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.

The authors have no conflicts of interest to disclose.

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-resolution intravital imaging, cytoskeletal proteins such as MTs could be analyzed in sufficient detail to analyze their dynamics during in situ GBM invasion.
The critical steps of the methodology lie in the preparation of the GBM cells and the microinjection procedure. Unhealthy and inadequately dissociated cells will stick together or to the capillary borders and block the flow of injection. In addition, cells need to be sufficiently concentrated in the capillary to minimize the injected volume and implant them in bulk. Injecting a higher volume of more diluted cells will result in multiple, sometimes intermixed tumor foci, whose invasive indexes become difficult to measure. In our hands, manual handling of the oil-based microinjector allows better control of the injection flow than a pressurized air-based electronic microinjector that has been used previously in a similar model19. This is critical to prevent excess flow pressure inside the brain, thereby avoiding subsequent tissue damage and ventricular aggregation of the injected cells.
Some limitations of this model include the necessity to perform the experiment at suboptimal temperatures for both species. Zebrafish are usually raised at 28 &#176;C, whereas human cells are cultured at 37 &#176;C. Above 32 &#176;C, zebrafish embryo development is altered and these changes can be lethal35. However, similar to what is done in adult zebrafish xenograft models36, sequentially acclimating the zebrafish larvae to a temperature of 32 &#176;C increases the survival of transplanted animals compared to the rapid change in temperature post transplantation from 28 &#176;C to 32 &#176;C. However, increasing the temperature further leads to increased animal deaths in accordance with the sensitivity of zebrafish embryos to temperatures above 32 &#176;C35.
Interpreting the in vivo MT dynamics data has to be done carefully as MT dynamics change when the temperature drops below 37 &#176;C37. Parallel in vitro measurement of MT dynamics at 37 &#176;C and 32 &#176;C in the same GBM cells with the same MTA treatment will help validate the differences seen between various GBM cells or between in vivo treatments. It should help confirm that the differences are not caused by variation in temperature sensitivity but by different regulation pathways (for GBM comparison analysis) or by MTA treatment (for MTA effect analysis). This will be of interest should the MT dynamics heterogeneities be linked to different invasion abilities.
Another limitation is the short time window during which invasion can be monitored (72 to 96 h), preventing the measurement of invasion plasticity driven by potential changes in MT dynamics38. After 96 h, we noticed a sharp decrease in GBM cell invasion. At 6 days post injection, the number of GBM cells declined rapidly, presumably due to a host immune response caused by the accumulation of neutrophils and macrophages in the tumor microenvironment39. Delivering MTAs to the whole brain is likely to affect nearby neuronal host cells, which depend on MTs for their activity and whose alteration might subsequently affect GBM invasion40. This approach needs to be complemented with shRNA or optogenetics assays restricting MT alteration to the GBM cells, but remains a good platform to screen for new anti-invasive compounds.
The orthotopic injection of MT-labeled GBM cells in the zebrafish brain is of particular interest to decipher the role of MTs during cancer cell invasion, as very few animal models permit in situ subcellular imaging of cancer cell migration in their tissue of origin15. To date, studies of MT functions during GBM migration rely mostly on in vitro and ex vivo assays and lack in vivo validation24,41,42,43. Coupled with gene-of-interest knock down or an unbiased gene-based screening approach, the assay presented here will help reveal new MT regulators that are important for GBM invasion in vivo.
GBMs are highly heterogeneous tumors whose invasive properties differ greatly between specimens44. Understanding the molecular mechanisms underlying their different mode of invasion will help define ad hoc therapeutic treatments to block GBM dissemination. Systematic measurement of the invasive index, mode of invasion, and cytoskeletal properties such as MT dynamics in various GBM samples will reveal new correlations between frequent genomic mutational profiles and cell invasion patterns relying on specific cytoskeletal properties. Revealing how these mutations affect the change in MT dynamics not only would add to our knowledge on MT regulation during cell migration45,46 but could also lead to long-awaited patient-specific, anti-invasive therapeutics.
The relative ease of microinjecting in zebrafish combined with the high number of larvae available and the ease of injection of drugs make this procedure suitable for personalized medicine47,48. Moreover, in contrast to intravital imaging of GBM xenografts in mice, that only occurs in the upper 500 &#181;m part of the cortex49,50, using zebrafish allows for the visualization of GBM infiltration in the whole CNS. The model presented here meets the criteria to become an invaluable tool for rapid analysis of glioblastoma&#39;s invasive capacities and its response to treatments.

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.

<table><tbody><tr><td>&lt;strong&gt;Glioblastoma cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Foetal calf serum</td><td>Eurobio</td><td>CVFSVF00-01</td><td>Reagent</td></tr><tr><td>MEM NEAA</td><td>Gibco</td><td>11140-050</td><td>Reagent</td></tr><tr><td>Modified Eagle&#039;s medium</td><td>Eurobio</td><td>CM1MEM18-01</td><td>Reagent</td></tr><tr><td>Penicillin&amp;ndash;streptomycin</td><td>Gibco</td><td>15140-122</td><td>Reagent</td></tr><tr><td>U-87 MG</td><td>ECACC</td><td>89081402-1VL</td><td>Cells</td></tr><tr><td>&lt;strong&gt;Lenitivirus production&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD FACSAria III</td><td>BD bioscience</td><td></td><td>Instrument</td></tr><tr><td>BD FACSDiva software v8.0</td><td>BD bioscience</td><td></td><td>Software</td></tr><tr><td>HEK-293T</td><td>Merck</td><td>12022001</td><td>Cells</td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>Plasmid #12259</td><td>Reagent</td></tr><tr><td>psPAX2</td><td>Addgene</td><td>Plasmid #12260</td><td>Reagent</td></tr><tr><td>Ultracentrifuge Optima XPN-80</td><td>Beckman Coulter</td><td></td><td>Instrument</td></tr><tr><td>&lt;strong&gt;Cell passaging and staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>dPBS</td><td>Gibco</td><td>14190-094</td><td>Chemical</td></tr><tr><td>&lt;em&gt;Hoechst 34580&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>63493</td><td>Chemical</td></tr><tr><td>Trypsin-EDTA (0,05%)</td><td>Gibco</td><td>25300-054</td><td>Reagent</td></tr><tr><td>&lt;strong&gt;Zebrafish husbandry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fluorescence stereomicroscope LEICA M165FC</td><td>LEICA</td><td>https://www.leica-microsystems.com/fr/produits/stereomicroscopes-et-macroscopes/informations-detaillees/leica-m165-fc/</td><td>Instrument</td></tr><tr><td>Methylene Blue hydrate</td><td>Sigma-Aldrich</td><td>M4159</td><td>Chemical</td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629-25G</td><td>Chemical</td></tr><tr><td>Transfer Pipettes fine tips</td><td>Samco Scientific</td><td>232</td><td>Equipment</td></tr><tr><td>Transfer Pipettes Large Bulb3mL</td><td>Samco Scientific</td><td>225</td><td>Equipment</td></tr><tr><td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td><td>Sigma-Aldrich</td><td>Cat#: A5040</td><td>Chemical</td></tr><tr><td>Volvic Source Water</td><td>DUTSCHER DOMINIQUE SAS</td><td>999556</td><td>Reagent</td></tr><tr><td>&lt;strong&gt;Xenotransplantation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>24-well plate</td><td>TPP</td><td>92024</td><td>Equipment</td></tr><tr><td>Borosilicate glass capillaries (1.0 ODx0.58IDx150L mm)</td><td>Harvard Apparatus</td><td>(#30-0017 GC100-15</td><td>Equipment</td></tr><tr><td>CellTram oil vario microinjector</td><td>Eppendorf</td><td>5176000.025</td><td>Instrument</td></tr><tr><td>Microloading pipet tips (Microloader) 20&amp;micro;L</td><td>Eppendorf</td><td>&amp;nbsp;5242956003</td><td>Equipment</td></tr><tr><td>Micromanipulator</td><td>NARISHIGE</td><td>https://products.narishige-group.com/group1/injection/english.html</td><td>Equipment</td></tr><tr><td>Mineral Oil</td><td>Sigma</td><td>M8410-100ml</td><td>Equipment</td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>KL 2500 LCD</td><td>Instrument</td></tr><tr><td>Universal capillary holder</td><td>Eppendorf</td><td>5176190002</td><td>Equipment</td></tr><tr><td>Vertical Pipette puller</td><td>KOPF (Roucaire)</td><td>Model 720</td><td>Instrument</td></tr><tr><td>&lt;strong&gt;Intravital Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3.5cm glass-bottom videoimaging dish</td><td>MatTek Life Sciences, MA, USA</td><td>P35G-1,5-14-C</td><td>Equipment</td></tr><tr><td>Acquisition software: NIS-Elements-AR version 5.21</td><td>Nikon</td><td></td><td>Software</td></tr><tr><td>Heat-Block</td><td>Techne</td><td>DRI-BLOCK DB-2D</td><td>Equipment</td></tr><tr><td>Microscope head Nikon Ti2E</td><td>Nikon</td><td></td><td>Instrument</td></tr><tr><td>sCMOS camera Prime 95B</td><td>Photometrics</td><td></td><td>Instrument</td></tr><tr><td>sCMOS camera Orca Flash 4</td><td>Hammatsu</td><td></td><td>Instrument</td></tr><tr><td>Ultrapure Low melting point agarose</td><td>Invitrogen</td><td>16520-050</td><td>Chemical</td></tr><tr><td>Yokagawa CSU-W1 spinning disk unit</td><td>Hammatsu</td><td></td><td>Instrument</td></tr><tr><td>&lt;strong&gt;Drug Treatment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650-100ML</td><td>Chemical</td></tr><tr><td>Nocodazole</td><td>Sigma-Aldrich</td><td>M1404-2MG</td><td>Chemical</td></tr><tr><td>&lt;strong&gt;Image Analysis&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Imaris 9.5.1 software</td><td>Oxford Instruments</td><td></td><td>Software</td></tr><tr><td>ImarisFileConverter 9.5.1</td><td>Oxford Instruments</td><td></td><td>Software</td></tr></tbody></table>

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 along growing and shrinking MTs (Figure 3C) or by manually tracking individual MT edges over time (Figure 3D). An example of drug treatment administered in the larvae medium and its reversible effect on MT network organization is given in Figure 4. Treatment with a low dose of nocodazole (200 nM) leads to progressive shrinkage of the MT network and disappearance of glioblastoma cell protrusion 4 h later (Figure 4A). Washing out the drug restored the capacity of glioblastoma cells to form protrusions. The cells resumed migrating along the vasculature 12 h after the washout (Figure 4B). These data suggest treatment with 200 nM nocodazole is sufficient to disrupt the MT network and rapidly blocks in vivo glioblastoma cell invasion. A 3 day-long analysis of the same treatment on global glioblastoma cell invasion reveals that nocodazole 200 nM halts long-term glioblastoma cell invasion in vivo, without affecting the general health of the fish, compared to a control (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64093/64093fig01.jpg" /> 
Figure 1: Protocol workflow diagram. Abbreviations: dpf = days post fertilization; hpi = hours post injection; MT = microtubule; MTA = microtubule-altering agent; GBM = glioblastoma multiforme. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64093/64093fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64093/64093fig02.jpg" /> 
Figure 2: Microinjecting glioblastoma cells into 3 dpf zebrafish larvae brains. (A) Photograph of the equipment used for xenotransplantation: 1, oil microinjector; 2, mechanical micromanipulator; 3, universal capillary holder; 4, glass capillary. The microinjection plate is under a stereomicroscope. (B) Photograph showing anesthetized 3 dfp zebrafish larvae aligned in a trench and ready to be microinjected. The tip of a microcapillary loaded with a red dye is visible on the right of the photograph. Details of the patterned trenches built in the agarose plate are seen in the inset on the bottom right corner. Scale bar = 3 mm. (C)&#160;Scheme of a representative microinjection plate. Ready-to-be-injected larvae are placed laterally (center of the plate). Note that cells are concentrated at the tip of the microcapillary (black arrow) before proceeding to the injection. Injected larvae are shown on the right of the plate, placed ventrally. (D)&#160;Scheme of a transversal slice in the microinjection plate (along the black dotted line in C) showing the trenches where the larvae are placed during injection. (E) Photograph showing the tip of the capillary ready to penetrate the optic tectum (dotted line). The ventricles are delineated by the white lines. Scale bar = 150 &#181;m. (F) Scheme of a 3 dpf fli1a:gfp larva expressing gfp in the endothelial cells, indicating the OT region where the cells are injected, just above the middle cerebral vein. (G)&#160;Fluorescence image of a 3 dpf gfap:gfp larva (gfp expressed in neural stem cells) placed laterally after microinjection of U87-mkate2 cells (white circle and white arrow). High autofluorescence in red is caused by the iridophores in the eyes. Scale bar = 100 &#181;m. (H) Confocal fluorescence image of a successfully injected fli1a:gfp larva at 16 hpi. Scale bar: 50 &#181;m. (I-K) Confocal fluorescence images of unsuccessfully injected fli1a:gfp larvae at 96 hpi (I) and 4 hpi (J,K).&#160;GBM cells have been injected in ventricles (I,J) or in multiple foci in the brain (K). Scale bars = 50 &#181;m. Abbreviations: dpf = days post fertilization; OT = optic tectum; MCeV = middle cerebral vein; gfp = green fluorescent protein; gfap = glial fibrillary acidic protein; hpi = hours post injection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64093/64093fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64093/64093fig03.jpg" /> 
Figure 3: Visualizing in vivo microtubule dynamics in glioblastoma cells. (A) Representative fluorescence image of xenografted U87 cells expressing tubulin-&#945;1-mkate2 in the OT of a fli1a:GFP zebrafish larva at 20 hpi. (B) Maximum intensity projected fluorescence image of the MT network in a single xenografted U87 cell. (C) Kymograph along the red dotted line in B, showing the growing, the pause, and the catastrophe phases of MT dynamic instability. (D) Time-lapse sequence of the boxed region in B highlighting the tracking of three MT + ends. Scale bars = 10 &#181;m. Abbreviations: OT = optic tectum; GFP = green fluorescent protein; hpi = hours post injection; MT = microtubule; G = growing phase; P = pause phase; C = catastrophe phase. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64093/64093fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64093/64093fig04.jpg" /> 
Figure 4: Visualizing the effects of the microtubule altering agent on glioblastoma invasion in vivo. (A) Time-lapse sequence of U87 cells expressing tubulin-&#945;1a-mkate2 in zebrafish larvae treated with nocadazole (200 nM). Arrows point to the extremity of the protrusion in two different cells invading the brain along a blood vessel. Note the retraction of the protrusion upon treatment with nocodazole. Scale bar = 10 &#181;m. (B) Time-lapse sequence representing the effect of nocodazole washout on U87 cell invasion. At 500 min after the washout, the cell marked with the white asterisk elongates an MT-based protrusion (white arrow), which permits its resumption of the invasion along a blood vessel. Scale bar = 20 &#181;m. (C) 3D representations of a xenografted larvae brain treated with DMSO or nocodazole (200 nM) for 72 h. U87 cells signal has been segmented (in red) and integrated to the fli1a-GFP fluorescence signal (in white). Note the decreased dissemination of U87 cells treated with nocodazole. Scale bar = 30 &#181;m. Abbreviations: GFP = green fluorescent protein; hpi = hours post injection; MT = microtubule. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64093/64093fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64093/64093fig05.jpg" /> 
Figure 5: Image analysis of in vivo glioblastoma invasion. (A) Fluorescent images of xenografted U87 cells expressing cytosolic mKate2 in fli1a-GFP zebrafish larvae, 4 hpi. White asterisks underline typical autofluorescence from eye iridophores. Scale bar = 40 &#181;m. (B) Fluorescent image of fli1a-GFP larvae coupled with the segmented surface corresponding to the U87 cells signal in A. The centroid of the tumor mass appears green. Scale bar = 40 &#181;m. (C) Fluorescent image representing the red channel signal in A and the newly defined &#34;distance to centroid&#34; channel (in blue). Scale bar = 30 &#181;m. (D)&#160;Red channel fluorescence image superimposed with colored spots, whose color represents the distance of the cell to the centroid (in green), violet being the closest to the centroid and white being the furthest apart.Scale bar = 20 &#181;m. (E) An example of sequential analysis of global GBM invasion. 3D distances are determined at 4 hpi and 72 hpi, and the invasion index (II) is calculated according to the formula in the inbox. Scale bar = 20 &#181;m. Abbreviations: GFP = green fluorescent protein; hpi = hours post injection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64093/64093fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</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 ...

live-imaging-microtubule-dynamics-glioblastoma-cells-invading

Nanyang Technological University

Research

JoVE Journal

Biology

2.6K Views.  Université Paris-Cité, Institut Pasteur, Équipe Labellisée Ligue Contre le Cancer. Deze methode is gewijd aan het onderzoek naar de rol van microtubuli tijdens de invasie van hersenkanker door de atopische injectie van hersenkankercellen in zebravissen te koppelen aan de subcellulaire intravitale beeldvorming. Het belangrijkste voordeel van de techniek ligt in de hoge ruimtelijke temporele resolutie waarbij het microtubulicytoskelet wordt verkregen. Het is uniek voor in vivo invasiemodellen voor hersenkanker en het maakt diepgaande microtubule dynamica-analyse mogelijk....

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

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neuroscience

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Cancer Research

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Developmental Biology

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Elucidating Microglia Behavior in Zebrafish Optic Tectum Development

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells' migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.

Author Spotlight: Using Zebrafish to Explore Microglia Migration During Brain Development

We demonstrate a method that takes advantage of fast scanning confocal microscopy to perform live imaging of microglia cells in the developing zebrafish optic tectum, allowing for the analysis of the dynamics of these cells in vivo.

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and ...

Immunology and Infection

In Vivo Whole-Brain Imaging of Zebrafish Larvae

In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy. The critical steps in the protocol are also discussed, including sample mounting and positioning, preventing bubble formation and dust in the agarose gel, and avoiding motion in images caused by incomplete solidification of the agarose gel and paralyzation of the fish. The protocol has been validated and confirmed in multiple settings. This protocol can be easily adapted for imaging other organs of a larval zebrafish.

Author Spotlight: In Vivo Whole-Brain Imaging of Zebrafish Larvae Using Three-Dimensional Fluorescence Microscopy  

Presented here is a protocol for in vivo whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy. The experimental procedure includes sample preparation, image acquisition, and visualization.

As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the ...

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

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