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Disruption of astrocyte-vascular coupling and the blood-brain barrier by invading glioma cells.

Watkins S, Robel S, Kimbrough IF, Robert SM, Ellis-Davies G, Sontheimer H - Nat Commun (2014)

Bottom Line: Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form.This causes a focal breach in the BBB.These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama at Birmingham, 1719 6th Avenue South, CIRC 425, Birmingham, Alabama 35294, USA [2].

ABSTRACT
Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of preexisting vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over the regulation of vascular tone through Ca(2+)-dependent release of K(+). These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

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Perivascular glioma cell co-option causes a breakdown of the blood-brain barrierThe tracer, Evans blue, permeates into the brain parenchyma in tumor-bearing (n=3 animals) (bottom), but not in sham mice 3 weeks post surgery (n=3 animals) (top), Scale 5 mm. (a). Immunofluorescence of CD31 and intravenously injected tracers, tetramethylrhodamine-albumin (TRITC-alb, white) or -cadaverine (TRITC-cad, white), and implanted eGFP-expressing human glioma cells (D54) allow analysis of tracer leakage in relation to tumor burden. The large MW 70 kDa TRITC-albumin (b), as well as the small MW 950 Da TRITC-cadaverine (d) can be found outside the vasculature (arrows) (b-f, Suppl. Fig.6). Note that leaked TRITC-cadaverine is taken up by nearby neurons (arrows) (d,e). No extravasation was seen in the absence of tumor cells (c,f). To assess extravasation of the tracer Cascade blue (MW 10 kDa), Aldh1l1-eGFP-scid (eGFP) immunodeficient mice, which were previously implanted with TdTomato-expressing human glioma cells (D54), were retro-orbitally injected with Alexa Fluor 633 hydrazide dye (633 hyd) and Cascade blue (g,h). Accumulation of Cascade blue occurs in the brain's parenchyma indicating breakdown of the blood-brain barrier (BBB), where perivascular astrocytes have been displaced by glioma cell co-option (g). Vessels lacking tumor cells do not show extravasation of the dye (h). Immunohistochemistry for the TJ proteins zonula occludens-1 (ZO-1) (i) and claudin-5 (k) show that these protein are lost from endothelial cells labeled with CD31 where tumor cells are present (enlarged panel, arrows), but colocalize with endothelial cells in tissue that has not been co-opted. Quantification of ZO-1 shows reduction of vessel coverage in areas co-opted by glioma cells. 8.86 ± 0.64% vessel area is covered by ZO-1 in control images, whereas this number was reduced to 5.1 ± 1.11% in areas where vessels were coopted by tumor cells (n=9 images each for control and tumor-covered vessels from 3 animals, paired t-test directly comparing control and tumor images in the same slice, p=0.037, error bars refer to SEM) (j). Black font was used to describe white labels in confocal images. Scale 20 μm. Statistical data: b,cn=10 animals; d-fn=7 animals; gn=2 animals; hn=4 animals; i,kn=6 animals.
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Figure 3: Perivascular glioma cell co-option causes a breakdown of the blood-brain barrierThe tracer, Evans blue, permeates into the brain parenchyma in tumor-bearing (n=3 animals) (bottom), but not in sham mice 3 weeks post surgery (n=3 animals) (top), Scale 5 mm. (a). Immunofluorescence of CD31 and intravenously injected tracers, tetramethylrhodamine-albumin (TRITC-alb, white) or -cadaverine (TRITC-cad, white), and implanted eGFP-expressing human glioma cells (D54) allow analysis of tracer leakage in relation to tumor burden. The large MW 70 kDa TRITC-albumin (b), as well as the small MW 950 Da TRITC-cadaverine (d) can be found outside the vasculature (arrows) (b-f, Suppl. Fig.6). Note that leaked TRITC-cadaverine is taken up by nearby neurons (arrows) (d,e). No extravasation was seen in the absence of tumor cells (c,f). To assess extravasation of the tracer Cascade blue (MW 10 kDa), Aldh1l1-eGFP-scid (eGFP) immunodeficient mice, which were previously implanted with TdTomato-expressing human glioma cells (D54), were retro-orbitally injected with Alexa Fluor 633 hydrazide dye (633 hyd) and Cascade blue (g,h). Accumulation of Cascade blue occurs in the brain's parenchyma indicating breakdown of the blood-brain barrier (BBB), where perivascular astrocytes have been displaced by glioma cell co-option (g). Vessels lacking tumor cells do not show extravasation of the dye (h). Immunohistochemistry for the TJ proteins zonula occludens-1 (ZO-1) (i) and claudin-5 (k) show that these protein are lost from endothelial cells labeled with CD31 where tumor cells are present (enlarged panel, arrows), but colocalize with endothelial cells in tissue that has not been co-opted. Quantification of ZO-1 shows reduction of vessel coverage in areas co-opted by glioma cells. 8.86 ± 0.64% vessel area is covered by ZO-1 in control images, whereas this number was reduced to 5.1 ± 1.11% in areas where vessels were coopted by tumor cells (n=9 images each for control and tumor-covered vessels from 3 animals, paired t-test directly comparing control and tumor images in the same slice, p=0.037, error bars refer to SEM) (j). Black font was used to describe white labels in confocal images. Scale 20 μm. Statistical data: b,cn=10 animals; d-fn=7 animals; gn=2 animals; hn=4 animals; i,kn=6 animals.

Mentions: Development of the BBB precedes astrocyte development and is induced by pericytes30, 31, yet maintenance of an intact barrier relies on soluble factors released by astrocytes and persistent contact between astrocytes and endothelial cells5, 32. Accordingly, pathological conditions that compromise astrocytes also present with an impaired BBB4. A leaky BBB is also typical for newly generated glioma vasculature, which lacks tight junctions33. This can be readily demonstrated by permeation of Evan's blue from the bloodstream into the brain parenchyma surrounding a large tumor mass (Fig. 3a).


Disruption of astrocyte-vascular coupling and the blood-brain barrier by invading glioma cells.

Watkins S, Robel S, Kimbrough IF, Robert SM, Ellis-Davies G, Sontheimer H - Nat Commun (2014)

Perivascular glioma cell co-option causes a breakdown of the blood-brain barrierThe tracer, Evans blue, permeates into the brain parenchyma in tumor-bearing (n=3 animals) (bottom), but not in sham mice 3 weeks post surgery (n=3 animals) (top), Scale 5 mm. (a). Immunofluorescence of CD31 and intravenously injected tracers, tetramethylrhodamine-albumin (TRITC-alb, white) or -cadaverine (TRITC-cad, white), and implanted eGFP-expressing human glioma cells (D54) allow analysis of tracer leakage in relation to tumor burden. The large MW 70 kDa TRITC-albumin (b), as well as the small MW 950 Da TRITC-cadaverine (d) can be found outside the vasculature (arrows) (b-f, Suppl. Fig.6). Note that leaked TRITC-cadaverine is taken up by nearby neurons (arrows) (d,e). No extravasation was seen in the absence of tumor cells (c,f). To assess extravasation of the tracer Cascade blue (MW 10 kDa), Aldh1l1-eGFP-scid (eGFP) immunodeficient mice, which were previously implanted with TdTomato-expressing human glioma cells (D54), were retro-orbitally injected with Alexa Fluor 633 hydrazide dye (633 hyd) and Cascade blue (g,h). Accumulation of Cascade blue occurs in the brain's parenchyma indicating breakdown of the blood-brain barrier (BBB), where perivascular astrocytes have been displaced by glioma cell co-option (g). Vessels lacking tumor cells do not show extravasation of the dye (h). Immunohistochemistry for the TJ proteins zonula occludens-1 (ZO-1) (i) and claudin-5 (k) show that these protein are lost from endothelial cells labeled with CD31 where tumor cells are present (enlarged panel, arrows), but colocalize with endothelial cells in tissue that has not been co-opted. Quantification of ZO-1 shows reduction of vessel coverage in areas co-opted by glioma cells. 8.86 ± 0.64% vessel area is covered by ZO-1 in control images, whereas this number was reduced to 5.1 ± 1.11% in areas where vessels were coopted by tumor cells (n=9 images each for control and tumor-covered vessels from 3 animals, paired t-test directly comparing control and tumor images in the same slice, p=0.037, error bars refer to SEM) (j). Black font was used to describe white labels in confocal images. Scale 20 μm. Statistical data: b,cn=10 animals; d-fn=7 animals; gn=2 animals; hn=4 animals; i,kn=6 animals.
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Figure 3: Perivascular glioma cell co-option causes a breakdown of the blood-brain barrierThe tracer, Evans blue, permeates into the brain parenchyma in tumor-bearing (n=3 animals) (bottom), but not in sham mice 3 weeks post surgery (n=3 animals) (top), Scale 5 mm. (a). Immunofluorescence of CD31 and intravenously injected tracers, tetramethylrhodamine-albumin (TRITC-alb, white) or -cadaverine (TRITC-cad, white), and implanted eGFP-expressing human glioma cells (D54) allow analysis of tracer leakage in relation to tumor burden. The large MW 70 kDa TRITC-albumin (b), as well as the small MW 950 Da TRITC-cadaverine (d) can be found outside the vasculature (arrows) (b-f, Suppl. Fig.6). Note that leaked TRITC-cadaverine is taken up by nearby neurons (arrows) (d,e). No extravasation was seen in the absence of tumor cells (c,f). To assess extravasation of the tracer Cascade blue (MW 10 kDa), Aldh1l1-eGFP-scid (eGFP) immunodeficient mice, which were previously implanted with TdTomato-expressing human glioma cells (D54), were retro-orbitally injected with Alexa Fluor 633 hydrazide dye (633 hyd) and Cascade blue (g,h). Accumulation of Cascade blue occurs in the brain's parenchyma indicating breakdown of the blood-brain barrier (BBB), where perivascular astrocytes have been displaced by glioma cell co-option (g). Vessels lacking tumor cells do not show extravasation of the dye (h). Immunohistochemistry for the TJ proteins zonula occludens-1 (ZO-1) (i) and claudin-5 (k) show that these protein are lost from endothelial cells labeled with CD31 where tumor cells are present (enlarged panel, arrows), but colocalize with endothelial cells in tissue that has not been co-opted. Quantification of ZO-1 shows reduction of vessel coverage in areas co-opted by glioma cells. 8.86 ± 0.64% vessel area is covered by ZO-1 in control images, whereas this number was reduced to 5.1 ± 1.11% in areas where vessels were coopted by tumor cells (n=9 images each for control and tumor-covered vessels from 3 animals, paired t-test directly comparing control and tumor images in the same slice, p=0.037, error bars refer to SEM) (j). Black font was used to describe white labels in confocal images. Scale 20 μm. Statistical data: b,cn=10 animals; d-fn=7 animals; gn=2 animals; hn=4 animals; i,kn=6 animals.
Mentions: Development of the BBB precedes astrocyte development and is induced by pericytes30, 31, yet maintenance of an intact barrier relies on soluble factors released by astrocytes and persistent contact between astrocytes and endothelial cells5, 32. Accordingly, pathological conditions that compromise astrocytes also present with an impaired BBB4. A leaky BBB is also typical for newly generated glioma vasculature, which lacks tight junctions33. This can be readily demonstrated by permeation of Evan's blue from the bloodstream into the brain parenchyma surrounding a large tumor mass (Fig. 3a).

Bottom Line: Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form.This causes a focal breach in the BBB.These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama at Birmingham, 1719 6th Avenue South, CIRC 425, Birmingham, Alabama 35294, USA [2].

ABSTRACT
Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumours that use the perivascular space for invasion and co-opt existing vessels as satellite tumour form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of preexisting vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over the regulation of vascular tone through Ca(2+)-dependent release of K(+). These findings have important clinical implications regarding blood flow in the tumour-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

Show MeSH
Related in: MedlinePlus