Name: live imaging of microtubule dynamics in glioblastoma cells invading the zebrafish brain
Uploaded: 2022-07-29T15:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain at JoVE.com

We are extremely grateful to Dr. P. Herbomel (Institut Pasteur, France) and his laboratory, especially Val&#233;rie Briolat, and Emma Colucci-Guyon for providing us with the zebrafish lines and the plastic mold for microinjection plates, and for their valuable expertise on zebrafish experimental procedures. We gratefully acknowledge the UtechS Photonic BioImaging (C2RT, Institut Pasteur, supported by the French National Research Agency France BioImaging, and ANR-10-INBS-04; Investments for the Future). This work was supported by the Ligue contre le cancer (EL2017.LNCC), the Centre National de la Recherche Scientifique, and Institut Pasteur and by the generous donations of Mrs.&#160;Marguerite MICHEL and Mr. Porquet.

Animal experiments were conducted according to European Union guidelines for the handling of laboratory animals. All protocols were approved by the Ethical Committee for Animal Experimentation of Institut Pasteur - CEEA 89 and the French Ministry of Research and Education (permit #01265.03). During injections or live imaging sessions, animals were anaesthetized with Tricaine.At the end of the experimental procedures, they were euthanized by anesthetic overdose. See the Table of Materials for details rela...

Imaging tumor xenografts at single-cell resolution is likely to become an indispensable tool to improve our understanding of GBM biology. Live imaging in mouse PDX models has led to valuable discoveries on how GBM collectively invades the brain tissue18. However, to date, the spatiotemporal resolution is not high enough to reveal the dynamics of proteins controlling GBM invasion. We reasoned that by coupling the orthotopic engrafting of GBM cells in transparent zebrafish larvae with high-resolutio...

Cell motility is a stereotyped process requiring polarity axis establishment and force-generating cytoskeletal rearrangements. Actin polymerization and its association with myosin are recognized as the main contributors to protrusive and contractile forces required for cell movement1. Microtubules are considered to be the main actors in cell polarization and directional persistence during migration2. In recent years, MTs have also been shown to create and stabilize protrusions to support mechanocompressive forces during cell invasion in 3D3. More recently, MTs have been directly involved in mechanotransduction at focal adhesions and mechanosensitive migration4. The dynamic instability that characterizes MT-plus end dynamics is made of repeated phases of polymerization (growth) and depolymerization (shrinkage), which are controlled by a plethora of microtubule-binding proteins and intracellular signaling cascades, such as those governed by RHO-GTPases5,6,7. The role of the MT network in cell migration and invasion has made the investigation of MT dynamics a key element to better understand the mechanisms of immune cell homing, wound healing, and cancer invasion.
The ability of cancer cells to escape the primary tumor core, spread in the tissues, and generate secondary tumors is a critical step in preventing global success in the war against cancer declared 50 years ago8,9. One of the biggest hurdles has been understanding how cancer cells actively invade the tissue. Key invasion mechanisms rely on the same principles as those governing non-tumorous cell migration10. However, cancer cell migration specificities have emerged11, triggering the need for better characterization of this type of migration. Specifically, because the tumor microenvironment appears as a key player in cancer progression12, observing and analyzing cancer cell invasion in a relevant physiological context is essential to unravel the mechanisms of cancer cell dissemination.
MTs are central to cancer progression, to sustain both proliferation and invasion. Precise analysis of MT dynamics in situ can help identify MT-altering agents (MTA) in both processes. MT dynamics vary drastically upon a change in environment. In vitro, treatment with MT-destabilizing agents such as nocodazole prevents cell protrusion formation when cells are embedded in gels in 3D, whereas it has little effect on 2D cell migration13,14. Although technically challenging, advances in intravital imaging permit in vivo analysis of MT dynamics during cancer cell invasion. For instance, the observation of MTs in subcutaneously xenografted fibrosarcoma cells in mice revealed that tumor-associated macrophages affect MT dynamics in tumor cells15. However, these mouse models involve extensive surgical procedures and remain unsatisfying for less accessible cancers, such as the highly invasive brain tumor, GBM.
Despite a dismal 15 month average survival time16, little is known about GBM&#39;s mode of dissemination within the brain parenchyma or the key molecular elements sustaining GBM cell invasion in the brain tissue. Improvement in the mouse orthotopic xenograft (PDX) model and the establishment of cranial windows offered new prospects for GBM cell invasion studies17,18. However, due to suboptimal imaging quality, this model has mostly permitted longitudinal imaging of superficial xenografts and has not been successfully used to study subcellular imaging of cytoskeleton proteins so far. Furthermore, in the wake of the &#34;3Rs&#34; injunction to reduce the use of rodents and replace them with lower vertebrates, alternative models have been established.
Taking advantage of the primitive immunity observed in zebrafish (Danio rerio) larvae, orthotopic injection of GBM cells in the fish brain was developed19,20,21. Injection in the vicinity of the ventricles in the developing midbrain recapitulates most of human GBM pathophysiology21, and the same preferred pattern of GBM invasion as in humans-vessel co-option-is observed22. Thanks to the transparency of the fish larvae, this model allows the visualization of GBM cells invading the brain from the peri-ventricular areas where most GBMs are thought to arise23.
Because MTs are essential for GBM cell invasion in vitro24,25, a better characterization of MT dynamics and the identification of key regulators during cell invasion is needed. However, to date, the data generated with the zebrafish orthotopic model has not included subcellular analysis of MT dynamics during the invasion process. This paper provides a protocol to study MT dynamics in vivo and determine its role during brain cancer invasion. Following stable microtubule labeling, GBM cells are microinjected at 3 dpf in zebrafish larvae&#39;s brains and imaged in real time at high spatio-temporal resolution during their progression in the brain tissue. Live imaging of fluorescent MTs allows the qualitative and quantitative analysis of MT plus-end dynamics. Furthermore, this model makes it possible to assess the effect of MTAs on MT dynamics and on the invasive properties of GBM cells in real time. This relatively non-invasive protocol combined with a large number of larvae handled at a time and the ease of drug application (in the fish water) makes the model an asset for preclinical testing.

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			<td>Modified Eagle&#39;s medium</td>
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			<td>Lenitivirus production</td>
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			<td>Fluorescence stereomicroscope LEICA M165FC</td>
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			<td>CellTram oil vario microinjector</td>
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live imaging of microtubule dynamics in glioblastoma cells invading the zebrafish brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

To analyze the role played by MTs during in vivo GBM invasion, we describe here the major steps to perform&#160;stable MT labeling in GBM cells by lentiviral infection, orthotopic xenotransplantation of GBM cells in 3 dpf zebrafish larvae, high-resolution intravital imaging of MT dynamics, MTA treatment and its effects on GBM invasion, and image analysis of MT dynamics and in vivo invasion (Figure 1). MT dynamics are measured either by building kymographs a...

Watch this Scientific Journal Video about Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain at JoVE.com

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...

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