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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glioblastoma cell culture</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&#39;s medium</td>
			<td>Eurobio</td>
			<td>CM1MEM18-01</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Penicillin&#8211;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>Lenitivirus production</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>
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		<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>Cell passaging and staining</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>Hoechst 34580</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>Zebrafish husbandry</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>Xenotransplantation</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&#181;L</td>
			<td>Eppendorf</td>
			<td>&#160;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>Intravital Imaging</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>Drug Treatment</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>Image Analysis</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. 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.

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

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 ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

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 ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

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

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Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

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

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

4D Imaging of Protein Aggregation in Live Cells

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury