1Department of Medical Biophysics, University of Western Ontario, 2London Regional Cancer Program, London Health Science Centre, 3Department of Pathology, Vanderbilt University, 4Translational Prostate Cancer Research Group, London Health Science Centre
Cho, C., Ablack, A., Leong, H., Zijlstra, A., Lewis, J. Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging. J. Vis. Exp. (52), e2808, doi:10.3791/2808 (2011).
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.
1. Inoculation of tumor into avian embryo CAM
2. Preparation of nanoparticles
3. Intravenous injection of fluorescently-labeled viral nanoparticles
4. Real time intravital imaging
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.
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.
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°C.
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.
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.
No conflicts of interest declared.
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.
|Fertilized leghorn eggs||Frey`s Hatchery, St. Jacobs||N/A|
|Dremel rotary tool||Dremel||Can used any model|
|Dremel cutoff wheels no. 36||Dremel||409|
|Sportsman hatcher||Berry Hill||1550HA|
|Sportsman incubator||Berry Hill||1502EA|
|Polystyrene weigh boats||VWR international||12577-01|
|Square Petri dishes||Simport||25378-115|
|Rubbermaid rubber container with lid||Guillevin||RH3-228-00-BLU||holes drilled into sides|
|Vertical pipette puller||David Kopf Instruments||model 720||Settings: 16.3 (heater) and 2.3 (solenoid)|
Sodium borosilicate glass capillary tubes
|Sutter Instrument Co.||BF100-58-10||
(OD, 1.0 mm; ID 0.58 mm; 10-cmlength)
|1X Dulbecco’s Modified Eagle Medium (DMEM)||Invitrogen||11995073|
|Phosphate-Buffered Saline (D-PBS) (1X), liquid|
|Trypsin, 0.05% (1X) with EDTA 4Na, liquid||Invitrogen||25300054|
|Fetal Bovine Serum||Invitrogen||12483-020||Heat inactivate|
|Fine-point forceps||VWR international||25607-856|
|Tygon R-3603 tubing||VWR international||63009-983||50 ft (1/32-inch inner diameter, 3/32-inch outer diameter, 1/32-inch wall thickness|
|Hypodermic needles for injections 18-gauge needles||BD Biosciences||305195||Box of 100|
|1 ml syringes for injections||BD Biosciences||309602||Box of 100|
|Fiber-optic microscope illuminator||Amscope||HL250-AY||150W|
|V. unguiculata seeds (California black-eye no. 5)||Burpee||51771A|
|Indoor growth lights||SunLite, Gardener’s Supply|
|Methyl-PEO4-NHS ester||Pierce, Thermo Scientific||PI22341|
|mPEG-NHS, PEG succinimidyl ester, MW 2000||NANOCS||PEG1-0002|
|Alexa Fluor 647 carboxylic acid (succinimidyl ester)||Invitrogen||A20006|
|Oregon Green 488 succinimidyl ester * 6-isomer *||Invitrogen||O-6149|
|Dimethyl sulfoxide (DMSO)||Sigma-Aldrich||D8418|
|Dibasic monohydrogen phosphate||Sigma-Aldrich||379980||K2HPO4 (for phopshate buffer)|
|Monobasic dihydrogen phosphate||P5655||KH2PO4 (for phopshate buffer)|
|Superose 6 size-exclusion column||GE Healthcare||17-0673-01|
|ÄKTA Explorer 100 Chromatograph||GE Healthcare||WS-AKTA100|
|ÄKTA high flow kit||GE Healthcare||18-1154-85|
|Ultracentrifuge||Beckman Coulter Inc.|
|SW 28 Ti rotor||Beckman Coulter Inc.||342204||Swing bucket|
|50.2 Ti rotor||Beckman Coulter Inc.||337901||Fixed angle|
|Amicon Ultra-15 Centrifugal Filter Units||EMD Millipore||UFC910008||100 kDa cut off|
|dextran, fluorescein, 70,000 MW, anionic||Invitrogen||D1823|
|Spinning disk confocal fluorescence microscope||Quorum Technologies||N/A|
|Epifluorescence wide-field microscope||Quorum; Zeiss Axio Examiner, Zeiss||N/A|
|Hamamatsu ImagEM 9100-12 EM-CCD camera||Quorum; Hamamatsu||N/A|
|Temperature enclosure unit for microscope||Precision Plastics||N/A|
|Vacuum grease||VWR international||59344-055|
|Circular glass coverslips no. 1 (18 mm)||VWR international||16004-300|
|Volocity software||PerkinElmer, Inc.|
|Chick embryo enclosure||custom fabricated|
|Delicate scissors||VWR international||25608-203|
|Formalin||BioShop Canada||FOR201.500||Use in fumehood|
|Optimal Cutting||Fisher Scientific||1437365|
|Plastic moulds||Fisher Scientific||22-038217|
|VWR VistaVision HistoBond Adhesive Slides||VWR international||16004-406|
|Prolong gold with DAPI||Invitrogen||P36931|
|Disposable blades for cryostat||Fisher Scientific||12-634-2|