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 border="1">
<tbody>
 <tr>
 <th>Reagent Name/Equipment</th>
 <th>Company</th>
 <th>Catalogue Number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>Fertilized leghorn eggs</td>
 <td>Frey`s Hatchery, St. Jacobs</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dremel rotary tool</td>
 <td>Dremel</td>
 <td>&nbsp;</td>
 <td>Can used any model</td>
 </tr>
 <tr>
 <td>Dremel cutoff wheels no. 36 </td>
 <td>Dremel</td>
 <td>409</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sportsman hatcher</td>
 <td>Berry Hill</td>
 <td>1550HA</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sportsman incubator </td>
 <td>Berry Hill</td>
 <td>1502EA</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>70% (vol/vol)</td>
 </tr>
 <tr>
 <td>Polystyrene weigh boats </td>
 <td>VWR</td>
 <td>12577-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Square Petri dishes</td>
 <td>Simport, VWR</td>
 <td>25378-115</td>
 <td>&nbsp;</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>Sodium borosilicate glass capillary tubes </td>
 <td>Sutter Instrument</td>
 <td>BF100-58-10</td>
 <td>(OD, 1.0 mm; ID 0.58 mm; 10-cm
 length)</td>
 </tr>
 <tr>
 <td>1X Dulbecco's Modified Eagle Medium (DMEM)</td>
 <td>Invitrogen</td>
 <td>11995073 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dulbecco'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>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
 <td>Invitrogen</td>
 <td>25300054</td>
 <td>&nbsp;</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, VWR</td>
 <td>15170-090</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Eppendorf model 5810R</td>
 <td>5811 000.010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fine-point forceps</td>
 <td>VWR</td>
 <td>25607-856</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tygon R-3603 tubing </td>
 <td>VWR</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</td>
 <td>305195</td>
 <td>Box of 100</td>
 </tr>
 <tr>
 <td>1 ml syringes for injections</td>
 <td>BD</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</td>
 <td>10805-905</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>V. unguiculata seeds (California black-eye no. 5)</td>
 <td>Burpee</td>
 <td>51771A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Indoor growth lights</td>
 <td>SunLite, Gardener's Supply</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Methyl-PEO4-NHS ester</td>
 <td>Pierce</td>
 <td>PI22341</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>mPEG-NHS, PEG succinimidyl ester, MW 2000</td>
 <td>NANOCS</td>
 <td>PEG1-0002</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Alexa Fluor 647 carboxylic acid (succinimidyl ester)</td>
 <td>Invitrogen</td>
 <td>A20006</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Oregon Green 488 succinimidyl ester * 6-isomer *</td>
 <td>Invitrogen</td>
 <td>O-6149</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dimethyl sulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D8418</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dibasic monohydrogen phosphate</td>
 <td>Sigma</td>
 <td>379980</td>
 <td>K2HPO4 (for phopshate buffer)</td>
 </tr>
 <tr>
 <td>Monobasic dihydrogen phosphate </td>
 <td>&nbsp;</td>
 <td>P5655</td>
 <td>KH2PO4 (for phopshate buffer)</td>
 </tr>
 <tr>
 <td>Superose 6 size-exclusion column</td>
 <td>GE healthcare life sciences</td>
 <td>17-0673-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&Auml;KTA Explorer 100 Chromatograph</td>
 <td>GE Amersham Pharmacia</td>
 <td>WS-AKTA100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&Auml;KTA high flow kit</td>
 <td>GE healthcare life sciences</td>
 <td>18-1154-85</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sucrose </td>
 <td>Sigma</td>
 <td>S0389</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ultracentrifuge</td>
 <td>Beckman</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SW 28 Ti rotor</td>
 <td>Beckman</td>
 <td>342204</td>
 <td>Swing bucket</td>
 </tr>
 <tr>
 <td>50.2 Ti rotor</td>
 <td>Beckman</td>
 <td>337901</td>
 <td>Fixed angle</td>
 </tr>
 <tr>
 <td>Amicon Ultra-15 Centrifugal Filter Units </td>
 <td>Millipore</td>
 <td>UFC910008</td>
 <td>100 kDa cut off</td>
 </tr>
 <tr>
 <td>Gradient former</td>
 <td>Biorad</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>dextran, fluorescein, 70,000 MW, anionic</td>
 <td>Invitrogen</td>
 <td>D1823</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spinning disk confocal fluorescence microscope</td>
 <td>Quorum; Yokogawa 
 CSU 10, Yokogawa</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Epifluorescence wide-field microscope </td>
 <td>Quorum; Zeiss Axio Examiner, Zeiss</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hamamatsu ImagEM 9100-12 EM-CCD camera</td>
 <td>Quorum; Hamamatsu</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Temperature enclosure unit for microscope </td>
 <td>Precision Plastics</td>
 <td>N/A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vacuum grease </td>
 <td>VWR</td>
 <td>59344-055</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Circular glass coverslips no. 1 (18 mm)</td>
 <td>VWR</td>
 <td>16004-300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Volocity software</td>
 <td>Perkin Elmer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chick embryo enclosure</td>
 <td>&nbsp;</td>
 <td>custom fabricated</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Delicate scissors</td>
 <td>VWR</td>
 <td>25608-203</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formalin</td>
 <td>Bioshop</td>
 <td>FOR201.500</td>
 <td>Use in fumehood</td>
 </tr>
 <tr>
 <td>Optimal Cutting </td>
 <td>Fisher; Tissue Tek</td>
 <td>1437365</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Temperature (OCT)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Plastic moulds</td>
 <td>Fisher</td>
 <td>22-038217</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>VWR VistaVision HistoBond Adhesive Slides</td>
 <td>VWR</td>
 <td>16004-406</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Prolong gold with DAPI</td>
 <td>Invitrogen</td>
 <td>P36931</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disposable blades for cryostat</td>
 <td>Fisher</td>
 <td>12-634-2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cryostat</td>
 <td>Leica CM 3050 S</td>
 <td>14047033518</td>
 <td>&nbsp;</td>
 </tr>
</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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Research

JoVE Journal

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

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

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 ...