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

Angiogenesis (2017) 20:533–546 DOI 10.1007/s10456-017-9565-6 ORIGINAL PAPER Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment Tonny Lagerweij1,2,3 • Sophie A. Dusoswa1,2,3,4 • Adrian Negrean7 • Esther M. L. Hendrikx4 • Helga E. de Vries4 • Jeroen Kole5 • Juan J. Garcia-Vallejo4 • Huibert D. Mansvelder7 • W. Peter Vandertop2,3 • David P. Noske1,2,3 • Bakhos A. Tannous9 • René J. P. Musters5 • Yvette van Kooyk4 • Pieter Wesseling1,2,6,8 • Xi Wen Zhao1,2,3 • Thomas Wurdinger1,2,3,9 Received: 22 May 2017 / Accepted: 27 June 2017 / Published online: 11 July 2017 Ó The Author(s) 2017. This article is an open access publication 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 Electronic supplementary material The online version of this article (doi:10.1007/s10456-017-9565-6) contains supplementary material, which is available to authorized users. 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 Vasculature
Imaging
3D
CLARITY
iDISCO
Multicellular network Tonny Lagerweij and Sophie A. Dusoswa have contributed equally to this work. & Thomas Wurdinger 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 Department of Physiology, VU University Medical Center, Amsterdam, The Netherlands 6 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 7 Integrative Neurophysiology, Centre for Neurogenomics and Cognitive Research, VU University, Amsterdam, The Netherlands 8 Princess Máxima Center for Pediatric Oncology, University Medical Center Utrecht, Utrecht, The Netherlands 9 Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 123 534 Angiogenesis (2017) 20:533–546 Introduction Methods Glioblastomas (GBMs) remain incurable, partly because of extensive, diffuse infiltration of the GBM cells into their surrounding microenvironment. GBM cell invasion and proliferation leads to changes in the microvasculature, tissue perfusion, and brain architecture. Increased awareness of spatial heterogeneity of the GBM cells, in relation to the microvasculature, and intervascular tissue microenvironment [1–4], has raised the need for 3D analyses of brain tumor tissues. Optical 3D analysis allows imaging of brain structures at cellular resolution and may serve as a bridge between CT, PET, or MRI and classical microscopic histology and immunohistochemistry [5, 6]. Intravital confocal microscopy enables 3D fluorescence imaging on a cellular level [7, 8], but its use is hampered by sedation time of the animal, limited imaging depth, small field of view, and limitations associated with fluorescent labeling [8]. These limitations do not apply to ex vivo optical imaging. For a long time, optical imaging of 3D structures was dependent on histological sectioning [3, 9, 10]. This sectioning is, however, a laborious and challenging task, since at least several dozens of histological slices have to be obtained and properly aligned for the creation of an informative 3D image. To avoid these laborious and error-prone approaches, optical slicing methods were developed. Optical slicing involves clearing of tissues to make them transparent, thus enabling deep-tissue fluorophore excitation and detection. Although optical clearing techniques were described already more than a century ago [11], the interest in this approach was boosted by the development of more advanced clearing techniques such as 3DISCO/iDISCO/ uDISCO, Scale, SeeDB, and CLARITY [12–20], which all have their specific advantages and disadvantages [18, 21]. Besides new clearing techniques, other major contributions to optical slicing methods were the development and improvement of equipment such as multi-photon microscopes and light sheet microscopes. Furthermore, numerous relevant software tools have been developed, including ImageJ, Vaa3D, Farsight, NeuronStudio, Amira, and Imaris [22–25]. Here, we employed optical clearance methods to study GBM cells in the mouse brain microenvironment. We demonstrate that optically cleared tissues can be imaged up to at least 2000 lm depth, at subcellular resolution. This allowed detailed 3D visualization of the brain tumor microenvironment and revealed patterns of networks of collectively invading GBM cells. Animal care guidelines 123 All animal experiments were approved by the VU University Medical Center Animal Welfare review board. Female, specific pathogen-free, athymic nude-Foxn1nu mice (6–8 weeks; Harlan/Envigo, The Netherlands) were kept in filter top cages and received food and water ad libitum. Intravital confocal imaging Application of a cranial window for intravital imaging of the mouse brain was based on the method as described by Mostany et al. [26]. Three mice were anesthetized by isoflurane inhalation and received temgesic (0.05 mg/kg) preoperatively and dexamethasone (0.2 mg/kg) with carprufen (5 mg/kg) postoperatively to prevent (...truncated)