This study was supported by CCSRI Grant #700537 and CIHR Grant #84535 to JDL and NIH/NCI grant #CA120711-01A1 and CA120711-01A1 to AZ. All experiments were performed in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee at the University of California San Diego, Animal Care and Use at the University of Western Ontario.

1. Inoculation of tumor into avian embryo CAM
<ol>
<li>Prepare shell-less fertilized chicken embryos as described8.</li>
<li>On Day 9 of embryonic development, assemble a micro-injector using an 18-gauge needle connected onto 1 mL syringe. Cut a 5-6 inches piece of Tygon tubing (1/32" inner diameter, 3/32" outer diameter,1/32" wall thickness) and carefully insert bevel of needle into the tubing. Approximately 4-5 inches of tubing should extend from the tip of the needle (Figure 1a).</li>
<li>Fill the syringe and tubing with cell suspension. Then, insert a microinjection glass needle at the end of the tubing and carefully remove any air bubbles.</li>
<li>Inject Day 9 embryos (Figure 1b) under a dissection scope with an illuminator with 10,000-100,000 cancer cells as a bolus within the CAM (Figure 1c). Slowly inject cells and carefully ensure that the needle is in the correct place for the cells to form a visible bolus within the CAM. Cells that drip onto the CAM surface can be cleaned using Kimwipe or other applicator. </li>
<li>Return embryos to humidified incubator at 38&deg;C at 60% humidity and allow tumor to grow and vascularize (up to 7 days).</li>
</ol>
2. Preparation of nanoparticles
<ol>
<li>To prepare fluorescently labeled CPMV nanoparticles for injection into the chicken embryo; dilute the viral nanoparticles (synthesized as in 8 in PBS, pH 7.4, to a concentration of 100 &mu;g/ml. Vortex mixture well before using and centrifuge for one minute to remove any aggregates. Sonication may also be useful depending on conjugates and degree of aggregation. Stocks of fluorescently labeled CPMVs are stable for at least 1 year when stored at 4&deg;C in the dark. Check stocks periodically by putting a couple drops onto a glass slide and checking for fluorescence. Transmission Electron Microscopy (TEM) can also be used to determine if the particles remained intact after conjugation. </li>
<li>Briefly, one microgram of viral nanoparticles (in a final volume of 2 &mu;l) can be added to copper coated grids. Next, add an equal volume of 2% uranyl acetate for 1 minute at electron microscope (Philips EM 410).</li>
</ol>
3. Intravenous injection of fluorescently-labeled viral nanoparticles
<ol>
<li>On Day 16, assemble micro-injector as described above. Draw up desired volume of viral nanoparticle through the tubing into the syringe (&gt; 200 &mu;L). Carefully remove any air bubbles. Insert microinjection glass needle at the end of the tubing. Ensure that the needle is as long and tapered as possible (Figure 2a). If the needle is too blunt, it will not be able to pierce through the ectoderm and will fail to penetrate the vessel. If the needle is too sharp, the tip will collapse when penetrating tissue.</li>
<li>Intravenously inject 100 &mu;l of 1 mg/ml fluorescein dextran (MW 70,000 Da, Invitrogen) into tumor bearing embryo distal from the desired site to be visualized (Figure 3d). Successful cannulation of CAM vein is evident by the clearing of blood in the path of the injection flow (Figure 2b). Assess vascular leak 1 hour after injection. </li>
<li>Intravenously inject 50 &mu;l of 1 mg/ml solution of CPMV-Alexa Fluor 647 (CPMV-AF 647) or PEGylated CPMV-Alexa Fluor 647 (CPMV-PEG-AF 647) into embryos containing tumors with vascular leak distal from the desired site to be visualized.</li>
<li>Image tumors immediately and every hour after injection using a Zeiss Examiner Z1 upright microscope fitted with a Yokogawa spinning disk, Hamamatsu ImagEM 9100-12 EM-CCD camera.</li>
</ol>
4. Real time intravital imaging
<ol>
<li>To assemble the embryo imaging unit, apply a thin layer of vacuum grease around the circumference of the imaging port on the underside of the lid of the embryo imaging unit, and fit an 18 mm glass coverslip onto the port.</li>
<li>Position the embryo such that the coverslip will cover the desired area for imaging. Slowly lower the lid until the coverslip just makes contact with the embryo, and then screw the lid onto the unit to hold it in place (Figure 2c).</li>
<li>Add water heated to 37&deg;C into the embryo imaging unit outside of the dish containing the embryo, and then place the entire unit onto the stage of an upright confocal microscope with the inside environmental chamber equilibrated to 37&deg;C. </li>
<li>Position the imaging unit containing the embryo under the Spinning disk confocal fluorescence microscope (Figure 2e). The imaging unit will hold the embryo in place and keep the field of view fixed while capturing images, allowing for both three-dimensional Z stacks and time-lapse images to be captured. We acquire and analyze three-dimensional time-lapse images using Perkin Elmer&#x2019;s (formerly Improvision) Volocity software package (Figure 3a). Acquire a high-resolution three-dimensional stack of the tumor and surrounding vasculature to visualize detailed structural analyses at specific time-points. Acquire three-dimensional stacks at regular time-points to map detailed structural changes in the tumor vasculature. Image tumors every hour after injection.</li>
<li>Quantitate the uptake of viral nanoparticles by calculating the mean Alexa Fluor (AF) 647 signal within selected regions in the tumor or in the stroma (non-tumor area) using image quantitation software such as Volocity (Perkin Elmer). The tumor to stroma ratio was calculated by dividing the mean AF 647 signal in the tumor over the mean AF 647 signal in the stroma. A tumor/stroma ratio higher than 1 indicates that the nanoparticle is being taken up by the tumor.</li>
</ol>
5. Representative results:
In the example described here, we injected HT-29 colon cancer cells to form a bolus approximately 1mm in size within the CAM of day 9 chicken embryos (Figure 1b). After inoculation, the embryos were cultured for 7 days in a humidified incubator to permit sufficient tumor growth and vascularization (Figure 1c). Embryos were injected intravenously with a low molecular weight dextran to confirm tumor vascularization, and the tumors were visualized under the Zeiss AxioExaminer Z1 upright microscope (Figure 2d).
After intravenous administration of CPMV-AF 647 or CPMV-PEG-AF 647 nanoparticles (Figure 3a and b), high-resolution real time confocal imaging (Figure 2e) revealed that both CPMV and CPMV-PEG nanoparticles rapidly labeled the entire vasculature, but the uptake of CPMV-PEG by the tumor was approximately 3 times higher than CPMV after 12 hours (Figure 3a). The relative tumor uptake of nanoparticles was determined using image analysis software (Volocity from Perkin Elmer). Regions of interest were selected within and outside the tumor (in the stromal compartment) and the mean fluorescence intensity of each was determined. Data is expressed as tumor/stroma ratio. 
<img src="/files/ftp_upload/2808/2808fig1.jpg" alt="Figure 1"/> 
Figure 1.Microinjection of tumors into the CAM of an avian embryo. (a) The microinjection apparatus is assembled from the components as indicated. (b) Avian embryos at day 9 are ready to be inoculated with tumor when the CAM has spread to cover the entire surface. (c) At day 16, the tumor will have grown to up to 1 cm in diameter (dashed line) and is ready for injection with nanoparticles.
<img src="/files/ftp_upload/2808/2808fig2.jpg" alt="Figure 2"/>
 Figure 2.Nanoparticle injection and intravital imaging. Injection under a dissection microscope showing (a) the tip of the microinjector needle ready to be injected into the CAM vein and (b) the microinjector needle inserted into the vein (indicated by arrow) and nanoparticles injected into the blood flow (seen by clearing of blood). (c) Imaging unit containing avian embryo with the coverslip interfaced directly with the CAM. (d) Prior to nanoparticle injection, tumor vascularization is assessed using intravital imaging after injection of fluorescein dextran. (e) Imaging unit containing the embryo positioned on the stage of an upright confocal microscope within a temperature-regulated enclosure set to 37&deg;C.
<img src="/files/ftp_upload/2808/2808fig3.jpg" alt="Figure 3"/>
 Figure 3.Intravital visualization of nanoparticle uptake in human tumors. Tumors are visualized 7 hours after injection of (a) CPMV-AF647 and (b) CPMV-PEG-AF647. d) Excision of tumor from avian embryo for subsequent analysis.

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	<li>Halin, C., Mora, J. R., Sumen, C., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+lymphocyte+trafficking.">In vivo imaging of lymphocyte trafficking.</a> Annu Rev Cell Dev Biol. 21, 581-603 (2005).</li><li>Judenhofer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+PET-MRI:+a+new+approach+for+functional+and+morphological+imaging.">Simultaneous PET-MRI: a new approach for functional and morphological imaging.</a> Nat Med. 14, 459-465 (2008).</li><li>Weissleder, R., Pittet, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+in+the+era+of+molecular+oncology.">Imaging in the era of molecular oncology.</a> Nature. 452, 580-589 (2008).</li><li>Chambers, A. F., Ling, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+for+experimental+metastatic+ability+of+heterologous+tumor+cells+in+the+chick+embryo+after+DNA-mediated+transfer.">Selection for experimental metastatic ability of heterologous tumor cells in the chick embryo after DNA-mediated transfer.</a> Cancer Res. 44, 3970-3975 (1984).</li><li>Cretu, A., Fotos, J. S., Little, B. W., Galileo, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+rat+glioma+growth,+invasion,+and+vascularization+in+a+novel+chick+embryo+brain+tumor+model.">Human and rat glioma growth, invasion, and vascularization in a novel chick embryo brain tumor model.</a> Clin Exp Metastasis. 22, 225-236 (2005).</li><li>Zijlstra, A., Lewis, J., Degryse, B., Stuhlmann, H., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibition+of+tumor+cell+intravasation+and+subsequent+metastasis+via+regulation+of+in+vivo+tumor+cell+motility+by+the+tetraspanin+CD151.">The inhibition of tumor cell intravasation and subsequent metastasis via regulation of in vivo tumor cell motility by the tetraspanin CD151.</a> Cancer cell. 13, 221-234 (2008).</li><li>Lewis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+nanoparticles+as+tools+for+intravital+vascular+imaging.">Viral nanoparticles as tools for intravital vascular imaging.</a> Nature medicine. 12, 354-360 (2006).</li><li>Leong, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+embryonic+and+tumor+neovasculature+using+viral+nanoparticles.">Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles.</a> Nature protocols. 5, 1406-1417 (2010).</li><li>Chatterji, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+addresses+on+an+addressable+virus+nanoblock;+uniquely+reactive+Lys+residues+on+cowpea+mosaic+virus.">New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus.</a> Chemistry &#38; biology. 11, 855-863 (2004).</li><li>Brunel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrazone+ligation+strategy+to+assemble+multifunctional+viral+nanoparticles+for+cell+imaging+and+tumor+targeting.">Hydrazone ligation strategy to assemble multifunctional viral nanoparticles for cell imaging and tumor targeting.</a> Nano letters. 10, 1093-1097 (2010).</li><li>Steinmetz, N. F., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated+viral+nanoparticles+for+biomedicine:+the+impact+of+PEG+chain+length+on+VNP+cell+interactions+in+vitro+and+ex+vivo.">PEGylated viral nanoparticles for biomedicine: the impact of PEG chain length on VNP cell interactions in vitro and ex vivo.</a> Biomacromolecules. 10, 784-792 (2009).</li><li>Steinmetz, N. F., Cho, C. F., Ablack, A., Lewis, J. D., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CPMV+nanoparticles+target+surface+vimentin+on+cancer+cells.">CPMV nanoparticles target surface vimentin on cancer cells.</a> Nanomedicine. , (2010).</li></ol>

No conflicts of interest declared.

The chorioallantoic membrane (CAM) of the avian embryo is a useful model to assess the vascular dynamics and pharmacokinetics of human tumors. The structure and position of the CAM allows high quality image acquisition and accommodates of many kinds of cancer xenografts without invasive surgical procedures. Moreover, cancer tumor xenografts implanted into the chorioallantoic membrane become vascularized within 7 days, offering a rapid, inexpensive and semi-high-throughput means to assess the accumulation of nanoparticles in tumor tissue. Since cancer xenografts implanted in the CAM of shell-less chicken embryos are accessible to the high-resolution optics of an upright epifluorescence or confocal microscope, contextual and temporal information regarding nanoparticle uptake in the tumor vasculature can be readily obtained. Cancer xenografts in this model tend to grow laterally across the CAM, resulting in tumors that are large while remaining less than 200 m in depth. This makes them particularly well-suited for intravital imaging because standard epifluorescence microscopes can effectively penetrate the entire tumor mass. In contrast, tumors implanted in either superficial or orthotopic sites within the mouse proliferate in three dimensions, making it difficult to accurately localize nanoparticles deep within these tumors by non-invasive techniques. We have utilized this model to assess the uptake of quantum dots, liposomes, and iron oxide nanoparticles in a number of human tumor xenografts, highlighting the potential for this model to be suitable for the in vivo analysis of a broad range of nanoparticle formulations.

<table><tbody><tr><td>Fertilized leghorn eggs</td><td>Frey`s Hatchery, St. Jacobs</td><td>N/A</td><td></td></tr><tr><td>Dremel rotary tool</td><td>Dremel</td><td></td><td>Can used any model</td></tr><tr><td>Dremel cutoff wheels no. 36 </td><td>Dremel</td><td>409</td><td></td></tr><tr><td>Sportsman hatcher</td><td>Berry Hill</td><td>1550HA</td><td></td></tr><tr><td>Sportsman incubator </td><td>Berry Hill</td><td>1502EA</td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td>70% (vol/vol)</td></tr><tr><td>Polystyrene weigh boats </td><td>VWR international</td><td>12577-01</td><td></td></tr><tr><td>Square Petri dishes</td><td>Simport</td><td>25378-115</td><td></td></tr><tr><td>Rubbermaid rubber container with lid </td><td>Guillevin</td><td>RH3-228-00-BLU</td><td>holes drilled into sides</td></tr><tr><td>Vertical pipette puller</td><td>David Kopf Instruments</td><td>model 720</td><td>Settings: 16.3 (heater) and 2.3 (solenoid)</td></tr><tr><td>&lt;p&gt;Sodium borosilicate glass capillary tubes &lt;/p&gt;</td><td>Sutter Instrument Co.</td><td>BF100-58-10</td><td>&lt;p&gt;(OD, 1.0 mm; ID 0.58 mm; 10-cm&lt;/p&gt; length)</td></tr><tr><td>1X Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Invitrogen</td><td>11995073</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s </td><td>Invitrogen</td><td>14190250</td><td>pH 7.4</td></tr><tr><td>Phosphate-Buffered Saline (D-PBS) (1X), liquid</td><td></td><td></td><td></td></tr><tr><td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td><td>Invitrogen</td><td>25300054</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen</td><td>12483-020</td><td>Heat inactivate</td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>15170-090</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5811 000.010</td><td></td></tr><tr><td>Fine-point forceps</td><td>VWR international</td><td>25607-856</td><td></td></tr><tr><td>Tygon R-3603 tubing </td><td>VWR international</td><td>63009-983</td><td>50 ft (1/32-inch inner diameter, 3/32-inch outer diameter, 1/32-inch wall thickness</td></tr><tr><td>Hypodermic needles for injections 18-gauge needles</td><td>BD Biosciences</td><td>305195</td><td>Box of 100</td></tr><tr><td>1 ml syringes for injections</td><td>BD Biosciences</td><td>309602</td><td>Box of 100</td></tr><tr><td>Fiber-optic microscope illuminator</td><td>Amscope</td><td>HL250-AY</td><td>150W</td></tr><tr><td>kimwipes</td><td>VWR international</td><td>10805-905</td><td></td></tr><tr><td>&lt;em&gt;V. unguiculata&lt;/em&gt; seeds (California black-eye no. 5)</td><td>Burpee</td><td>51771A</td><td></td></tr><tr><td>Indoor growth lights</td><td>SunLite, Gardener&amp;rsquo;s Supply</td><td></td><td></td></tr><tr><td>Methyl-PEO4-NHS ester</td><td>Pierce, Thermo Scientific</td><td>PI22341</td><td></td></tr><tr><td>mPEG-NHS, PEG succinimidyl ester, MW 2000</td><td>NANOCS</td><td>PEG1-0002</td><td></td></tr><tr><td>Alexa Fluor 647 carboxylic acid (succinimidyl ester)</td><td>Invitrogen</td><td>A20006</td><td></td></tr><tr><td>Oregon Green 488 succinimidyl ester * 6-isomer *</td><td>Invitrogen</td><td>O-6149</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Dibasic monohydrogen phosphate</td><td>Sigma-Aldrich</td><td>379980</td><td>K2HPO4 (for phopshate buffer)</td></tr><tr><td>Monobasic dihydrogen phosphate </td><td></td><td>P5655</td><td>KH2PO4 (for phopshate buffer)</td></tr><tr><td>Superose 6 size-exclusion column</td><td>GE Healthcare</td><td>17-0673-01</td><td></td></tr><tr><td>&amp;Auml;KTA Explorer 100 Chromatograph</td><td>GE Healthcare</td><td>WS-AKTA100</td><td></td></tr><tr><td>&amp;Auml;KTA high flow kit</td><td>GE Healthcare</td><td>18-1154-85</td><td></td></tr><tr><td>Sucrose </td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter Inc.</td><td></td><td></td></tr><tr><td>SW 28 Ti rotor</td><td>Beckman Coulter Inc.</td><td>342204</td><td>Swing bucket</td></tr><tr><td>50.2 Ti rotor</td><td>Beckman Coulter Inc.</td><td>337901</td><td>Fixed angle</td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Units </td><td>EMD Millipore</td><td>UFC910008</td><td>100 kDa cut off</td></tr><tr><td>Gradient former</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>dextran, fluorescein, 70,000 MW, anionic</td><td>Invitrogen</td><td>D1823</td><td></td></tr><tr><td>Spinning disk confocal fluorescence microscope</td><td>Quorum Technologies</td><td>N/A</td><td></td></tr><tr><td>Epifluorescence wide-field microscope </td><td>Quorum; Zeiss Axio Examiner, Zeiss</td><td>N/A</td><td></td></tr><tr><td>Hamamatsu ImagEM 9100-12 EM-CCD camera</td><td>Quorum; Hamamatsu</td><td>N/A</td><td></td></tr><tr><td>Temperature enclosure unit for microscope </td><td>Precision Plastics</td><td>N/A</td><td></td></tr><tr><td>Vacuum grease </td><td>VWR international</td><td>59344-055</td><td></td></tr><tr><td>Circular glass coverslips no. 1 (18 mm)</td><td>VWR international</td><td>16004-300</td><td></td></tr><tr><td>Volocity software</td><td>PerkinElmer, Inc.</td><td></td><td></td></tr><tr><td>Chick embryo enclosure</td><td></td><td>custom fabricated</td><td></td></tr><tr><td>Delicate scissors</td><td>VWR international</td><td>25608-203</td><td></td></tr><tr><td>Formalin</td><td>BioShop Canada</td><td>FOR201.500</td><td>Use in fumehood</td></tr><tr><td>Optimal Cutting </td><td>Fisher Scientific</td><td>1437365</td><td></td></tr><tr><td>Temperature (OCT)</td><td></td><td></td><td></td></tr><tr><td>Plastic moulds</td><td>Fisher Scientific</td><td>22-038217</td><td></td></tr><tr><td>VWR VistaVision HistoBond Adhesive Slides</td><td>VWR international</td><td>16004-406</td><td></td></tr><tr><td>Prolong gold with DAPI</td><td>Invitrogen</td><td>P36931</td><td></td></tr><tr><td>Disposable blades for cryostat</td><td>Fisher Scientific</td><td>12-634-2</td><td></td></tr><tr><td>Cryostat</td><td>Leica Microsystems</td><td>14047033518</td><td></td></tr></tbody></table>

evaluation of nanoparticle uptake in tumors in real time using intravital imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

Watch this Scientific Journal Video about Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging at JoVE.com

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

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Medicine

18.2K Views.  University of Western Ontario. We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model. 

Video: Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Cancer Research

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Bioengineering

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Real-Time Tracking of Nanoparticles in Brain Tumors Using 2P-IVM in Mice

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Author Spotlight: Multimodal Imaging Strategies for Optimizing Drug Delivery and Early Detection in Glioblastoma Treatment

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

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Chapters in this video

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Viral Nanoparticles for In vivo Tumor Imaging

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice