Name: validation of nanobody and antibody based in vivo tumor xenograft nirf-imaging experiments in mice using ex vivo flow cytometry and microscopy
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Description: Watch this Scientific Journal Video about Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy at JoVE.com

This work was supported by the graduate school &#8216;Inflammation and regeneration&#8217; of the Collaborative Research Centre 841 of the Deutsche Forschungsgemeinschaft (Alexander Lenz, Valentin Kunick, William Fumey), by the Collaborative Research Centre 877 of the Deutsche Forschungsgemeinschaft (Friedrich Koch-Nolte), by the Werner Otto Foundation (Peter Bannas), by the Wilhelm Sander Foundation (Peter Bannas, Friedrich Koch-Nolte), and by the Deutsche Forschungsgemeinschaft (Martin Trepel, Friedrich Haag and Friedrich Koch-Nolte). We thank the University Cancer Center Hamburg (UCCH) In Vivo Optical Imaging Core Facility and staff at UKE for consultation and their high quality service. The Core Facility was supported in part by grants from Deutsche Krebshilfe (German Cancer Aid).

NOTE: Experiments were performed in accordance with international guidelines on the ethical use of animals and were approved by the local animal welfare commission of the&#160;University Medical Center, Hamburg.
1. Preparation of Tumor Cells, Mice, and Antibody Constructs
<ol>
	<li>Preparation of Lymphoma Cells and Aliquoting of Basement Matrix (Matrigel).
	<ol>
		<li>The day before injection of tumor cells put a sterilized tip box (1,000 &#181;l tips) and the appropriate pipette in -20 &#176;C freezer.</li>
		<li>Thaw the bottle with the basement matrix on ice in the 4 &#176;C fridge O/N.</li>
		<li>On the day of injection fill an ice bucket and place the basement matrix along with pipette, tips, and 1 ml syringes with 30 G&#160;needles on ice.</li>
		<li>Aliquot lymphoma cells in a volume of 100 &#181;l of RPMI medium in 1.5 ml microcentrifuge tubes and mix carefully with 100 &#181;l of the basement matrix. Draw up into pre-cooled syringes and put on ice until injection. 
		NOTE: Use good sterile technique and to work on ice the whole time to prevent clogging of the basement matrix.</li>
	</ol>
	</li>
	<li>Mice Preparation
	<ol>
		<li>Use 8-10 week old athymic nude mice (NMRI-Foxn1nu).</li>
		<li>To reduce autofluorescence of the intestine keep mice on an alfalfa-free diet for 1 week prior to in vivo imaging.</li>
		<li>For injection of lymphoma cells anesthetize mice to effect with 2% isoflurane in an induction chamber. Maintain 1-2% isoflurane for the duration of the procedure using an isofluorane manifold.</li>
		<li>Inject lymphoma cells subcutaneously into the shoulder flanks. For direct intra-individual comparison inject antigen-positive and antigen-negative cells on the right and left side, respectively.
		<ol>
			<li>Use a thumb and index finger to pinch the skin of the mouse and pull it away from the body of the mouse. Inject slowly and evenly into the pouch created by the fingers, creating a single cluster of cells beneath the skin. The basement matrix helps keeping injected cells in place.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of Antibody Constructs
	<ol>
		<li>Label monoclonal antibodies and single domain nanobodies with a commercially available protein labelling kit to the fluorescent dye AlexaFluor-680 (AF680) (excitation wavelength = 679 nm, emission wavelength = 702 nm) according the manufacturer&#8217;s instructions. Calculate the number of dyes per nanobody and conventional antibody using molar extinction coefficients of 15,720 mol-1 cm-1 and 203,000 mol-1 cm-1, respectively. Assess the purity of antibody constructs by SDS-PAGE size fractionation and Coomassie brilliant blue stain.</li>
		<li>Ensure the specificity of the labeled conjugates by conducting a series of in vitro experiments before the actual imaging studies. Test the specificity of labeled antibody constructs in vitro through blocking studies and unspecific isotype controls 8,20. 
		NOTE: In step 1.2.4 cell numbers for antigen-negative and antigen-positive cells (or different cells lines) may have to be adapted according to different growth rates. For these experiments, 0.5x106 DC27.10 ARTC2 negative and 1.5x106 DC27.10 ARTC2 positive cells were used to obtain tumors of similar size. In step 1.3.1 the process of labeling and labeling efficacy of antibody probes with fluorophores may differ by the labeling kit used.</li>
	</ol>
	</li>
</ol>
2. In Vivo Imaging
<ol>
	<li>After 7-9 days, when tumors reach ~8 mm in diameter, inject 50 &#181;g of AlexaFluor680 labelled antibody constructs in a volume of 200 &#181;l saline intravenously into the tail vein of the mouse (mAb-AF680: 50 &#181;g with 2 dyes/molecule &#8776; ~4^14 fluorochromes &#8776; ~0.8 &#181;g fluorochromes; Nanobody-AF680: 50 &#181;g with 0.3 dyes/molecule &#8776; ~5.6^14 fluorochromes &#8776; ~1.1 &#181;g fluorochromes).</li>
	<li>Initialize the imaging system and anesthetize mice to effect with 3% isoflurane using an XGI-8 anesthesia system in the induction chamber prior to imaging. Maintain 1-2% isoflurane for the duration of the imaging procedure using the isoflurane manifold housed in the imaging chamber.
	<ol>
		<li>Position mice on heated imaging stage with tumors directed towards the camera. Monitor the proper anesthetization by pinching the toe or tail of the animal; any reaction of the animal indicates that anesthesia is too light.</li>
		<li>Monitor the respiration rate; the anesthesia is too light if the respiration rate is increased and too deep if the respiration rate is decreased, deep or irregular. Protect animal&#8217;s corneas with an eye ointment to prevent dryness while under anesthesia.</li>
	</ol>
	</li>
	<li>Choose fluorescent filter sets of 615-665 nm for excitation, 695-770 nm for emission, and 580-610 nm excitation for background subtraction with a 51 2x 512 pixel matrix size. Set exposure time to auto, pixel binning to medium and F/Stop to 2. 
	NOTE: Shorter exposure times enable faster frame rates; longer exposure times provide greater sensitivity. Binning controls the pixel size on the CCD camera. Increasing the binning increases the pixel size, sensitivity, and frame rate, but reduces spatial resolution. F/Stop sets the size of the camera lens aperture. The aperture size controls the amount of light detected and the depth of field.Note that settings can differ by imaging device and experimental set up. Consult the manufacturer&#8217;s manual for optimal results.
	<ol>
		<li>In the imaging software optimize exposure time, F/stop and pixel binning based on the expression level of the cell line. Change these settings at any time during an experiment without impacting the quantitative result. Alternatively, let the Imaging Wizard software automatically determine the parameters.</li>
	</ol>
	</li>
	<li>Image mice before injection and 6 hr after injection of antibody constructs. 
	NOTE: The labelling efficacy may affect the required dose needed for optimal imaging results. Therefore the amount of required antibody-construct for optimal imaging results has to be determined empirically. It may vary depending on labelling efficacy and size of the construct as well as the tumor model and the target-antigen expression.</li>
</ol>
3. Harvesting and Preparation of Tumors
<ol>
	<li>Fill an ice bucket and place 4%, paraformaldehyde (PFA) phosphate buffered saline (PBS)/protease inhibitor (AEBSF) and PBS/0.2% bovine serum albumin (BSA) on ice.</li>
	<li>Once the imaging session is finished, increase the concentration of isoflurane to 4%. After animal ceases breathing, remove it from the imaging stage and perform cervical dislocation.</li>
	<li>Mount mouse on a Styrofoam block and spray with 70% ethanol.</li>
	<li>Use scissors and forceps to cut the outer skin and pull it back carefully to expose tumors. Remove tumors with scalpel to ensure tumor tissue remains intact.</li>
	<li>Cut tumors in half using a scalpel. Place one half in a collection tube with PBS/AEBSF for FACS analyses and the other half in a 50 ml tube with 4% PFA for immunohistochemistry.</li>
	<li>Preparation for immunohistochemistry
	<ol>
		<li>Keep tumors in 4% PFA in the fridge at 4 &#176;C for 24 hr. Transfer tumors to a tube with PBS/30% sucrose and keep in the fridge at 4 &#176;C until the tumor sinks to the bottom of the tube.</li>
		<li>Cut the tumors in appropriate pieces for the cryomolds. Put the tumors in cryomolds and fill with OCT compound so that the tumors are covered.</li>
		<li>Put molds on dry ice and wait until the compound is completely frozen. Transfer frozen tumors to -80 &#176;C freezer and store for immunohistochemistry.</li>
		<li>Cut frozen tumors in sections of 8 &#956;m with a microtome. Use standard immunohistochemistry protocol to stain with antibody against CD31-AF488 to visualize blood vessels and diamidino-phenylindole (DAPI) to visualize nuclei. 
		NOTE: Tumor sections are very sensitive to light as they already contain fluorescently labelled antibodies. Minimize exposure to light as much as possible.</li>
	</ol>
	</li>
	<li>Preparation for FACS analysis
	<ol>
		<li>Place petri dish on ice and remove the plunger from a 2.0 ml syringe. Put the tumor in the cell strainer and cut it gently into 3-4 pieces using a scalpel. Pour the initial PBS in the petri dish and use the flat end of the plunger to mash the tumor within the cell strainer.</li>
		<li>Flush the cell strainer with the initial PBS to wash out all cells with a 10 ml pipette. Transfer cell suspension in a new tube and spin at 500 x g for 5 min. Discard the supernatant and resuspend the cells in 10 ml PBS/0.2% BSA. Count cells.</li>
		<li>Aliquot 1-5 x 106 lymphoma cells in a 5 ml FACS tube. Spin cells again (500 x g), discard supernatant and resuspend in 100 &#181;l PBS/0.2% BSA.</li>
		<li>Optionally, block FcR using an anti-CD16/CD32-mAb (FcgR3/2) that binds to Fc&#947;R on ice for 10 min. Wash cells once with PBS/0.2% BSA.</li>
		<li>Add anti-CD45-mAb to discriminate leucocytes from other cells. Incubate for 20 min on ice in the dark, followed by two washes with PBS/0.2% BSA.</li>
		<li>Right before FACS analysis stain with propidium iodide for 15 min on ice to discern dead cells, followed by two washes with plain PBS. Resuspend in 150 &#181;l for FACS analysis. 
		NOTE: In step 3.7.4 and step 3.7.5 other antibodies may be used depending on the tumor entity.</li>
	</ol>
	</li>
</ol>
4. FACS&#160;Analysis
<ol>
	<li>Use a series of gating tools to gate the population of interest in the form of large scatter, doublet discrimination, exclusion of dead cells, gating for CD45 positive cells, and gating for antigen-positive cells.
	<ol>
		<li>At first, gate out cell debris in a forward scatter (FSC-A) versus side scatter (SSC-A) using a two-parameter plot. Next discard cell doublets. Finally, gate out non CD45-antigen-positive (CD45) and dead/dying cells (LD &#8211; live/dead-stain).</li>
	</ol>
	</li>
	<li>Record samples using the same template for all experiments. 
	NOTE: Refer to the manufacturer&#8217;s protocol for technical advice regarding the use of hardware and analytic software.</li>
</ol>
5. Microscopic Analysis
<ol>
	<li>Analyze stained tumor cryosections using a confocal microscope with an oil immersion lens (40X objective). Use a He-Neon 633 nm laser excitation of AF680, an Argon Laser for AF488, and a 405-Diode for DAPI.</li>
	<li>Process the raw image data using a software compatible with the microscope. Perform background-correction and noise filtering if necessary. Perform additional image adjustments such as sectioning, cropping as well as brightness and contrast adjustments.
	<ol>
		<li>Finally generate a single composite overlay image from all detection channels. Adjust the brightness and contrast of the individual layers. The composite images will show localization of cells (DAPI-stain, blue), distribution of labelled antibody constructs (AF680-stain, red) and blood vessels (AF488-stain, green).</li>
	</ol>
	</li>
</ol>
6. In Vivo Imaging Analysis
<ol>
	<li>Open image files in imaging software and create an overlay image by combining photograph image data with fluorescence image data. Optimize image display by removing tissue autofluorescence background signal from the specific fluorescent signal. This can be done by subtracting the image acquired with a background filter set from the image acquired with the filter set specific for the tracer.</li>
	<li>Draw identical circular measurement regions of interest (ROI) around the antigen-positive tumor and the antigen-negative tumor. To determine background signal intensity, place a circular ROI in an area of the animal where fluorescence signal is expected to be low (e.g., hind leg).
	<ol>
		<li>Use the same ROIs for all animals at all time points. For positioning, use the photographic black-and-white images to identify tumor margins.</li>
	</ol>
	</li>
	<li>Display ROI data in a measurement table. Use average radiant efficiency data, which enables a more quantitative comparison of fluorescent signals for further statistical analyses.
	<ol>
		<li>Display and compare data as absolute signal intensities or calculate the signal-to-background ratio using measured ROI data from targeted tissue and background tissue. Calculate the tumor-to-background ratio by dividing the tumor uptake value by the background value determined from the hind limb.</li>
		<li>As controls, also analyze antigen-negative and antigen-positive tumors in the same animals as well as animals injected with labelled isotype controls to assess the specificity of signals in vivo.</li>
	</ol>
	</li>
</ol>

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Friedrich Koch-Nolte and Friedrich Haag receive a share of antibody and protein sales via MediGate GmbH, a wholly owned subsidiary of the University Medical Center Hamburg-Eppendorf.

We used near-infrared fluorophore labelled nanobodies and conventional monoclonal antibodies directed against the same target on lymphoma cells for a multimodal comparison of in vivo and ex vivo analyses. We showed that nanobodies are well suited as diagnostic tools for rapid and specific in vivo detection of lymphomas.
In vivo, s+16a680 allowed a fast and more specific detection of ARTC2-positive xenografts. Apart from the different kinetics for best tumor visualization in vivo, the major drawback of Nika102680 was the high nonspecific signal from ARTC2-negative tumors and nonspecific background signals.
Ex vivo flow cytometric analyses of dispersed cells from dissected tumors showed no nonspecific binding to ARTC2-negative lymphoma cells of injected AF680-conjugates. Ex vivo fluorescence revealed strong and almost homogenous staining of cells in ART2C-positive tumor sections in case of nanobody s+16a, confirming that the nanobody was able to reach even remote areas within the tumor after 6 h. In contrast, the monoclonal antibody showed weaker and inhomogeneous staining of cells in ARTC2-positive tumors after 6 hr. Better imaging results with the conventional antibody can be achieved after 24 hr or 48 hr (data not shown). In order to perform a thorough comparison of two differently sized constructs, imaging at different time points (serial-imaging) has to be performed to identify the optimal imaging time point for each construct.
Like other previous studies, the results reported here emphasize that in vivo molecular imaging with labelled nanobodies allows rapid and specific same-day tumor imaging with high tumor-to-background ratios 12-15,17-19. Contrariwise, conventional antibodies result in low tumor-to-background ratios and nonspecific signals from antigen-negative tumors early after injection due to their slow clearance from the body. In order to obtain optimal imaging results with conventional antibodies, imaging time points 24 hr or even 48 hr after injection are commonly needed. These findings are in accord with previous studies that have suggested that conventional antibodies with proven therapeutic benefit have limited utility in molecular imaging 17,19,26. Therefore conventional antibodies might be rather suited for therapeutic purposes due to their long plasma half-life while nanobodies are rather suited for imaging purposes due to their rapid clearance from the circulation. These differences are due to the fact that any excess of the smaller nanobodies (15-17 kDa) is rapidly cleared via renal elimination while excess of larger conventional antibodies (150 kDa) is retained in the circulation. So the major advantage of nanobodies for molecular imaging is the low background signal at early imaging time points regardless of the injected dose. This allows same day imaging and could be translatable to the clinical setting. Contrariwise, conventional antibodies have to be exactly titrated to minimize nonspecific background signals, while maintaining enough specific signal from the targeted tissue (unpublished data).
One of the limitations of the in vivo NIRF-imaging technique is the low penetration depth which generally allows only imaging of subcutaneous but not of orthotopic tumor models. However, this limitation might be overcome in an experimental setting by the recently developed tomographic photo-acoustic techniques that allow whole-body imaging of living mice 27. Another limitation of the NIRF-imaging technique is the assessment of the tissue dose as compared to radionuclide-mediated imaging. However, the nanobodies may be radiolabelled for positron emission tomography (PET) imaging of xenograft models and exact quantitative assessment of tracer biodistribution. Indeed, our NIRF-imaging results are in accordance with a recent study that compared nanobodies and conventional antibodies for PET imaging. The authors also came to the conclusion that nanobodies allow same-day imaging with high tumor-to-background ratios 15.
However, only the labelling of antibody constructs with the near-infrared fluorescent dye AF680 allowed us the comprehensive in vitro, in vivo and ex vivo near-infrared fluorescence imaging comparison using flow cytometry, fluorescence microscopy, and NIRF-imaging. For this reason, and because it is nonradioactive, highly sensitive, inexpensive, and uses comparatively easy-to-produce targeted probes, we advocate the use of the NIRF-imaging technique for evaluation of new antibody constructs in preclinical molecular imaging.

In the present report, we describe the implementation of near-infrared fluorophore labelled probes for validation of in vivo xenograft imaging experiments by using ex vivo flow cytometry and fluorescence microscopy of the dissected xenograft tumors. We compare a single domain nanobody (s+16a, 17 kDa) 1 and a monoclonal antibody (Nika102, 150 kDa) 2,3 directed to the same target antigen for specific in vivo near-infrared fluorescence imaging in a lymphoma xenograft model. The target antigen ADP-ribosyltransferase ARTC2.2 is expressed as a GPI-anchored cell surface ecto-enzyme by lymphoma cells 4-9.
Nanobodies derived from camelid heavy-chain-only antibodies are the smallest available antigen-binding fragments 10,11. With only ~15 kDa, these small antibody fragments are soluble, very stable and are renally cleared from circulation 8,10. These properties make them particularly suited for specific and efficient targeting of tumor antigens in vivo 12-20. Common antigen targets of available nanobodies are the epidermal growth factor receptor (EGFR1 or HER-1), human epidermal growth factor type 2 (HER-2 or CD340), carcinoembryonic antigen (CEA) and vascular cell adhesion molecule-1 (VCAM-1) 21. Nanobody conjugates are promising tools for cancer immunotherapy and treatment of inflammatory diseases 22.
Recent studies have shown that nanobodies allow higher tumor-to-background (T/B)-ratios than conventional antibodies in in vivo molecular imaging applications 8,17,19. This is explained mainly by the relatively poor and slow tissue penetration of conventional antibodies, slow clearance from circulation, and long retention in non-targeted tissues 23. Moreover, excess of conventional antibodies leads to non-specific accumulation in target antigen-negative tumors caused by the enhanced permeability and retention (EPR) effect 24,25. This means that higher doses of conventional antibodies may increase not only specific signals but also nonspecific signals, thus reducing the maximum achievable tumor-to-background ratio. In contrast, increasing the dose of nanobodies increases the signals of antigen-positive tumors but not of normal tissue or antigen-negative tumors (unpublished data).
Beyond the comparison of nanobodies and conventional antibodies, we outline an intraindividual assessment of antigen-positive and -negative xenografts in the same mice for direct comparison of specific and nonspecific signals due to the EPR effect. The near-infrared fluorophore conjugated probes allowed us to exploit a single probe in vivo and ex vivo&#160;using near-infrared fluorescence imaging, flow cytometry, and fluorescence microscopy. Applying our protocols allows for nonradioactive, highly sensitive, and inexpensive optimization of in vivo molecular imaging experiments such as evaluation of new antibody constructs for specific tumor targeting.
The aim of this tutorial study is to highlight the use of NIRF-imaging for evaluation of new antibody constructs in preclinical molecular imaging.
In this protocol, all experiments were performed with a small-animal NIRF-Imaging system, a fluorescence activated cell sorter (FACS) flow cytometer, and a confocal microscope.

<table><tbody><tr><td>AF680 protein labelling kit</td><td>Invitrogen</td><td>A20172</td><td></td></tr><tr><td>Anti-CD16/CD32-antibody</td><td>BioXCell</td><td>BE0008</td><td></td></tr><tr><td>Anti-CD31-antibody</td><td>Santa Cruz</td><td>sc-1506</td><td>labeled with secondary antibody with AF488</td></tr><tr><td>Anti-CD45-antibody V450</td><td>BD Biosciences</td><td>560501</td><td></td></tr><tr><td>AxioVision LE software</td><td>Zeiss</td><td>www.zeiss.com</td><td></td></tr><tr><td>Basement membrane matrix</td><td>BD Biosciences</td><td>354234</td><td>Alternative product can be used</td></tr><tr><td>Cell strainer 70 &amp;micro;m</td><td>Corning</td><td>431751</td><td>Alternative product can be used</td></tr><tr><td>Confocal microscope</td><td>Leica&amp;nbsp;</td><td>www.leica-microsystems.com</td><td>Leica SP5 with the following lasers: He-Neon for AF680, Argon Laser for AF488, and a 405-Diode for DAPI</td></tr><tr><td>DAPI</td><td>Molcular Probes</td><td>D1306</td><td></td></tr><tr><td>DC27.10 cells&amp;nbsp;</td><td>laboratory specific</td><td></td><td>Other cells with different surface targets can be used</td></tr><tr><td>DPBS</td><td>Sigma Aldrich</td><td>D8662</td><td></td></tr><tr><td>FACS Canto II</td><td>BD Biosciences</td><td>www.bdbiosciences.com</td><td></td></tr><tr><td>Flourescence mircoscope</td><td>Zeiss</td><td>www.zeiss.com</td><td>Zeiss Axiovert 200 with Filter Set #32 for AF680: 000000-1031-354</td></tr><tr><td>ImageJ software</td><td>NIH</td><td>http://imagej.nih.gov/ij/</td><td></td></tr><tr><td>IVIS 200</td><td>Perkin Elmer</td><td>www.perkinelmer.com</td><td>Alternative in vivo imaging system can be used</td></tr><tr><td>Leica LAS software</td><td>Leica</td><td>www.leica-microsystems.com</td><td>Software specific to microscope used</td></tr><tr><td>Living Image software</td><td>Perkin Elmer</td><td>www.perkinelmer.com</td><td>Software specific to imaging system used</td></tr><tr><td>Needles 30 G</td><td>BD Biosciences</td><td>305128</td><td>Alternative product can be used</td></tr><tr><td>Nika102-antibody AF680</td><td>laboratory specific</td><td></td><td>Other antibodies against different surface targets can be used</td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td>Potential hazards: carcinogenic, can irritate the eyes and skin, contact may cause drying of the skin and/or allergic dermatitis</td></tr><tr><td>s+16a-nanobody AF680</td><td>laboratory specific</td><td></td><td>Other antibodies against different surface targets can be used</td></tr><tr><td>Syringes 1 ml</td><td>Braun</td><td>916 1406 V</td><td>Alternative product can be used</td></tr></tbody></table>

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.

Fluorescently labelled probes allow for the combination of different NIRF-imaging techniques (Figure 1A). We aimed to perform in vivo NIRF-imaging, flow cytometry, and fluorescence microscopy sequentially in order to compare fluorescently labelled nanobodies and monoclonal antibodies for specific in vivo imaging (Figure 1B).
Mice were injected with 50 &#956;g of nanobody and monoclonal antibody to evaluate the specificity of the fluorescently labelled constructs for in vivo imaging. The results showed specific labeling of antigen-positive tumors with both nanobody and monoclonal antibody at 6 hr after injection (Figure 2). ROI analyses of the antigen-positive tumors showed a much higher T/B ratio of ~12 for the nanobody compared to ~6 for the monoclonal antibody. Moreover, the nanobody showed no nonspecific signal in the antigen-negative tumors, whereas the monoclonal antibody showed nonspecific confounding signals in the antigen-negative tumors.
Besides the nonspecific signal of the negative tumors, the monoclonal antibody also induced nonspecific background signals in the entire animal. This is likely due to excessive free circulating antibodies, which are too large to be renally excreted. Contrariwise, animals injected with nanobodies showed nonspecific signals only in the kidneys due to the renal elimination of the small nanobodies.
Flow cytometry analyses of tumor cell suspensions showed specific labeling of antigen-positive tumor cells with both AF680-conjugates 6 hr after injection. The stronger fluorescence signal of the nanobody labelled cells compared to monoclonal antibody labelled cells reflects the in vivo NIRF-imaging results. Importantly, the flow cytometric analyses reveal that there is no nonspecific labelling of antigen-negative cells with either of the two constructs (Figure 3).
Fluorescence microscopy of tumor cryosections showed a strong and almost homogenous labeling of antigen-positive cells with the nanobody 6 hr after injection. Contrariwise, the monoclonal antibody showed a much weaker and rather inhomogeneous staining (Figure 4A). Antigen-negative tumors show no staining 6 hr after injection of the nanobody, whereas antigen-negative tumors injected with the conventional antibody show nonspecific scattered staining in the interstitial space (Figure 4B).
<img alt="Figure 1" src="/files/ftp_upload/52462/52462fig1highres.jpg" /> 
Figure 1: Flourescence imaging and antibody constructs. (A) Imaging setup for evaluation of AF680-conjugates: In vivo NIRF-imaging followed by flow cytometry and fluorescence microscopy. (B) Schematic of AlexaFluor680 labelled nanobody s+16a (red) and monoclonal antibody Nika102 (blue). Orange stars indicate the AlexaFluor680 fluorochromes.
<img alt="Figure 2" src="/files/ftp_upload/52462/52462fig2highres.jpg" /> 
Figure 2: In vivo NIRF-imaging. Images of the fluorescence signal of antigen-positive (+) and antigen-negative (-) tumors in mice that have been injected with nanobody s+16a (A) and monoclonal antibody Nika102 (B). In vivo imaging was performed before (0 h) and 6 h after injection. Signal intensities are displayed as radiant efficiency (p/sec/cm2/sr) / (&#181;W/cm2).
<img alt="Figure 3" src="/files/ftp_upload/52462/52462fig3highres.jpg" /> 
Figure 3: Ex vivo FACS analyses of cell bound antibody constructs from tumor cell suspensions. (A) Gating strategy for FACS analyses of tumor cells. (B) Histograms display the amount of the intravenously injected AF680-conjugated nanobody s+16a and antibody Nika102 specifically bound to the tumor cells in vivo. Antigen-negative tumors are displayed as unfilled histograms and antigen-positive tumors are displayed as filled histograms.
<img alt="Figure 4" src="/files/ftp_upload/52462/52462fig4highres.jpg" /> 
Figure 4: Ex vivo fluorescence microscopy. (A) Overview fluorescence microscopy of entire antigen-positive tumor cryosections 6 h after injection of s+16a680 or Nika102680. Signal intensities of the in vivo intravenously injected AF680-conjugates without any secondary-labelling agents are displayed in red. Ex vivo counterstained nuclei are displayed in blue and vessels in green. Dotted lines indicate outer margins of the entire tumors. (B) Close-up fluorescence microscopy of antigen-positive and antigen-negative tumors.

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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.

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 ...

validation-nanobody-antibody-based-vivo-tumor-xenograft-nirf-imaging

Universidad San Sebastian

Research

JoVE Journal

Medicine

11.6K Views.  University Medical Center, Hamburg. 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.

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

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

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.

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 ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

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

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

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 ...

Fluorescence Lifetime Macro Imager for Biomedical Applications

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ranging from solid-state, O2-sensitive coatings to live animal tissue samples stained with soluble O2-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O2 phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability.
The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O2 levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O2-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O2 concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

This paper describes the use of a new, fast optical imager for the macroscopic photoluminescence lifetime imaging of long decay emitting samples. The integration, image acquisition, and analysis procedures are described, along with the preparation and characterization of the sensor materials for the imaging and the application of the imager in studying biological samples.

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...

Biology

In Vivo Fluorescence Imaging to Localize Antibodies in a Mouse Tumor Xenograft Model

Source: Shivange, G. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody.</a>&#160;J. Vis. Exp. (2020)
The video demonstrates a fluorescence imaging method for in vivo antibody localization in a mouse tumor xenograft model. It involves the injection of tumor cells in a mouse to generate solid xenografts beneath the skin. Subsequent injection of fluorescent antibodies confirms specific localization through enhanced fluorescence signals within the tumor xenograft.

1. Expression and purification of antibodies

	Maintenance of CHO cells
	
		Grow CHO cells in FreeStyle CHO Media supplemented with commercially available ...

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

Summary

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In Vivo Fluorescence Imaging to Localize Antibodies in a Mouse Tumor Xenograft Model