GPIHBP1 expression in gliomas promotes utilization of lipoprotein-derived nutrients

Abstract

GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) within the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1-bound LPL is essential for the margination of triglyceride-rich lipoproteins (TRLs) along capillaries, allowing the lipolytic processing of TRLs to proceed. In peripheral tissues, the intravascular processing of TRLs by the GPIHBP1–LPL complex is crucial for the generation of lipid nutrients for adjacent parenchymal cells. GPIHBP1 is absent from the capillaries of the brain, which uses glucose for fuel; however, GPIHBP1 is expressed in the capillaries of mouse and human gliomas. Importantly, the GPIHBP1 in glioma capillaries captures locally produced LPL. We use NanoSIMS imaging to show that TRLs marginate along glioma capillaries and that there is uptake of TRL-derived lipid nutrients by surrounding glioma cells. Thus, GPIHBP1 expression in gliomas facilitates TRL processing and provides a source of lipid nutrients for glioma cells.

Introduction

GPIHBP1, a GPI-anchored protein of capillary endothelial cells, is required for lipoprotein lipase (LPL)–mediated processing of triglyceride-rich lipoproteins (TRLs) (Beigneux et al., 2007). The principal function of GPIHBP1 is to capture LPL within the interstitial spaces, where it is secreted by parenchymal cells, and then to shuttle this enzyme to the luminal surface of capillary endothelial cells (Davies et al., 2010). GPIHBP1 is a long-lived protein (Young et al., 2011; Olafsen et al., 2010) that moves bidirectionally across endothelial cells, with each trip to the abluminal plasma membrane representing an opportunity to capture LPL and bring it to the capillary lumen (Davies et al., 2012). When GPIHBP1 is absent or defective, LPL is stranded within the interstitial spaces, where it remains bound to sulfated proteoglycans near the surface of cells (Young et al., 2011; Davies et al., 2010; Allan et al., 2017a; Fong et al., 2016). The inability of LPL to reach the capillary lumen in the absence of GPIHBP1 expression profoundly impairs TRL processing, resulting in severe hypertriglyceridemia (chylomicronemia) (Beigneux et al., 2007; Davies et al., 2010; Goulbourne et al., 2014).

GPIHBP1 is expressed in the capillary endothelial cells of peripheral tissues, with particularly high levels of expression in heart and brown adipose tissue (Beigneux et al., 2007; Davies et al., 2010; Fong et al., 2016). Most of the LPL within those tissues is bound to GPIHBP1 on capillaries (Beigneux et al., 2007; Davies et al., 2010; Davies et al., 2012; Allan et al., 2017a; Fong et al., 2016; Allan et al., 2017b; Allan et al., 2016), and the processing of TRLs in these tissues is robust, generating fatty acid nutrients for nearby parenchymal cells (Fong et al., 2016; Jiang et al., 2014a; He et al., 2018a). By contrast, GPIHBP1 is absent from capillaries of the brain (Young et al., 2011; Davies et al., 2010; Olafsen et al., 2010), a tissue that depends on glucose for fuel (Mergenthaler et al., 2013). When wild-type mice are injected intravenously with a GPIHBP1-specific antibody, the antibody rapidly binds to GPIHBP1-expressing capillaries in peripheral tissues and disappears from the plasma (Davies et al., 2010; Olafsen et al., 2010). By contrast, there is no antibody binding to the capillaries of the brain (Davies et al., 2010; Olafsen et al., 2010).

For the lipolytic processing of TRLs to proceed, lipoproteins in the bloodstream must marginate along the luminal surface of capillaries (Goulbourne et al., 2014). TRL margination along capillaries depends on GPIHBP1, more specifically on GPIHBP1-bound LPL (Goulbourne et al., 2014). In GPIHBP1-deficient mice, TRLs never stop along heart capillaries and instead simply ‘flow on by’ in the bloodstream (Goulbourne et al., 2014). In wild-type mice, TRLs marginate along heart capillaries, but TRL margination is absent along capillaries of the brain (Goulbourne et al., 2014).

Even though GPIHBP1 is not found in brain capillaries, there is ample evidence for LPL expression within the brain (Ben-Zeev et al., 1990; Bessesen et al., 1993; Goldberg et al., 1989; Vilaró et al., 1990; Yacoub et al., 1990; Eckel and Robbins, 1984). Several groups have found LPL in the rat brain, specifically in neurons of the dentate gyrus and hippocampus, in pyramidal cells of the cortex, and in Purkinje cells of the cerebellum (Ben-Zeev et al., 1990; Bessesen et al., 1993; Goldberg et al., 1989; Vilaró et al., 1990; Eckel and Robbins, 1984). Using single-cell RNA sequencing, Zhang et al. (2014) found Lpl transcripts in the resident macrophages of the brain (microglia), with lower levels in astrocytes, neurons, and oligodendrocytes. Using the same approach, Vanlandewijck et al. (2018) found LPL expression in brain smooth muscle cells and in perivascular fibroblasts (at even higher levels than in microglial cells). Given the absence of GPIHBP1 expression in brain capillaries and the absence of TRL margination along brain capillaries, we have proposed that the LPL in the brain probably has an extravascular function, presumably to hydrolyze glycerolipids within the extracellular spaces (Young et al., 2011; Adeyo et al., 2012).

Despite the absence of GPIHBP1 expression in brain capillaries, we were curious about whether GPIHBP1 might be expressed in the capillaries of gliomas. Glioma capillaries are morphologically distinct from normal brain capillaries (Yuan et al., 1994; Hobbs et al., 1998; Monsky et al., 1999; Bullitt et al., 2005), and the blood–brain barrier is often defective (Zhang et al., 1992). Electron microscopy has suggested that glioblastoma capillaries resemble capillaries in peripheral tissues (Vaz et al., 1996).

If GPIHBP1 were to be expressed in glioma capillaries, it could be relevant to glioma metabolism. The GPIHBP1 might capture locally produced LPL, allowing for TRL margination and TRL processing, and thereby providing a source of lipid nutrients for glioma cells. Interestingly, Dong et al. (2017) documented LPL expression in gliomas. Also, several studies have raised the possibility that glioma cells use fatty acids for fuel (Lin et al., 2017; Guo et al., 2011; Guo et al., 2009a; Guo et al., 2009b; Guo et al., 2013) and that levels of free fatty acids are higher in gliomas than in normal brain tissue (Guo et al., 2013; Gopal et al., 1963).

In the current study, we sought to determine whether glioma capillaries express GPIHBP1 and, if so, whether it binds LPL and facilitates TRL margination and the lipolytic processing of TRLs. In our study, we took advantage of NanoSIMS imaging, a high-resolution mass spectrometry–based imaging modality that makes it possible to visualize TRL margination and TRL processing in tissue sections (He et al., 2018a; Jiang et al., 2014a; Jiang et al., 2014b; He et al., 2017a; He et al., 2017b; He et al., 2018b; He et al., 2018c). This imaging modality allowed us to visualize TRL margination in glioma capillaries as well as the entry of TRL-derived nutrients into tumor cells.

Results

GPIHBP1 is expressed in the endothelial cells of human gliomas

We sectioned 20 human gliomas (Table 1) and screened them for GPIHBP1 expression by confocal microscopy with three GPIHBP1-specific monoclonal antibodies (mAbs) — RF4, which binds to residues 27–44 downstream from GPIHBP1’s acidic domain (Kristensen et al., 2018); and RE3 and RG3, which both bind to GPIHBP1’s LU (Ly6/uPAR) domain (Hu et al., 2017). GPIHBP1 in capillary endothelial cells was detected in 14 of 20 gliomas (Table 1) and colocalized with von Willebrand factor, an endothelial cell marker (Figure 1). GPIHBP1 expression in glioma capillaries did not appear to correlate with glioma grade, 1p/19q co-deletions, or IDH1 mutations (Table 1). GPIHBP1 was not detectable in the capillaries of human brain specimens (Figure 1). The GPIHBP1 in glioma capillaries could be detected with all three GPIHBP1-specific mAbs (Figure 2A). To be confident in the specificity of the antibodies, we performed studies in which recombinant human GPIHBP1 was added to the GPIHBP1-specific mAbs before incubating the solution with the glioma sections. As expected, the presence of recombinant GPIHBP1 eliminated binding of the GPIHBP1-specific mAbs to glioma capillaries (Figure 2B). GPIHBP1 expression in glioma capillaries could also be detected by immunoperoxidase staining (Figure 1—figure supplement 1).

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 diagnosis, location, 1p/19q co-deletion, and IDH1 mutation status, as well as the presence of GPIHBP1.

Sample ID	Tissue diagnosis	Location	1p/19q co-deletion	IDH1 mutation	GPIHBP1
1	Glioblastoma (GBM)	Right frontal, parietal	No	Negative	Yes
2	GBM	Left temporal	No	Negative	Yes
3	GBM	Right occipital	No	Negative	Yes
4	GBM	Left frontal	No	Negative	Yes
5	Oligodendroglioma Grade II	Left anterior temporal, left posterior temporal	Yes	Negative	Yes
6	Oligoastrocytoma Grade III	Right temporal	No	Negative	Yes
7	GBM + oligodendroglial component	Left frontal	Yes	Negative	Yes
8	GBM + extensive oligodendroglial component	Right frontal	No	Negative	Yes
9	Oligodendroglioma Grade III	Left frontal	Yes	+R132H	Yes
10	Oligodendroglioma Grade III	Left frontal	Yes	+R132H	Yes
11	Oligoastrocytoma	Right parietal	No	Negative	Yes
12	Oligodendroglioma Grade III	Right parietal	Yes	+R132H	Yes
13	Oligodendroglioma Grade III	Right parietal	Yes	Negative	Yes
14	Oligoastrocytoma Grade III	Left temporal	No	+R132H	Yes
15	Oligoastrocytoma Grade III	Right temporal	No	+R132G	No
16	Oligoastrocytoma Grade III	Right frontal	No	+R132H	No
17	Oligodendroglioma Grade III	Left frontal	Yes	Negative	No
18	Oligodendroglioma Grade III	Left frontal	Yes	+R132H	No
19	Oligodendroglioma Grade III	Left temporal	Yes	Negative	No
20	Oligodendroglioma Grade III	Right temporal	Yes	+R132H	No

Figure 1. 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 frontal lobe specimens. GPIHBP1 (detected with a combination of the mAbs RE3 and RF4, 10 μg/ml each [red]) colocalized with von Willebrand factor (vWF, a marker for endothelial cells [green]), but not with glial fibrillary acidic protein (GFAP, a marker for astroglial cells [magenta]). DNA was stained with DAPI (blue). Three sections of each tumor and normal brain were evaluated and representative images are shown. Scale bar, 50 μm.

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 incubation with the primary antibody was omitted. Images were recorded with 40× and 100× objectives. The boxed region in the 40× image (upper left) is shown at higher magnification in the 100× image (lower left). Three sections of tumor were evaluated and representative images are shown. Scale bar, 20 μm.

Figure 2. 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 (mAbs). Tissue sections were fixed with 3% PFA and then stained with mAbs against human GPIHBP1 (RF4, RE3, or RG3, 10 μg/ml [red]), an antibody against von Willebrand factor (vWF[green]), and an antibody against glial fibrillary acidic protein (GFAP [magenta]). All three GPIHBP1-specific mAbs detected GPIHBP1 in capillaries, colocalizing with von Willebrand factor. DNA was stained with DAPI (blue). Scale bar, 50 μm. (B) Immunofluorescence confocal microscopy studies on human glioma sample 5, performed with mAbs RF4 and RE3 (10 μg/ml) in the presence or absence of 50 μg of recombinant soluble human GPIHBP1 (hGPIHBP1). Adding recombinant hGPIHBP1 to the antibody incubation abolished binding of the GPIHBP1-specific mAbs to GPIHBP1 on glioma capillaries. Images show GPIHBP1 (red), vWF (green), GFAP (magenta), and DAPI (blue). Three sections of tumors were evaluated; representative images are shown. Scale bar, 50 μm.

GPIHBP1 is present in the capillary endothelial cells of mouse gliomas

To determine whether GPIHBP1 is expressed in a mouse model of glioblastoma, spheroids of syngeneic C57BL/6 mouse CT-2A glioma cells (Seyfried et al., 1992; Oh et al., 2014), modified to express a blue fluorescent protein (BFP) (Mathivet et al., 2017), were engrafted into the brains of mice harboring an endothelial cell–specific Pdgfb-iCreER^T2 transgene (Claxton et al., 2008) and a ROSA^mT/mG reporter allele (Muzumdar et al., 2007). ROSA^mT/mG is a two-color fluorescent, membrane-targeted Cre-dependent reporter allele. In the absence of Cre, all cells express a membrane-localized tdTomato and fluoresce red. In the setting of Cre expression, cells express membrane-localized EGFP (rather than tdTomato) and fluoresce green. Before tumor implantation, mice were injected with tamoxifen to induce Pdgfb-driven Cre expression in endothelial cells; thus, the endothelial cells of the mice expressed EGFP and fluoresced green. Mice harboring gliomas (after three weeks of growth) were injected intravenously with an Alexa Fluor 647–conjugated antibody against mouse GPIHBP1 (11A12) (Beigneux et al., 2009). Mice were perfused with PBS and then perfusion-fixed with 2% PFA, and tumor sections were processed for confocal immunofluorescence microscopy. GPIHBP1 was detected in endothelial cells of the gliomas, colocalizing with EGFP (brain endothelial cells), but GPIHBP1 was absent from capillaries in the adjacent normal brain (Figure 3, Figure 3—figure supplement 1). Using transmission electron microscopy, we observed large and irregularly shaped capillaries in gliomas, with numerous villus-like structures on the luminal surface of endothelial cells (Figure 3—figure supplement 2), similar to findings reported for capillaries in human gliomas (Vaz et al., 1996; Coomber et al., 1987; Weller et al., 1977).

Figure 3. GPIHBP1 is expressed by capillary endothelial cells in mouse gliomas.

Confocal microscopy images of a BFP-tagged CT-2A glioma implanted in a ROSA^mT/mG::Pdgfb-iCreER^T2 mouse, revealing the expression of GPIHBP1 in capillary endothelial cells of the glioma but not those of normal brain. Tamoxifen was administered prior to implantation of the glioma spheroid to activate membrane-targeted EGFP in endothelial cells (green). After three weeks of glioma growth, mice were anesthetized and injected via the tail vein with an Alexa Fluor 647–labeled antibody against mouse GPIHBP1 (11A12; red). The mice were then perfused with PBS and perfusion-fixed with 2% PFA in PBS. Glioma and adjacent normal brain were harvested, and 200-μm-thick sections were imaged by confocal microscopy. GPIHBP1 was present on endothelial cells of the glioma (blue) but was absent from normal brain. High-magnification images of the boxed area are shown on the right. Three mice were evaluated; representative images are shown. Scale bar, 50 μm.

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 ROSA^mT/mG::Pdgfb-iCreER^T2 mouse, revealing GPIHBP1 in the capillary endothelial cells of the glioma but not those of normal brain. Before implantation of glioma spheroids, mice were treated with tamoxifen to activate membrane-targeted EGFP expression in endothelial cells (green). Non-tumor cells expressed membrane-targeted tdTomato (red). Mice were injected intravenously (via the tail vein) with an Alexa Fluor 647–labeled antibody against mouse GPIHBP1 (11A12; white), then perfused with PBS and perfusion-fixed with 2% PFA. Glioma and normal brain were harvested, sectioned (200-μm-thick), and imaged by confocal microscopy. GPIHBP1 (white) colocalized with endothelial cells (green) in glioma (blue) but not in normal brain (red). High-magnification images of the boxed areas are shown below. Three mice were evaluated; representative images are shown. Scale bar, 200 μm.

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 micrographs of the glioma revealed irregularly shaped and large capillaries with villus-like structures on the luminal surface of capillary endothelial cells. Higher magnification images of the boxed areas are shown to the right. Scale bar, 1 μm.

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 known to be upregulated by VEGF (Angpt2, Cxcr4, and Dusp5) were measured by qRT-PCR (n = 3 replicates). VEGF increased transcript levels for Angpt2, Cxcr4, Dups5, and Gpihbp1. (B) Gpihbp1:Cd31 transcript ratio in the absence of VEGF (Control) and in the presence of VEGF. Differences were assessed with a Student’s t-test. *p<0.01; **p<0.001. ns, not significant.

The factors that regulate Gpihbp1 expression in the capillary endothelial cells of peripheral tissues and gliomas are incompletely understood. However, a recent study found that Gpihbp1 transcript levels in rat aortic endothelial cells are upregulated by vascular endothelial growth factor (VEGF) (Chiu et al., 2016), an angiogenic factor known to be expressed at high levels by glioma cells (Plate et al., 1994; Pietsch et al., 1997; Christov et al., 1998). We found that Gpihbp1 expression in the mouse brain endothelial cell line bEnd.3 is upregulated by recombinant VEGF (Figure 3—figure supplement 3).

GLUT1 is expressed in the capillaries of gliomas and normal brain

We used immunofluorescence microscopy to examine the expression of GPIHBP1 and GLUT1 (the main glucose transporter in brain capillaries [Maher et al., 1994; Pardridge et al., 1990]) in mouse gliomas and adjacent normal brain. GPIHBP1 expression was detected in gliomas but was absent in the normal brain. The signal for GLUT1 was strong in the endothelial cells of the normal brain and was easily detectable in the capillaries of gliomas (Figure 4, Figure 4—figure supplements 1–2). Consistent findings were observed in single-cell RNA-seq studies on vascular cells of gliomas (Ken Matsumoto, manuscript in preparation) and normal brain vascular cells (Vanlandewijck et al., 2018; He et al., 2018d). Endothelial cells of gliomas (identifed by high von Willebrand factor [vWF] expression) exhibit high expression of Gpihbp1 and somewhat lower levels of Glut1 expression (e.g., Endothelial cell cluster 5 in Figure 4—figure supplement 3). In normal brain, Glut1 was highly expressed in endothelial cells, whereas Gpihbp1 expression was absent (Figure 4—figure supplement 3). In Gpihbp1-deficient mice, GLUT1 expression was detectable in the capillaries of gliomas and normal brain (Figure 4—figure supplement 4).

Figure 4. 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; green), then perfused with PBS and perfusion-fixed with 2% PFA. Glioma and adjacent normal brain tissue were harvested, then 200-μm thick sections cut, fixed with 4% PFA, and stained with an antibody against GLUT1 (red). GPIHBP1 was present in the capillaries of mouse gliomas (blue) but absent from the capillaries of the normal brain. High-magnification images in the boxed region are shown below. Three mice were evaluated; representative images are shown. Scale bar, 50 μm.

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% PFA in PBS and then stained with antibodies against GLUT1. GPIHBP1 (green) is expressed in glioma capillaries; GLUT1 (red) expression was also detectable in glioma capillaries. Higher magnification images of the boxed regions are shown on the right. Three mice were evaluated; representative images are shown. Scale bar, 20 μm.

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 antibodies against CD31 (white), GLUT1 (red), and GPIHBP1 (green). GPIHBP1 was absent from the capillaries of normal brain but colocalized with CD31 and GLUT1 in the capillaries of gliomas. DNA was stained with DAPI (blue). Glioma and brain from three mice were used for this study; representative images are shown. Scale bar, 20 μm.

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. Glut1 was expressed in normal brain endothelial cells and in endothelial cells of gliomas. Lpl was not detected in the endothelial cells of normal brain or gliomas but was expressed in fibroblasts, microglia, and smooth muscle cells of normal brain and in macrophages of gliomas. RNA expression data for normal mouse brain vascular cells were obtained from an online database established by the Betsholtz laboratory (Vanlandewijck et al., 2018; He et al., 2018d) (http://betsholtzlab.org/VascularSingleCells/database.html). The glioma single-cell RNA-seq data were analyzed in a similar fashion, showing gene-expression levels in individual cells within each cluster.

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 adjacent normal brain tissue were harvested, then 200-μm thick sections were cut, fixed with 4% PFA, and stained with an antibody against GLUT1 (red). GLUT1 was detectable in endothelial cells in glioma and normal brain in both wild-type and Gpihbp1^–/– mice. Two mice per genotype were evaluated; representative images are shown. Scale bar, 100 μm.

LPL is present on GPIHBP1-expressing capillaries of mouse gliomas

Most of the LPL in peripheral tissues (e.g., heart or brown adipose tissue) is bound to GPIHBP1 on capillaries; consequently, LPL and GPIHBP1 colocalize in tissue sections (Young et al., 2011; Davies et al., 2010; Davies et al., 2012; Allan et al., 2017a; Fong et al., 2016; Allan et al., 2017b; Allan et al., 2016). We hypothesized that GPIHBP1-expressing endothelial cells of gliomas could capture LPL. Several observations prompted us to consider this hypothesis. First, as noted earlier, there is ample evidence for LPL expression in the brain (Ben-Zeev et al., 1990; Bessesen et al., 1993; Goldberg et al., 1989; Vilaró et al., 1990; Yacoub et al., 1990; Zhang et al., 2014), and it seemed reasonable that some of that LPL would reach high-affinity GPIHBP1-binding sites on endothelial cells. Second, gliomas contain large numbers of macrophages (F4/80-expressing cells; Figure 5—figure supplement 1), and macrophages are known to express LPL (Mahoney et al., 1982). We found that LPL could be detected in peritoneal macrophages from wild-type mice but not in macrophages harvested from Lpl^–/– mice carrying a skeletal muscle–specific human LPL transgene (Lpl^–/–MCK-hLPL) (Levak-Frank et al., 1995) (Figure 5—figure supplement 2). We also found that LPL could be detected in some of the macrophages in mouse gliomas and in normal brain of wild-type mice, but not in the brain of Lpl^–/–MCK-hLPL mice (Figure 5—figure supplement 3). These findings were consistent with single-cell RNA-seq data from glioma and normal brain, in which Lpl transcripts were found in the macrophages of gliomas and microglia of normal brain (Figure 4—figure supplement 3). Lpl transcripts are not present in capillary endothelial cells. Third, the most highly upregulated fatty acid metabolism gene in human gliomas, compared to normal brain tissue, is LPL (Figure 5—figure supplement 4). The second most perturbed gene in gliomas is CD36, which encodes a putative fatty acid transporter (Figure 5—figure supplement 4).

To determine whether LPL is bound to GPIHBP1-expressing capillaries of gliomas, we performed immunohistochemical studies, taking advantage of an affinity-purified goat antibody against mouse LPL (Page et al., 2006). These studies revealed colocalization of GPIHBP1 and LPL in glioma capillaries (Figure 5, Figure 5—figure supplement 5). LPL was not present in the capillaries of the normal brain or in the capillaries of gliomas from Gpihbp1^–/– mice (Figure 5, Figure 5—figure supplement 5). As expected, the binding of the goat LPL antibody to tissues of Lpl^–/–MCK-hLPL mice was low (Figure 5, Figure 5—figure supplement 5), whereas mouse LPL was easily detectable in the heart capillaries of wild-type mice (colocalizing with GPIHBP1) (Figure 5—figure supplement 6). Consistent with earlier publications (Ben-Zeev et al., 1990; Vilaró et al., 1990), we observed a strong mouse LPL signal in the hippocampal neurons of wild-type mice but not of Lpl^–/–MCK-hLPL mice (Figure 5—figure supplement 7). Of note, LPL was undetectable in ‘secondary antibody–only’ experiments (i.e., when the incubation of the primary antibody with tissue sections was omitted) (Figure 5, Figure 5—figure supplement 5–7).

Figure 5. 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 transgene (MCK). Glioma and brain sections (10-μm-thick) were fixed with 3% PFA and then stained with a mAb against mouse GPIHBP1 (11A12; green), a goat antibody against mouse LPL (red), and a rabbit antibody against CD31 (white). LPL colocalizes with GPIHBP1 and CD31 in the capillaries of gliomas; GPIHBP1 and LPL were absent from normal brain capillaries and from glioma capillaries in Gpihbp1^–/– mice. DNA was stained with DAPI (blue). No LPL was detected in the capillaries of Lpl-deficient mice (MCK) or when the incubation with primary antibodies was omitted (Secondary Only). Staining of all tissue sections was performed simultaneously, and all images were recorded with identical microscopy settings. Three mice per genotype were evaluated; representative images are shown. Scale bar, 50 μm.

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 cells were stained with an antibody against CD31 (green), and DNA was stained with DAPI (blue). High-magnification images of boxed regions are shown on the right. Four tumors were evaluated; representative images are shown. Scale bar, 50 μm.

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 from wild-type mice but not in macrophages from Lpl^–/–MCK-hLPL mice. ALO-D4 (a modified cytolysin that binds ‘accessible cholesterol’ [Das et al., 2014; Das et al., 2013; Gay et al., 2015]) (green) was used to visualize the macrophage plasma membrane. DNA was stained with DAPI (blue). Two mice per genotype were evaluated; representative images are shown. Scale bar, 10 μm.

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]) was present in macrophages (identified with an antibody against F4/80[green]) in brain and glioma of wild-type mice, but was absent from the brain of MCK mice. White arrows point to areas of LPL and F4/80 colocalization. Images were recorded with 20× (left) and 100× objectives (right). DNA was stained with DAPI (blue). Two mice per genotype were evaluated; representative images are shown. Scale bar, 20 μm.

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 most upregulated gene (related to fatty acid metabolism) in gliomas.

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 LPL, a mAb against GPIHBP1 (11A12), and an antibody against CD31. LPL (red) colocalized with GPIHBP1 (green) and CD31 (white) in the glioma capillaries of wild-type mice but not in the glioma capillaries in Gpihbp1^–/– mice or in the capillaries of normal brain in wild-type, Gpihbp1^–/–, or MCK mice. There was no LPL signal when the primary antibody was omitted (Secondary Only). DNA was stained with DAPI (blue). Staining of all tissue sections was performed simultaneously, and images at the same magnification were recorded using the same microscope settings. Three mice per genotype were evaluated; representative images are shown. Scale bar, 20 μm.

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 (11A12), and an antibody against CD31. LPL (red) colocalized with GPIHBP1 (green) and CD31 (cyan) in the heart of wild-type mice, but LPL was absent in the heart and brain of Lpl^–/–MCK-hLPL mice. No LPL was detected when primary antibodies were omitted (Secondary Only). DNA was stained with DAPI (blue). Staining of all tissue sections was performed simultaneously, and images at the same magnification were recorded with the same laser settings. Two mice per genotype were evaluated; representative images are shown. Scale bar, 20 μm.

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 low-magnification image recorded with a 20× objective; the right panel shows a single image recorded with a 20× objective. DNA was stained with DAPI (blue). LPL was detected in the hippocampus (note the high density of neurons). LPL was absent from the hippocampus of Lpl^–/–MCK-hLPL mice. No LPL was detectable when primary antibodies were omitted. Staining of all tissue sections was performed simultaneously, and images at the same magnification were recorded with the same microscope settings. Two mice per genotype were evaluated; representative images are shown. Scale bar, 50 μm.

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 tissue.

There is little reason to suspect that the expression of LPL influences the expression of GPIHBP1 in capillaries. The overexpression of human LPL in the skeletal muscle of Lpl^–/–MCK-hLPL mice did not alter levels of Gpihbp1 expression (Figure 5—figure supplement 8).

Margination of TRLs along glioma capillaries and uptake of TRL-derived nutrients in glioma cells

Given the presence of GPIHBP1-bound LPL on glioma capillaries, we suspected that we might find evidence of TRL margination and processing in gliomas. To test this idea, TRLs that were heavily labeled with deuterated lipids ([²H]TRLs) (He et al., 2018a) were injected intravenously into mice harboring CT-2A gliomas (after three weeks of glioma growth). After allowing the [²H]TRLs to circulate for either 1 min or 30 min, the mice were euthanized, extensively perfused with PBS, and perfusion-fixed with carbodiimide/glutaraldehyde. Heart, brain, and glioma specimens were harvested and processed for NanoSIMS imaging. ¹²C¹⁴N^– or ¹H^– images were used to visualize tissue morphology, and ²H/¹H images were used to identify regions of ²H enrichment. The scale in the ²H/¹H images of brain and glioma specimens ranges from 0.00018 to 0.0003 (i.e., from levels slightly above ²H natural abundance to levels twice as high as ²H natural abundance). The scale in the heart ²H/¹H images ranges from 0.00018 to 0.0006. In mice euthanized 1 min after the [²H]TRLs injection, [²H]TRL margination was visualized along the luminal surface of glioma and heart capillaries, but not along the capillaries of normal brain (Figure 6A–B). After 1 min, deuterated lipids from the [²H]TRLs had already entered glioma cells and were even found in cytosolic neutral lipid droplets of those cells (Figure 6B). By contrast, ²H enrichment was virtually absent in normal brain. As expected (He et al., 2018a), we observed substantial amounts of [²H]TRL-derived lipids in cardiomyocytes, including in cytosolic lipid droplets. In gliomas harvested 30 min after the injection of [²H]TRLs, we observed similar findings: TRL margination along capillaries of gliomas and heart and the uptake of TRL-derived nutrients by glioma cells and cardiomyocytes (Figure 7). Again, [²H]TRL margination was absent in capillaries of the normal brain at the 30-min time point, and we did not find ²H enrichment in the parenchymal cells of the normal brain. We did, however, observe very low levels of ²H enrichment in capillary endothelial cells of normal brain. Given the absence of TRL margination in normal brain capillaries, we speculate that the very low amounts of ²H enrichment in brain capillary endothelial cells may relate to [²H]TRL processing in the periphery, followed by the uptake of unesterified [²H]fatty acids by endothelial cells of the brain.

Figure 6. 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 carbodiimide/glutaraldehyde. Tissue sections were processed for NanoSIMS imaging. (A) NanoSIMS images showing margination of [²H]TRLs in glioma capillaries. ¹H^– images were created to visualize tissue morphology (upper panels). Composite ²H/¹H (red) and ¹H^– (blue) images reveal [²H]TRLs (white arrows) in glioma and heart capillaries (middle and lower panels). The lower panels are close-up images of the regions outlined in the middle panels. ²H/¹H ratio scales were set to show marginated TRLs. Scale bars, 4 μm. (B) NanoSIMS images showing ²H enrichment in glioma tissue. ¹²C¹⁴N^– images were generated to visualize tissue morphology. ²H/¹H ratio images reveal margination of [²H]TRLs within the capillary lumen and ²H-enriched lipid droplets in gliomas and heart. There was no ²H enrichment in normal brain tissue. Scale bars, 4 μm. The bar graph shows the average fold change ± SD in the ²H/¹H ratio above natural abundance. The experiment was performed in two mice with a minimum of seven images analyzed for each sample. Differences were assessed using a Student’s t-test with Welch’s correction.

Figure 7. 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 carbodiimide/glutaraldehyde. Sections of glioma, brain, and heart were processed for NanoSIMS imaging. ¹²C¹⁴N^– images were created to visualize tissue morphology. ²H/¹H ratio images reveal margination of [²H]TRLs along the capillary lumen (white arrows) and ²H enrichment in glioma and heart, including in cytosolic lipid droplets. Images of normal brain revealed slight ²H enrichment in capillary endothelial cells. Scale bars, 4 μm. The bar graph shows the average fold change ± SD in the ²H/¹H ratio above natural abundance. The experiment was performed in two mice, with a minimum of seven images analyzed for each sample. Differences were assessed with a Student’s t-test with Welch’s correction.

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 carbodiimide/glutaraldehyde, and glioma and brain were processed for NanoSIMS imaging. ¹²C¹⁴N^– images were generated to visualize tissue morphology. The ²H/¹H ratio images revealed the absence of ²H enrichment in glioma and brain. One mouse was evaluated. Scale bars, 7 μm.

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 glutaraldehyde; and sections of heart and brain were prepared for NanoSIMS imaging. ¹²C¹⁴N^– images were generated to visualize tissue morphology. The ²H/¹H ratio images revealed ²H enrichment in the heart of the wild-type mouse but minimal ²H enrichment in the heart of the Gpihbp1^–/– mouse (²H enrichment in the cytosolic lipid droplets of the heart of the Gpihbp1^–/– mouse was very low, ~10% greater than ²H natural abundance; p<0.005). ²H enrichment was not detected in the brains of the wild-type mouse or the Gpihbp1^–/– mouse. One mouse per genotype was evaluated. Scale bars, 4 μm.

At both the 1-min and 30-min time points, we observed heterogeneity in ²H enrichment in glioma cells, with occasional perivascular cells exhibiting striking ²H enrichment. We do not know the identity of the highly enriched perivascular cells (i.e., whether they are tumor cells, pericytes, or macrophages), nor do we understand why some cells within the glioma took up more [²H]TRL-derived lipids than other cells.

As an experimental control, we injected a mouse with PBS alone rather than with [²H]TRLs. As expected, there was no ²H enrichment in the tissues of that mouse (Figure 7—figure supplement 1).

We performed an additional study in which [²H]TRLs were injected intravenously into a wild-type mouse and a Gpihbp1^–/– mouse. After 15 min, the hearts and brains from these mice were harvested and processed for NanoSIMS imaging. The ²H/¹H ratio images revealed ²H enrichment in the heart of the wild-type mouse but negligible ²H enrichment in the heart of the Gpihbp1^–/– mouse (²H enrichment in cardiomyocyte lipid droplets was only ~10% greater than natural abundance) (Figure 7—figure supplement 2). In hindsight, the negligible amounts of ²H enrichment in the heart of the Gpihbp1^–/– mouse was probably not surprising, given the very large pool of unlabeled triglycerides in the bloodstream of Gpihbp1^–/– mice (~50–100-fold higher than that in wild-type mice). At the 15-min time point, we were unable to detect ²H enrichment in the brain of either the wild-type mouse or the Gpihbp1^–/– mouse (Figure 7—figure supplement 2).

¹³C enrichment in gliomas following administration of ¹³C-labeled fatty acids or ¹³C-labeled glucose by gastric gavage

In addition to studies of gliomas after an intravenous injection of [²H]TRLs, we performed NanoSIMS imaging after administering ¹³C-labeled fatty acids or ¹³C-labeled glucose by gastric gavage (three doses over 36 hr) (Figure 8). In the case of the ¹³C-labeled fatty acid experiments, it is likely that most of the ¹³C-labeled lipids entered the bloodstream in chylomicrons. Once again, ¹²C¹⁴N^– images were useful for tissue morphology, and the ¹³C/¹²C ratio images were useful to identify regions of ¹³C enrichment. The scale for the ¹³C/¹²C images ranges from 0.0115 to 0.0150 (from slightly above ¹³C natural abundance to ~36% greater than natural abundance). After administering ¹³C-labeled fatty acids, ¹³C enrichment was observed in both glioma cells and in the capillary endothelial cells of gliomas (Figure 8A). In some images, ¹³C-enriched cytosolic lipid droplets were visible in glioma cells (Figure 8—figure supplement 1). ¹³C enrichment was virtually absent from normal brain (Figure 8A). However, after adjusting the scale of the NanoSIMS images, a small amount of ¹³C enrichment was observed in capillary endothelial cells within the brain parenchyma (Figure 8—figure supplement 2). As expected (He et al., 2018a), we observed substantial amounts of ¹³C enrichment in cardiomyocytes (Figure 8A).

Figure 8. 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). ¹²C¹⁴N^– images were generated to visualize tissue morphology; ¹³C/¹²C ratio images were used to visualize ¹³C enrichment in tissues. Scale bars, 4 μm. (B) NanoSIMS images revealing ¹³C enrichment in tissues following oral administration of ¹³C-labeled glucose to mice (three 75-mg doses given 12-h apart). ¹²C¹⁴N^– images were generated to visualize tissue morphology; ¹³C/¹²C ratio images were generated to assess ¹³C enrichment in tissues. Scale bars, 4 μm. The bar graphs show the average ¹³C/¹²C ratio ± SD multiplied by 10,000 for fatty acids (left) and glucose (right). Each experiment was performed in two mice, with a minimum of seven images analyzed for each sample. Differences were assessed using a Student’s t-test with Welch’s correction.

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 were euthanized; perfusion-fixed with carbodiimide/glutaraldehyde; and sections of glioma were prepared for NanoSIMS imaging. ¹²C¹⁴N^– images were generated to visualize tissue morphology. The ¹³C/¹²C ratio images depict ¹³C enrichment in glioma cells; white arrows point to cytosolic lipid droplets in glioma cells. Two mice were evaluated and representative images are shown. Scale bars, 3 μm.

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 to visualize tissue morphology; ¹³C/¹²C ratio images show ¹³C enrichment (white arrows) in brain capillary endothelial cells. Two mice were evaluated and representative images are shown. Scale bar, 4 μm.

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 carbodiimide/glutaraldehyde; and sections of glioma and brain were prepared for NanoSIMS imaging. ¹²C¹⁴N^– images were generated to visualize tissue morphology. The ¹³C/¹²C ratio images show an absence of ¹³C enrichment in glioma and brain. One mouse was evaluated. Scale bars, 7 μm.

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 animals was assessed by measuring luciferase activity in the blood. (A) Luciferase activity in the blood. No significant differences were observed. (B) Body weights at weekly intervals; no significant differences were observed. (C) Survival curves of mice (mice were euthanized when they lost > 20% of their body weight). No significant differences were observed. (D) Tumor weight as a percentage of total brain weight at the time that the mice were euthanized. A Student’s t-test was performed, assuming both equal variance and unequal variance. Both tests yielded a p-value of 0.33. Means ± SDs are shown.

After administering [¹³C]glucose to mice, ¹³C enrichment was easily detectable in normal brain but was even ~20% higher in gliomas (Figure 8B). We also observed ¹³C enrichment in cardiomyocytes (Figure 8B). As expected, there was no ¹³C enrichment in the tissues of a mouse that was administered PBS alone (Figure 8—figure supplement 3).

To determine whether an absence of GPIHBP1 expression would influence the growth of glioma tumors, CT-2A glioma cells that had been stably transfected with a Gaussia luciferase reporter were injected into the brains of wild-type and Gpihbp1^–/– mice (n = 11/group). Tumor burden was assessed in live animals by measuring luciferase activity in the blood (Mai et al., 2017; Tannous, 2009). We observed no statistically significant differences in tumor growth, tumor size, or survival between wild-type and Gpihbp1^–/– mice (Figure 8—figure supplement 4). This result was not particularly surprising, given that gliomas have a robust capacity to utilize glucose-derived nutrients (Figure 8B).

Discussion

We sought to determine whether GPIHBP1, despite its complete absence from the capillaries of the brain, might nevertheless be expressed in the capillaries of gliomas. Using standard immunohistochemistry procedures, we documented GPIHBP1 expression in capillary endothelial cells of human gliomas and CT-2A-derived mouse gliomas. The expression of GPIHBP1 in glioma capillaries was intriguing, but the crucial issue is whether LPL would be bound to the GPIHBP1. Additional immunohistochemistry studies on mouse gliomas revealed that LPL colocalizes with GPIHBP1 on glioma capillaries, just as LPL colocalizes with GPIHBP1 in the capillaries of heart and brown adipose tissue (Young et al., 2011; Davies et al., 2010; Davies et al., 2012; Allan et al., 2017a; Fong et al., 2016; Allan et al., 2017b; Allan et al., 2016). The binding of LPL to GPIHBP1 was specific: the LPL-specific goat antibody did not detect LPL in the capillaries of gliomas in Gpihbp1^–/– mice, nor did it detect any LPL in macrophages or hippocampal neurons of Lpl^–/–MCK-hLPL mice. The colocalization of GPIHBP1 and LPL in the capillaries of gliomas implied that we might find evidence for TRL margination and processing in these tumors. Indeed, we observed both [²H]TRL margination along glioma capillaries and the entry of TRL-derived nutrients into glioma cells. Consistent with results of earlier studies (Goulbourne et al., 2014; He et al., 2018a), TRL margination was absent from the capillaries of normal brain, and we found no ²H enrichment in the brain parenchyma. We did, however, find very low levels of ²H enrichment in capillary endothelial cells of normal brain, perhaps as a result of the uptake of fatty acids that are derived from TRL processing in peripheral tissues. We observed consistent findings after administering [¹³C]fatty acids to mice by gastric gavage. In those experiments, we observed strong ¹³C enrichment in gliomas but no ¹³C enrichment in the normal brain (except for low levels of enrichment in capillary endothelial cells). After administering [¹³C]glucose by gavage, ¹³C enrichment was observed in both gliomas and normal brain. It is important to note that the [¹³C]fatty acids and the [¹³C]glucose were administered in three doses over 36 hr before harvesting tissues for NanoSIMS analyses, allowing ample time for the labeled nutrients to be utilized as fuel or to be converted into other nutrients (e.g., nonessential amino acids) (He et al., 2018a; Sidossis et al., 1995; Schneider and Potter, 1957). Thus, after administering ¹³C-labeled fatty acids or glucose to mice, the ¹³C in glioma cells was probably present in a variety of macromolecules (e.g., glucose, lipids, proteins, and nucleic acids).

Documenting GPIHBP1 and LPL in glioma capillaries, combined with the discovery that TRL-derived nutrients are taken up and utilized by glioma cells, opens a new chapter in glioma metabolism research (Figure 9). Laboratories that are interested in glioma metabolism have typically focused on the intrinsic metabolic properties of glioma cells and on how metabolic pathways in gliomas differ from those in normal brain (Lin et al., 2017; Guo et al., 2009a; Guo et al., 2009b; Guo et al., 2013; Gopal et al., 1963; Strickland and Stoll, 2017; Agnihotri and Zadeh, 2016). There have been suggestions, based on indirect observations of substrate utilization, that glioma tumors are capable of utilizing fatty acids for fuel and for anabolic processes (Lin et al., 2017; Guo et al., 2013; Mashimo et al., 2014; Ru et al., 2013; Zaidi et al., 2013). In those studies, however, the assumption was that the fatty acids probably originated from the tumor cells by de novo lipogenesis (Guo et al., 2011; Guo et al., 2009a; Guo et al., 2009b). No one, as far as we are aware, had ever considered the possibility that gliomas might be capable of taking up and utilizing nutrients from LPL-mediated intravascular processing of TRLs.

Figure 9. 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 that LPL remains within the interstitial spaces of the brain (i.e., that it has an extravascular function) (Adeyo et al., 2012; Young et al., 2011). In gliomas (right panel), GPIHBP1 is expressed in capillary endothelial cells, allowing GPIHBP1 to capture locally produced LPL and to shuttle it to the capillary lumen. Intravascular processing of triglyceride-rich lipoproteins in gliomas provides a source of lipid nutrients for glioma cells. HSPGs, heparan sulfate proteoglycans.

In an ultrastructural study of human gliomas, Vaz et al. (1996) commented that the morphology of endothelial cells in gliomas resembles that of capillary endothelial cells in peripheral tissues, with euchromatin-rich nuclei, occasional fenestrations, and numerous pinocytotic vesicles within the cytoplasm. The expression of GPIHBP1 (a hallmark of capillary endothelial cells in peripheral tissues) in gliomas provides biochemical support for the notion that glioma capillaries resemble capillaries in peripheral tissues (Vaz et al., 1996). Our electron microscopy studies confirmed that the morphological features of glioma capillaries and normal brain capillaries differ substantially.

We have relatively few insights into the molecular basis for GPIHBP1 expression in glioma capillaries. One possibility is that the absence of a blood–brain barrier in glioma capillaries (Dubois et al., 2014; Wolburg et al., 2012; Liebner et al., 2000; Sage and Wilson, 1994) permits the exposure of endothelial cells to a paracrine factor that activates GPIHBP1 expression. Another possibility is that GPIHBP1 expression is stimulated by the expression of VEGF that is produced by glioma cells (Plate et al., 1994; Pietsch et al., 1997; Christov et al., 1998). In our studies, VEGF increased GPIHBP1 expression in the mouse brain endothelial cell line bEnd.3.

In the past, other laboratories have reported that glioma tumor cells can transdifferentiate into endothelial cells, thereby augmenting the vascular supply to tumors (Wang et al., 2010; Ricci-Vitiani et al., 2010; Soda et al., 2011). For example, endothelial cells in human glioblastomas were reported to harbor the same genetic alterations as the tumor cells, implying that at least some of the glioblastoma endothelial cells originate from stem cells within the tumor (Wang et al., 2010; Ricci-Vitiani et al., 2010). In another model (Soda et al., 2011), a Cre recombinase (Cre)-loxP–controlled lentiviral vector encoding activated forms of H-Ras and Akt was injected into the hippocampus of GFAP-Cre p53 mice, eliciting glioblastomas. In that model, the oncogenes were expressed in the GFAP+ cells, and the resulting tumors expressed GFP, H-Ras, and Akt and the loss of p53. Some GFP+ endothelial cells were observed in tumors, implying that these endothelial cells had originated from tumor cells. Furthermore, implanting a tumor cell line (generated from tumors induced with the same lentiviral vector) into the brain of immunocompromised mice was reported to yield tumors containing GFP+ endothelial cells. In our current studies, we observed no evidence of the differentiation of glioma cells into capillary endothelial cells. The glioma cell line that we used expressed blue fluorescent protein (BFP), but we did not find BFP expression in the capillary endothelial cells of gliomas.

Mass spectrometry–based analyses of homogenized tissue extracts from mouse gliomas and normal brain tissue, along with similar analyses of tumors from human patients, suggested differences in acetate oxidation in gliomas vs. normal brain (Mashimo et al., 2014). Although these studies of tissue extracts have been useful, they obviously cannot provide anatomical insights into metabolism. We have argued that NanoSIMS imaging studies are particularly useful when the goal is to understand metabolism at an anatomic level (cellular or subcellular) (He et al., 2018a). In the current studies, NanoSIMS imaging provided anatomic insights into glioma metabolism. For example, we observed TRL margination along the capillaries of gliomas but not in the capillaries of adjacent normal brain tissue. We also showed that the transport of TRL-derived nutrients across glioma capillaries and into glioma cells is rapid, occurring within 1 min, and that there is heterogeneity in nutrient uptake by different cells within the tumor. We found no uptake of TRL-derived nutrients by normal brain 1 or 15 min after the injection of [²H]TRLs and only very small amounts (confined to capillary endothelial cells) after 30 min. In addition, following the administration of [¹³C]glucose, we found more ¹³C enrichment in gliomas than in normal brain. As far as we are aware, our study is the first to use NanoSIMS analyses to investigate cancer metabolism in vivo. As we look to the future, we have little doubt that NanoSIMS imaging will be an important tool for understanding tumor metabolism, making it possible to investigate metabolic heterogeneity in tumor cells along with the metabolic properties of vascular cells, fibroblasts, and macrophages within the tumor. Nevertheless, it is important to point out that NanoSIMS imaging is not high-throughput, at least with the current instruments, and for that reason NanoSIMS imaging is best used (as in this study) to address discrete anatomic issues in metabolism. Examining large numbers of tumors or large numbers of mice would be difficult. Also, NanoSIMS imaging is very expensive.

Our studies have provided fresh insights into the uptake of lipid nutrients by gliomas, but many issues remain to be investigated. For example, in the current studies, we found numerous macrophages within gliomas, but we did not address differences in nutrient uptake by macrophages and glioma cells. In future studies, it should be possible to examine the uptake of TRL-derived nutrients into tumor cells, macrophages, and other immune cells within gliomas (by identifying specific cell types with ¹⁵N-labeled monoclonal antibodies or antibodies tagged with different lanthanide metals [Waentig et al., 2012; Kanje et al., 2016; Angelo et al., 2014; Keren et al., 2018]). It would also be desirable to determine whether the uptake of TRL-derived nutrients in gliomas correlates with the levels of GPIHBP1 and LPL in glioma capillaries (as quantified with LPL- and GPIHBP1-specific antibodies tagged with different lanthanide metals). Finally, it would be desirable to investigate whether the presence of GPIHBP1 and LPL in glioma capillaries could be exploited for patient care. For example, it is conceivable that fluorescently labeled GPIHBP1 antibodies or DiI-labeled TRLs could guide the surgical resection of tumors. In addition, a localized injection of GPIHBP1-specific monoclonal antibodies conjugated to chemotherapeutic agents into gliomas might be useful in targeting tumor vasculature (Schrama et al., 2006). A localized injection of gold-conjugated GPIHBP1-specific monoclonal antibodies could augment the efficacy of external beam radiotherapy (Haume et al., 2016; Hainfeld et al., 2004; Hainfeld et al., 2008).

Materials and methods

Key resources table.

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (M. musculus)	Gpihbp1–/–	Beigneux et al., 2007	RRID: MGI:3771172	Dr. Stephen G Young (UCLA)
Genetic reagent (M. musculus)	Lpl–/–MCK-hLPL	Levak-Frank et al., 1995	RRID: MGI:3624988	Dr. Rudolph Zechner (Graz University)
Genetic reagent (M. musculus)	ROSAmT/mGPdgfb-iCreT2	Mathivet et al., 2017		Dr. Holger Gerhardt (VIB KU-Leuven)
Cell line (M. musculus)	CT-2A	Seyfried et al., 1992		Dr. Thomas Seyfried (Boston College)
Cell line (M. musculus)	CT-2A–BFP	PMID: 24658686		Dr. Holger Gerhardt (VIB KU-Leuven)
Cell line (M. musculus)	bEnd.3	ATCC	Catalog No. CRL-2299 RRID: CVCL_0170
Transfected construct (lentiviral plasmid)	plenti-GLuc-IRES-EGFP	Targeting Systems	Catalog No. GL-GFP
Antibody	Rat monoclonal anti-mouse GPIHBP1 (11A12)	Beigneux et al., 2009		Dr. Stephen G Young (UCLA); IHC (10 μg/ml)
Antibody	Mouse monoclonal anti-human GPIHBP1 (RE3)	Hu et al., 2017		Dr. Stephen G Young (UCLA); IHC (10 μg/ml)
Antibody	Mouse monoclonal anti-human GPIHBP1 (RF4)	Hu et al., 2017		Dr. Stephen G Young (UCLA); IHC (10 μg/ml)
Antibody	Mouse monoclonal anti-human GPIHBP1 (RG3)	Hu et al., 2017		Dr. Stephen G Young (UCLA); IHC (10 μg/ml)
Antibody	Rabbit polyclonal anti-vWF	Dako	Catalog No. A0082 RRID: AB_2315602	IHC (1:200)
Antibody	Goat polyclonal anti-GFAP	Abcam	Catalog No. ab53554 RRID: AB_880202	IHC (1:200)
Antibody	Rabbit polyclonal anti-GLUT1	Millipore-Sigma	Catalog No. 07–1401 RRID: AB_1587074	IHC (1:200)
Antibody	Rabbit polyclonal anti-CD31	Abcam	Catalog No. ab28364 RRID: AB_726362	IHC (1:50)
Antibody	Rat monoclonal anti-F4/80	Abcam	Catalog No. ab6640 RRID: AB_1140040	IHC (10 μg/ml)
Antibody	Goat polyclonal anti-mouse LPL	Page et al., 2006		Dr. André Bensadoun (Cornell); IHC (12 μg/ml)
Antibody	Alexa Fluor 488, 568, 647 secondaries	ThermoFisher Scientific		IHC (1:500)
Commercial assay or kit	ImmPRESS Excel Staining Kit	Vector Laboratory	Catalog No. MP-7602
Sequence-based reagent	Mouse Gpihbp1 primers			5′-AGCAGGGACAGAGCACCTCT-3′ and 5′-AGACGAGCGTGATGCAGAAG-3′
Sequence-based reagent	Mouse Cd31 primers			5′-AACCGTATCTCCAAAGCCAGT-3′ and 5′-CCAGACGACTGGAGGAGAACT-3′
Sequence-based reagent	Mouse Angpt2 primers			5′-AACTCGCTCCTTCAGAAGCAGC-3′ and 5′-TTCCGCACAGTCTCTGAAGGTG-3′
Sequence-based reagent	Mouse Dusp5 primers			5′-TCGCCTACAGACCAGCCTATGA-3′ and 5′-TGATGTGCAGGTTGGCGAGGAA-3′
Sequence-based reagent	Mouse Cxcr4 primers			5′-GACTGGCATAGTCGGCAATGGA-3′ and 5′-CAAAGAGGAGGTCAGCCACTGA-3′
Sequence-based reagent	Mouse Lpl primers			5′-AGGTGGACATCGGAGAACTG-3′ and 5′-TCCCTAGCACAGAAGATGACC-3′
Sequence-based reagent	Human LPL primers			5′-TAGCTGGTCAGACTGGTGGA-3′ and 5′-TTCACAAATACCGCAGGTG-3′
Recombinant DNA reagent	ALO-D4 plasmid	Gay et al., 2015		Dr. Arun Radhakrishnan (UT Southwestern)
Chemical compound, drug	N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (carbodiimide)	Millipore-Sigma	Catalog No. 03449
Chemical compound, drug	Glutaraldehyde25% solution	Electron Microscopy Sciences	Catalog No. 16220
Chemical compound, drug	Osmium tetroxide 4% solution	Electron Microscopy Sciences	Catalog No. 18459
Chemical compound, drug	Paraformaldehyde 16% solution	Electron Microscopy Sciences	Catalog No. 15170
Chemical compound, drug	EMbed 812	Electron Microscopy Sciences	Catalog No. 14120
Chemical compound, drug	Sodium cacodylate trihydrate	Electron Microscopy Sciences	Catalog No. 12300
Chemical compound, drug	Uranyl acetate	SPI-Chem	Catalog No. 02624AB
Chemical compound, drug	DAPI	ThermoFisher Scientific	Catalog No. 1306	IHC (3 μg/ml)
Chemical compound, drug	Mouse VEGF	Millipore-Sigma	Catalog No. V4512
Software, algorithm	LIMMA	Ritchie et al., 2015	RRID: SCR_010943
Other	D-GLUCOSE (U-13C6, 99%)	Cambridge Isotope Laboratories	Catalog No. CLM-1396-PK
Other	Mixed fatty acids (U-D, 96–98%)	Cambridge Isotope Laboratories	Catalog No. DLM-8572-PK
Other	Mixed fatty acids (13C, 98%+)	Cambridge Isotope Laboratories	Catalog No. CLM-8455-PK

Immunohistochemical studies on human glioma specimens

Frozen surgical glioma specimens were obtained from the UCLA Department of Neurosurgery. Frozen autopsy control brain samples (frontal lobe, occipital lobe, and cerebellum) were obtained from the UCLA Section of Neuropathology. Samples were sectioned to 8 μm and placed on glass slides. All samples were fixed with 3% paraformaldehyde (PFA) in PBS/Ca/Mg and permeabilized in 0.2% Triton X-100 in PBS/Ca/Mg. Tissues were blocked with PBS/Ca/Mg containing 5% donkey serum and 0.2% bovine serum albumin (BSA) and incubated overnight at 4°C with one or more mouse monoclonal antibodies (mAbs) against human GPIHBP1 (RF4, RE3, RG3; 10 μg/ml) (Hu et al., 2017), a rabbit polyclonal antibody against von Willebrand factor (vWF) (Dako; 1:200), and a goat polyclonal antibody against human glial fibrillary acidic protein (GFAP) (Abcam; 1:500). In some experiments, recombinant soluble human GPIHBP1 (50 μg) was added to the primary antibody incubation. After washing the slides, 1-hr incubations were performed with an Alexa Fluor 647–conjugated donkey anti–mouse IgG (ThermoFisher Scientific; 1:500), an Alexa Fluor 488–conjugated donkey anti–rabbit IgG (ThermoFisher Scientific; 1:500), and an Alexa Fluor 568–conjugated donkey anti–goat IgG (ThermoFisher Scientific; 1:500). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were taken with an LSM700 confocal microscope with an Axiovert 200M stand and processed with Zen 2010 software (Zeiss).

Immunoperoxidase staining was performed with the ImmPRESS Excel Staining Kit (Vector Laboratories). Endogenous peroxidase activity was quenched with BLOXALL Blocking Solution (Vector Laboratories). After incubating sections in 10% normal horse serum, sections were incubated for 1 hr with mAb RF4 (5 μg/ml), followed by a 15-min incubation with a goat anti-mouse IgG (10 μg/ml, Vector Laboratories). Slides were then incubated for 30 min with a horseradish peroxidase–conjugated horse anti–goat IgG (ImmPRESS Excel Reagent, Vector Laboratories). After washing, the slides were incubated with ImmPACT DAB EqV (Vector Laboratories) until a color change was evident (~30 s). Finally, sections were counterstained with hematoxylin and mounted with Vectashield Mounting Media (Vector Laboratories). Images were recorded with a Nikon Eclipse E600 microscope (Plan Fluor 40×/0.50 NA or 100×/0.75 NA objectives) equipped with a DS-Fi2 camera (Nikon).

Genome dataset and gene-expression analyses

Cohorts for RNA-seq analysis were obtained from two databases: The Cancer Genome Atlas (TCGA) for tumor samples and Genotype-Tissue Expression (GTEx) for normal brain samples. Samples from TCGA (n = 157) and GTEx (n = 283) were processed with the TOIL pipeline as described (Vivian et al., 2017). A differential expression analysis of fatty acid metabolism genes was carried out with a linear model RNA-seq analysis software (LIMMA) (Ritchie et al., 2015). Genes were considered differentially expressed if the p-values were <0.05 and the log₂ changes were >twofold. A heatmap was generated with the software R (Kolde, 2015).

Animal procedures and glioma implantation

Mice on a C57BL/6 background expressing both the ROSA^mT/mG Cre-reporter (Muzumdar et al., 2007) and tamoxifen-inducible Pdgfb-iCreER^T2 alleles (Claxton et al., 2008) were generated by breeding. In those mice, the administration of tamoxifen induces Cre recombinase expression in Pdgfb-positive cells. The recombination event results in the expression of EGFP in endothelial cells; all other cells express tdTomato. For the glioma implantation studies, mice (8–12-weeks-old) were injected intraperitoneally with tamoxifen (65 μg/g body weight, 4 injections in 2 weeks) before surgery. Mice were anesthetized with ketamine/xylazine, and a craniotomy was performed by drilling a 5‐mm hole between the lambdoid, sagittal, and coronal sutures. A blue fluorescent protein (BFP)-tagged CT‐2A glioblastoma spheroid (250-μm in diameter) (Seyfried et al., 1992; Oh et al., 2014) was injected into the cortex and sealed by cementing a glass coverslip on the skull. The CT-2A cell line was generated by Seyfried and coworkers through chemical induction with 20-methylcholanthrene in the brain of C57BL/6 mice and has been characterized extensively (Seyfried et al., 1992). In other experiments, CT-2A glioblastoma spheroids were implanted into the cortex of C57BL/6 wild-type mice and Gpihbp1^–/– mice (Beigneux et al., 2007). Those procedures were performed as described previously (Stanchi et al., 2019).

Immunohistochemical studies on mouse gliomas

Mice harboring BFP-expressing CT-2A gliomas (Seyfried et al., 1992; Oh et al., 2014) were anesthetized with ketamine/xylazine and then injected intravenously (via the tail vein) with 100 μg of an Alexa Fluor 647–conjugated antibody against mouse GPIHBP1 (11A12) (Beigneux et al., 2009). After 1 min, the mice were perfused through the heart with 15 ml of PBS, followed by 10 ml of 2% PFA in PBS. Brain and glioma tissues were harvested and fixed overnight in 4% PFA. Tissue sections (200-μm-thick) were prepared with a vibratome. For immunofluorescence microscopy studies, the sections were fixed with 4% PFA in PBS and blocked and permeabilized in TNBT (0.1 M Tris, pH 7.4, 150 mM NaCl, 0.5% blocking reagent from Perkin Elmer, 0.5% Triton X‐100) for 4 hr at room temperature. Tissues were incubated with an antibody against GLUT1 (Millipore; 1:200) diluted in TNBT buffer overnight at 4°C, washed in TNT buffer (0.1 M Tris pH 7.4; 150 mM NaCl, 0.5% Triton X‐100) and incubated with an Alexa Fluor 488–conjugated donkey anti–rabbit IgG (ThermoFisher Scientific; 1:200). Tissues were washed and mounted in fluorescent mounting medium (Dako). Images were obtained with a Leica TCS SP8 confocal microscope.

For the analysis of tissues from mice that were not injected with anti-GPIHBP1 antibodies, tissues were embedded in OCT medium, and 10-µm sections were cut with a cryostat. Sections were fixed with 3% PFA in PBS/Ca/Mg, permeabilized with 0.2% Triton X-100 in PBS/Ca/Mg, and blocked with PBS/Ca/Mg containing 5% donkey serum and 0.2% BSA. Tissue sections were incubated overnight at 4°C with a rabbit antibody against CD31 (Abcam; 1:50), a goat antibody against mouse LPL (12 μg/ml) (Page et al., 2006), an Alexa Fluor 488–conjugated antibody against F4/80, or an Alexa Fluor 647–conjugated antibody against mouse GPIHBP1 (11A12, 10 μg/ml). After removing non-bound antibodies and washing the sections, unlabeled primary antibodies were detected with an Alexa Fluor 488–conjugated donkey anti–rabbit IgG (ThermoFisher Scientific; 1:500) or an Alexa Fluor 568–conjugated donkey ant–goat IgG (ThermoFisher Scientific; 1:500). DNA was stained with DAPI, and tissues were mounted with ProLong Gold mounting media (ThermoFisher Scientific). Images were recorded on an LSM 800 confocal microscope (Zeiss).

Immunocytochemistry studies on mouse peritoneal macrophages

Macrophages were collected by peritoneal lavage of C57BL/6 wild-type and Lpl^–/–MCK-hLPL mice. Cells were centrifuged at 400 × g for 5 min at 4°C, washed with 5 ml of red blood cell lysing buffer (Sigma) for 5 min, washed twice with cold PBS, and then plated onto FBS-coated Petri dishes. Cells were cultured overnight in macrophage medium (Dulbecco Modified Eagle Medium with 10% FBS, 1% glutamine, and 1% sodium pyruvate). On the next day, macrophages were lifted with cold PBS containing 5 mM EDTA for 30 min at 4°C. Cells were then plated onto poly-D-lysine–coated glass coverslips (75,000 cells/coverslip) and incubated overnight in macrophage media. On the following day, the cells were washed three times for 10 min in PBS/Ca/Mg containing 0.2% BSA and then incubated with Alexa Fluor 568–labeled ALO-D4 (a modified cytolysin that binds to ‘accessible cholesterol’ in the plasma membrane) (Das et al., 2014; Das et al., 2013; Gay et al., 2015) for 2 hr at 4°C. Samples were washed three times for 1 min with PBS/Ca/Mg, fixed with 3% PFA in PBS/Ca/Mg, permeabilized with 0.2% Triton X-100 in PBS/Ca/Mg, and blocked with PBS/Ca/Mg containing 5% donkey serum and 0.2% BSA. Cells were then incubated with a goat antibody against mouse LPL (12 μg/ml) (Page et al., 2006) for 1 hr at room temperature followed by a 30-min incubation with an Alexa Fluor 647–labeled donkey anti–goat IgG (ThermoFisher Scientific; 1:500). DNA was stained with DAPI, and coverslips were mounted onto glass slides in ProLong Gold mounting media (ThermoFisher Scientific). Images were recorded with a Zeiss LSM700 confocal microscope.

Administration of [¹³C]fatty acids, [¹³C]glucose, and [²H]TRLs to mice

C57BL/6 mice with CT-2A gliomas (of three-week duration) were given 80 μl of [¹³C]fatty acids (~1 mg/μl; Cambridge Isotope Laboratories) or 80 μl of [¹³C]glucose (3 mg/kg body weight; Cambridge Isotope Laboratories) by oral gavage every 12 hr for 36 hr (three doses). To study TRL metabolism, mice were injected intravenously with a single bolus of [²H]TRLs (40 μg triglycerides in 100 µl) via the tail vein. The [²H]TRLs were isolated from the plasma of Gpihbp1^–/– mice after administering deuterated fatty acids by gastric gavage (He et al., 2018a). After allowing the [²H]TRLs to circulate for 1 min or 30 min, the mice were perfused through the heart with 15 ml of ice-cold PBS/Ca/Mg at 3 ml/min (10 ml though the left ventricle and 5 ml through the right ventricle). Next, the mice were perfusion-fixed through the left ventricle with 10 ml of ice-cold 4% N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (‘carbodiimide;’ Sigma-Aldrich) (mass/vol) and 0.4% glutaraldehyde (Electron Microscopy Sciences) (vol/vol) in 0.1 M phosphate buffer. The heart, brain, and glioma tumors were collected and placed in 0.1 M phosphate buffer containing 4% carbodiimide and 2.5% glutaraldehyde for 2 hr at 4°C. Tissues were cut into 1-mm³ pieces and fixed overnight in 2.5% glutaraldehyde, 3.7% PFA, and 2.1% sucrose in 0.1 M sodium cacodylate (pH 7.4).

Preparation of tissue sections for NanoSIMS imaging and electron microscopy

After fixation, 1-mm³ pieces of tissue were rinsed three times (10 min each) in 0.1 M sodium cacodylate buffer (pH 7.4) and fixed with 2% OsO₄ (Electron Microscopy Sciences) in 0.1 M sodium cacodylate on ice for 90 min. The samples were rinsed three times (10 min each) with distilled water and stained overnight with 2% uranyl acetate at 4°C. On the following day, the samples were rinsed three times for 10 min each with distilled water and then dehydrated with increasing amounts of ethanol (30%, 50%, 70%, 85%, 95%, and 100%; 3 × 10 min) before infiltration with Embed812 resin (Electron Microscopy Sciences) diluted in acetone (33% for 2 hr; 66% overnight; 100% for 3 hr). The samples were embedded in polyethylene molds (Electron Microscopy Sciences) with fresh resin and polymerized in a vacuum oven at 65°C for 48 hr. The polymerized blocks were then removed from the molds, trimmed, and sectioned.

For transmission electron microscopy, 65-nm sections were cut and collected on freshly glow-discharged copper grids (Ted Pella) that were coated with formvar and carbon. Sections were then stained with Reynold’s lead citrate solution for 10 min. Images were acquired with an FEI T12 transmission electron microscope set to 120 kV accelerating voltage and a Gatan 2K × 2K digital camera (Electron Imaging Center).

For NanoSIMS analyses, 500-nm sections were cut with a Leica UC6 ultramicrotome and collected on silicon wafers. Sections of tissue were coated with ~5 nm of platinum and analyzed with NanoSIMS 50L or NanoSIMS 50 instruments (CAMECA). Samples were scanned with a 16-KeV ¹³³Cs⁺ beam, and secondary electrons (SEs) and secondary ions (¹H^–, ²H^–, ¹²C^–, ¹³C^–, ¹²C¹⁴N^–) were collected. A 50 × 50-μm region of the section was pre-sputtered with a ∼1.2-nA beam current (primary aperture D1 = 1) to reach a dose of ~1 × 10¹⁷ ions/cm² to remove the platinum coating and implant ¹³³Cs⁺ in order to ensure a steady state of secondary ion release. A ∼40 × 40-μm region was imaged with an ∼3-pA beam current (primary aperture D1 = 2) and a dwell time of ~10 ms/pixel per frame for multiple frames. Both 256 × 256–pixel and 512 × 512–pixel images were obtained. Images were prepared using the OpenMIMS plugin in ImageJ. For image quantification, ²H/¹H and ¹³C/¹²C ratios in the regions of interests were calculated with the OpenMIMS plugin and processed by GraphPad Prism 7.0.

Tumor studies in wild-type and Gpihbp1-deficient mice

Three-month-old C57BL/6 wild-type (five females, six males) and Gpihbp1^–/– mice (six females, five males) were injected intracranially with CT-2A glioma cells stably expressing a Gaussia luciferase reporter gene (4 × 10⁵ cells/mouse). Cells were injected 1 mm posterior and 2 mm lateral to the bregma at a depth of 2 mm. Tumor burden was monitored every three days by measuring Gaussia luciferase in the blood (Mai et al., 2017; Tannous, 2009). Mice were weighed at weekly intervals and were euthanized when they lost >20% of their body weight. After the mice were euthanized, their tumors and brains were weighed. All studies were approved by UCLA’s Animal Research Committee.

Gaussia luciferase measurements

To measure the levels of secreted Gaussia luciferase (sGluc), blood was obtained from the tail vein of mice and mixed with 50 mM EDTA to prevent coagulation. 5 μl of blood was transferred to a 96-well plate, and sGluc activity was measured by chemiluminescence after injecting 100 μl of 100 μM coelentarazine (Nanolight) (Mai et al., 2017; Tannous, 2009). Data were plotted as relative light units (RLU).

Quantifying mouse and human transcripts by qRT-PCR

C57BL/6 wild-type mice and Lpl^–/–MCK-hLPL mice were anesthetized with isoflurane and perfused with PBS. Heart, brown adipose tissue (BAT), and quadricep were harvested and flash-frozen in liquid nitrogen. RNA was isolated with TRI reagent (Molecular Research), and quantitative (q)RT-PCR measurements were performed in triplicate with a 7900HT Fast real-time PCR system (Applied Biosystems). Gene expression was calculated with a comparative CT method and normalized to levels of cyclophilin A expression. Primers for mouse Gpihbp1, mouse Lpl, and human LPL are described in the 'Key Resources Table'.

VEGF treatment of brain endothelial cells

Mouse brain microvascular endothelial cells (bEnd.3; ATCC #CRL-2299) were plated into 6-well plates and grown in DMEM media containing 10% FBS, 1% glutamine, and 1% sodium pyruvate overnight. On the next day, cells were rinsed with PBS and incubated in medium containing recombinant mouse VEGF (100 ng/ml; Sigma) for another 24 hr. RNA was isolated with TRI reagent (Molecular Research), and qRT-PCR measurements were performed in triplicate with a 7900HT Fast real-time PCR system (Applied Biosystems). Gene expression was calculated with a comparative CT method and normalized to cyclophilin A expression. Primers for mouse Gpihbp1, Cd31, Angpt2, Cxcr4, and Dusp5 are described in the 'Key Resources Table'.

Cell lines

CT-2A cells were obtained originally from the Seyfried laboratory and have been extensively tested and characterized (Seyfried et al., 1992). These cells also robustly expressed GFAP. The bEnd.3 cells were obtained from ATCC with a proper ‘certificate of analysis’. All cell lines were negative for mycoplasma contamination.

Statistics

Statistical analyses of data were performed with GraphPad Prism 7.0 software. All data are shown as the means ± standard deviations. Differences were assessed using a Student’s t-test with Welch’s correction.

Study approval

All tissue samples from patients were obtained after informed consent and with approval from the UCLA Institutional Review Board (IRB; protocol 10–000655). Animal housing and experimental protocols were approved by UCLA’s Animal Research Committee (ARC; 2004-125-51, 2016–005) and the Institutional Animal Care and Research Advisory Committee of the KU Leuven (085/2016). The animals were housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International)-approved facility and cared for according to guidelines established by UCLA’s Animal Research Committee. The mice were fed a chow diet and housed in a barrier facility with a 12 hr light-dark cycle.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

• National Heart, Lung, and Blood Institute HL090553 to Stephen G Young.

• National Heart, Lung, and Blood Institute HL087228 to Stephen G Young.

• National Heart, Lung, and Blood Institute HL125335 to Stephen G Young.

• Fondation Leducq 12CVD04 to Stephen G Young.

• National Institutes of Health Ruth L Kirschstein National Research Service Award (T32HL69766) to Xuchen Hu.

• National Institute of General Medical Sciences GM008042 to Xuchen Hu.

• National Cancer Institute Brain Tumor SPORE grant (P50-CA211015) to Linda M Liau.

• Stichting Tegen Kanker 2012‐181 to Holger Gerhardt.

• Stichting Tegen Kanker 2018-074 to Holger Gerhardt.

• Japan Society for the Promotion of Science Postdoctoral Fellowship to Ken Matsumoto.

• University of California, Los Angeles Medical Scientist Training Program to Xuchen Hu.

• Australian Research Council Discovery Early Career Researcher Award to Haibo Jiang.

• Cancer Council Western Australia Early Career Investigator Grant to Haibo Jiang.

Data availability

All data generated during this study are included in the manuscript and supporting files.

The following previously published dataset was used:

He L, Vanlandewijck M, Mae MA, Andrae J, Ando K, Del Gaudio F. 2018. Molecular atlas of vascular and vessel-associated cell types in the mouse brain and lung. NCBI Gene Expression Omnibus. GSE98816