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Angiogenesis. 2017 Nov;20(4):533-546. doi: 10.1007/s10456-017-9565-6. Epub 2017 Jul 11.

Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment.

Author information

1
Neuro-oncology Research Group, VU University Medical Center, CCA Room 3.60, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.
2
Brain Tumor Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands.
3
Department of Neurosurgery, VU University Medical Center, Amsterdam, The Netherlands.
4
Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands.
5
Integrative Neurophysiology, Centre for Neurogenomics and Cognitive Research, VU University, Amsterdam, The Netherlands.
6
Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands.
7
Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
8
Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands.
9
Princess Máxima Center for Pediatric Oncology, University Medical Center Utrecht, Utrecht, The Netherlands.
10
Neuro-oncology Research Group, VU University Medical Center, CCA Room 3.60, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands. t.wurdinger@vumc.nl.
11
Brain Tumor Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands. t.wurdinger@vumc.nl.
12
Department of Neurosurgery, VU University Medical Center, Amsterdam, The Netherlands. t.wurdinger@vumc.nl.
13
Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. t.wurdinger@vumc.nl.

Abstract

BACKGROUND:

Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and migration of cells in the brain of mice, however, with limited imaging depth. To enable comprehensive analysis of GBM and the brain microenvironment, in-depth 3D imaging methods are needed. Here, we employed methods for optical tissue clearing prior to 3D microscopy to visualize the brain microvasculature and routes of invasion of GBM cells.

METHODS:

We present a workflow for ex vivo imaging of optically cleared brain tumor tissues and subsequent computational modeling. This workflow was used for quantification of the microvasculature in relation to nuclear or cellular density in healthy mouse brain tissues and in human orthotopic, infiltrative GBM8 and E98 glioblastoma models.

RESULTS:

Ex vivo cleared mouse brain tissues had a >10-fold imaging depth as compared to intravital imaging of mouse brain in vivo. Imaging of optically cleared brain tissue allowed quantification of the 3D microvascular characteristics in healthy mouse brains and in tissues with diffuse, infiltrative growing GBM8 brain tumors. Detailed 3D visualization revealed the organization of tumor cells relative to the vasculature, in both gray matter and white matter regions, and patterns of multicellular GBM networks collectively invading the brain parenchyma.

CONCLUSIONS:

Optical tissue clearing opens new avenues for combined quantitative and 3D microscopic analysis of the topographical relationship between GBM cells and their microenvironment.

KEYWORDS:

3D; CLARITY; Imaging; Multicellular network; Vasculature; iDISCO

PMID:
28699046
PMCID:
PMC5660146
DOI:
10.1007/s10456-017-9565-6
[Indexed for MEDLINE]
Free PMC Article

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