence-based reagentMouse Angpt2 primers5′-AACTCGCTCCTTCAGAAGCAGC-3′ and 5′-TTCCGCACAGTCTCTGAAGGTG-3′
Sequence-based reagentMouse Dusp5 primers5′-TCGCCTACAGACCAGCCTATGA-3′ and 5′-TGATGTGCAGGTTGGCGAGGAA-3′
Sequence-based reagentMouse Cxcr4 primers5′-GACTGGCATAGTCGGCAATGGA-3′ and 5′-CAAAGAGGAGGTCAGCCACTGA-3′
Sequence-based reagentMouse Lpl primers5′-AGGTGGACATCGGAGAACTG-3′ and 5′-TCCCTAGCACAGAAGATGACC-3′
Sequence-based reagentHuman LPL primers5′-TAGCTGGTCAGACTGGTGGA-3′ and 5′-TTCACAAATACCGCAGGTG-3′
Recombinant DNA reagentALO-D4 plasmidGay et al., 2015Dr. Arun Radhakrishnan (UT Southwestern)
Chemical compound, drugN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (carbodiimide)Millipore-SigmaCatalog No. 03449
Chemical compound, drugGlutaraldehyde25% solutionElectron Microscopy SciencesCatalog No. 16220
Chemical compound, drugOsmium tetroxide 4% solutionElectron Microscopy SciencesCatalog No. 18459
Chemical compound, drugParaformaldehyde 16% solutionElectron Microscopy SciencesCatalog No. 15170
Chemical compound, drugEMbed 812Electron Microscopy SciencesCatalog No. 14120
Chemical compound, drugSodium cacodylate trihydrateElectron Microscopy SciencesCatalog No. 12300
Chemical compound, drugUranyl acetateSPI-ChemCatalog No. 02624AB
Chemical compound, drugDAPIThermoFisher ScientificCatalog No. 1306IHC (3 μg/ml)
Chemical compound, drugMouse VEGFMillipore-SigmaCatalog No. V4512
Software, algorithmLIMMARitchie et al., 2015RRID:
SCR_010943
OtherD-GLUCOSE (U-13C6, 99%)Cambridge Isotope LaboratoriesCatalog No. CLM-1396-PK
OtherMixed fatty acids (U-D, 96–98%)Cambridge Isotope LaboratoriesCatalog No. DLM-8572-PK
OtherMixed fatty acids (13C, 98%+)Cambridge Isotope LaboratoriesCatalog No. CLM-8455-PK

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