We thank Dr. Andrew Olson (Stanford Neuroscience Microscopy Service) for discussions and equipment use. We thank Dr. Juergen K. Willmann for his mentorship. This study was supported by NIH R21EB022214 grant (KEW), NIH R25CA118681 training grant (KEW), and NIH K99EB023279 (KEW). The Stanford Neuroscience Microscopy Service was supported by NIH NS069375.

All methods described here have been approved by the Institutional Administrative Panel on Laboratory Animal Care (APLAC) of Stanford University.
1. Transgenic mouse model of breast cancer development
<ol>
	<li>Observe mice from the desired cancer model for the appropriate tumor growth via palpation or caliper measurement before proceeding. 
	NOTE: Here, the transgenic murine model of breast cancer development (FVB/N-Tg(MMTV-PyMT)634Mul/J) (MMTV-PyMT) was used. These animals spontaneously develop invasive breast carcinomas between 6 and 12 weeks of age in each mammary gland20. Normal mammary glands were used as controls from the transgene-negative, age-matched littermates.</li>
</ol>
2. Intravenous injection of specific and nonspecific antibody agents
<ol>
	<li>Purify rabbit anti-mouse B7-H3 and rabbit IgG isotype control antibodies on a desalting column (e.g., PD-10) to remove preservatives and storage buffers following manufacturer instructions. 
	NOTE: Some antibodies may need further purification with Protein-A agarose beads based protocol21.</li>
	<li>Aliquot dosages of 33 &#181;g of each antibody conjugate in individual microcentrifuge tubes. 
	NOTE: Dosage of the administered agent may vary depending on the application and matches the dosages at which the antibody conjugate is routinely used. The concentration of the antibody solutions is required if the volume is more than 100 &#181;L for animal safety.</li>
	<li>At the desired time point before the tissue collection, here 96 h, anesthetize the tumor bearing animal with 2% isoflurane flowing in oxygen at 2 L/min, and place on a 37 &#176;C heated stage.&#160;Do a toe pinch to make sure the proper level of anesthsia was reached prior to the procedure.</li>
	<li>To prepare for the tail vein inoculation of the antibody solutions, disinfect the tail of the animal by wiping three times with an alcohol wipe. Dilate the tail veins by warming with a heat pad for approximately 30 s. Avoid heating the entire animal.&#160;Wipe the tail once more with an alcohol wipe after removing the heat pad.</li>
	<li>Using a 27G tail vein catheter, insert the butterfly needle into one of the two lateral tail veins and carefully fix the tail with the inserted needle to the stage with a piece of surgical tape. 
	NOTE: Visible blood backflow into the catheter indicates the proper location of the needle within the tail vein.</li>
	<li>Flush the catheter with 25 &#181;L of sterile phosphate buffered saline (PBS), then inject the antibody solution into the catheter using insulin syringes. Flush the catheter once again with 25 &#181;L of sterile PBS.</li>
	<li>Remove the needle from the tail and apply pressure to stop any bleeding.</li>
	<li>Turn off anesthesia and observe the animal until fully awake for any signs of distress.</li>
</ol>
3. Collection and preparation of target tumor tissues
<ol>
	<li>At the desired time point, humanely euthanize the animal according to the acceptable institutional procedure, here, by gradual inhalation of 100% CO2 from a compressed gas tank with a chamber displacement flow rate of 10-30% volume/min. 
	NOTE: Some applications of the IVIL technique require euthanasia by cardiac perfusion with PBS to remove freely circulating antibody19,22.</li>
	<li>After confirmation of humane euthanasia via cessation of respiratory and cardiac movement, lack of toe pinch response, and greying of mucous membranes, excise tumor tissues using surgical scissors and forceps as follows:
	<ol>
		<li>Lay the mouse in the supine position and grasping only the outer layer of skin between the set of mammary glands closest to the tail (5th) with forceps, make a small incision with a pair of surgical scissors.</li>
		<li>Introduce the closed scissors into the cut and slowly open the tip to carefully separate the skin from the underlying abdominal wall membrane keeping it intact.</li>
		<li>Make a vertical incision up the abdomen, continuing to separate the skin from the inner membrane. Between the 3rd and 4th mammary glands, make a horizontal cut across the abdomen to allow retraction of the skin and visualization of the mammary glands. 
		NOTE: Mammary tumors and glands are located superficially under the skin.</li>
		<li>Grasping each tumor or normal gland with forceps, carefully trim away the attached skin using surgical scissors.</li>
	</ol>
	</li>
	<li>Place excised tissues into tissue disposable base molds, prelabeled and filled with optimal cutting temperature (OCT) embedding medium and freeze the molds quickly by placement on dry ice. In order to study off-target delivery, excise other tissues or organs of interest (e.g., the liver or lungs). 
	NOTE: To pause the protocol at this point, store frozen tissue blocks at -80 &#176;C until ready to proceed.</li>
	<li>Using a cryostat, section frozen tissue blocks at 10 &#181;m thickness and place adjacent sections onto prelabeled adhesion glass slides. 
	NOTE: To pause the protocol at this point, store slides at -80 &#176;C until ready to proceed.</li>
</ol>
4. Ex vivo staining protocol
NOTE: For the quantitative comparison between fluorescence microscopy images, all slides are stained at the same time with the same prepared solutions.
<ol>
	<li>Rinse frozen tissue slides with room temperature PBS for 5 min to remove OCT.</li>
	<li>Demarcate tissue sections with a hydrophobic barrier pen to reduce the volume of solutions needed during staining. 
	NOTE: Be careful not to allow the pen to run over the tissue samples as it may either remove the tissue from the slide or prevent proper staining on the affected tissue portion. Do not allow the tissue sections to dehydrate at any point.</li>
	<li>Fix the tissue sections with 4% paraformaldehyde solution for 5 min.</li>
	<li>Rinse slides in PBS for 5 min.</li>
	<li>Permeabilize tissue sections with 0.5% Triton-X 100 in PBS for 15 min.</li>
	<li>Rinse slides in PBS for 5 min.</li>
	<li>Block the tissues with 3% w/v bovine serum albumin (BSA) and 5% v/v goat serum, both in PBS (blocking solution) for 1 h at room temperature. 
	NOTE: Match the blocking solution serum to the secondary antibody host animal.</li>
	<li>Rinse slides in PBS for 5 min.</li>
	<li>Incubate sections with record-keeping primary antibodies as desired, possibly a common nuclear (e.g., DAPI), vascular (e.g., CD31), or cytoplasmic marker (e.g., actin). Here, rat anti-mouse CD31 (vascular marker) at a 1:100 dilution was used according to manufacturer&#39;s instructions in the blocking solution overnight at 4 &#176;C protected from dehydration on a slide tray. 
	NOTE: Do not add additional primary antibody or antibody conjugate. The conjugate that was injected and allowed to accumulate in the tissues in vivo act as the&#160;primary antibody.</li>
	<li>Rinse slides in PBS for 5 min three times, changing the PBS each time.</li>
	<li>Incubate slides with secondary antibodies to label primary antibodies. For this application, visualize anti-B7-H3 antibody using AlexaFluor-546 conjugated goat anti-rabbit antibody (1:200 dilution, optimized according to manufacturer&#39;s instructions) and CD31 with AlexaFluor-488 goat anti-rat secondary antibody (1:200 dilution, optimized according to manufacturer&#39;s instructions) in blocking solution, protected from light and dehydration on a slide tray, for 1 h at room temperature. 
	NOTE: Secondary antibodies are from the same host animal but match the affinity of the secondary antibodies to the host species of the respective primary antibody. Slides are protected from light from this point onwards.</li>
	<li>Rinse slides in PBS for 5 min three times, changing the PBS each time.</li>
	<li>Apply one drop of the mounting medium into the center of the tissue slice and carefully place a coverslip avoiding entrapment of air bubbles.</li>
	<li>Seal the edges of the coverslip with clear nail polish and allow to dry. 
	NOTE: To pause the protocol for up to a week at this point, store slides at -20 &#176;C until ready to proceed.</li>
</ol>
5. Confocal microscopy imaging and quantitative image analysis
NOTE: Preparing the confocal microscope and imaging parameters will depend on the confocal system used. The microscope used here was purchased commercially (e.g., Zeiss LSM 510 Meta system) and the associated acquisition software was used (e.g., Zen 2009). However, many of these steps will apply to any confocal microscope and assume basic confocal microscopy knowledge.
<ol>
	<li>After the system has been turned on and warmed up, select the desired objective; here a 20x (numerical aperture = 0.8) objective was used.</li>
	<li>Load a positive control slide, coverslip down, to allow for setting the optimization for the brightest signal. Imaging in the red channel, focus the system on the sample in Live imaging mode.</li>
	<li>For each laser channel used, optimize the laser intensity, master gain, and pinhole size as follows:
	<ol>
		<li>Switch to Continuous mode for imaging.</li>
		<li>Optimize the Laser Intensity (which controls the laser power) and Gain (Master) (which controls the voltage for photomultiplier tube) slide bars while monitoring the Look Up Table (LUT) histogram. Adjust these two settings until the dynamic range of the histogram is filled without saturating pixels. 
		NOTE: If the laser intensity is too high photobleaching will occur. If the Gain (Master) is too high, the image will become noisy. Ideally, the Gain (Master) will be in the middle of its range.</li>
		<li>Set the pinhole to 1 airy unit (AU), which gives the highest resolution and the thinnest z-slice.</li>
		<li>Slide the Digital Offset bar to minimize the noise floor on the LUT histogram for a true black background. 
		NOTE: Once microscopy settings are optimized for each laser channel and objective, keep them constant throughout the imaging session and for imaging of all slides, to allow for the quantitative comparison between slides.</li>
	</ol>
	</li>
	<li>Under the Acquisition Mode tab, under Averaging, select the desired Number and Bit Depth. Click the Optimal button to set the optimal pixel size.</li>
	<li>Use the Snap acquisition button to collect a high-quality image. Here, random fields of view were selected from within the tumor, but other areas of interest may apply for different applications (vessels, tumor margins, penetration depth, etc.)</li>
	<li>Save image files in the format used by the confocal software (here, &#34;.lsm&#34;) for offline processing and quantification. 
	NOTE: If not all slides are imaged during the same session, save the settings and reload them on subsequent visits, though reimaging the same slide is not recommended due to photobleaching.</li>
	<li>Perform the quantitative fluorescence intensity measurements. Open Fiji (Fiji Is Just ImageJ software)19,23 and load an .lsm image file by dragging onto the status bar.</li>
	<li>Split the color channel data. Go to Image &#62; Stacks &#62; Split Channels.</li>
	<li>Preprocess the fluorescence images as needed, i.e., subtract the background signal (Process &#62; Subtract Background) or reduce the noise via a filtering method.</li>
	<li>Segment the color channel corresponding to the reference protein (vascular, nuclear, cellular stain) by setting a threshold on the signal intensity (Image &#62; Adjust &#62; Threshold). 
	NOTE: Manual thresholding introduces subjectivity into the image analysis, therefore, using an automated thresholding algorithm or referencing image histograms makes image analysis results less biased.</li>
	<li>Use this threshold to create a binary mask (Process &#62; Binary &#62; Convert to Mask).</li>
	<li>Measure and label ROIs within the mask (Analyze &#62; Analyze Particles &#62; Check Add to Manager &#62; OK).</li>
	<li>Apply ROIs to the color channel corresponding to the antibody of interest (Click channel image, Analyze &#62; Tools &#62; ROI Manager &#62; Measure). This will apply the mask ROIs to the antibody image and provide image measurements for label sections in the Results window. Save results window (File &#62; Save).</li>
	<li>Calculate the desired statistic of interest, such as the mean fluorescence intensity as described here24.</li>
	<li>Perform all processing steps identically to each image within a set of slides. Create a macro to automatically do this for large image batches23.</li>
	<li>For image display only after quantitative measurements, apply qualitative image adjustments to optimize visualization of biodistribution patterns by adjusting minimum, maximum, brightness, and contrast to the same levels on all slides (Image &#62; Adjust &#62; Brightness/Contrast).</li>
	<li>Convert the image type (Image &#62; Type &#62; RGB Color) and save files in a lossless image type such as .tiff (File &#62; Save As &#62; Tiff&#8230;) for use in presentations and publications.</li>
</ol>

<ol>
	<li>Forthal, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+Antibodies.">Functions of Antibodies.</a> Microbiology Spectrum. 2 (4), 1-17 (2014).</li><li>Boder, E. T., Midelfort, K. S., Wittrup, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+evolution+of+antibody+fragments+with+monovalent+femtomolar+antigen-binding+affinity.">Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (20), 10701-10705 (2000).</li><li>K&#246;hler, G., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cultures+of+fused+cells+secreting+antibody+of+predefined+specificity.">Continuous cultures of fused cells secreting antibody of predefined specificity.</a> Nature. 256 (5517), 495-497 (1975).</li><li>Lonberg, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-specific+human+antibodies+from+mice+comprising+four+distinct+genetic+modifications.">Antigen-specific human antibodies from mice comprising four distinct genetic modifications.</a> Nature. 368 (6474), 856-859 (1994).</li><li>McCafferty, J., Griffiths, A. D., Winter, G., Chiswell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+antibodies:+filamentous+phage+displaying+antibody+variable+domains.">Phage antibodies: filamentous phage displaying antibody variable domains.</a> Nature. 348 (6301), 552-554 (1990).</li><li>Ferlay, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+incidence+and+mortality+worldwide:+sources,+methods+and+major+patterns+in+GLOBOCAN+2012.">Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012.</a> International Journal of Cancer. 136 (5), E359-E386 (2015).</li><li>Reichert, J. M., Valge-Archer, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+trends+for+monoclonal+antibody+cancer+therapeutics.">Development trends for monoclonal antibody cancer therapeutics.</a> Nature Reviews Drug Discovery. 6 (5), 349-356 (2007).</li><li>Kircher, M. F., Willmann, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Body+Imaging:+MR+Imaging,+CT,+and+US.+Part+I.+Principles.">Molecular Body Imaging: MR Imaging, CT, and US. Part I. Principles.</a> Radiology. 263 (3), 633-643 (2012).</li><li>Fleuren, E. D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theranostic+applications+of+antibodies+in+oncology.">Theranostic applications of antibodies in oncology.</a> Molecular Oncology. 8 (4), 799-812 (2014).</li><li>Forsstr&#246;m, B., Bis&#322;awska Axn&#228;s, B., Rockberg, J., Danielsson, H., Bohlin, A., Uhlen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+Antibodies+with+Regards+to+Linear+and+Conformational+Epitopes.">Dissecting Antibodies with Regards to Linear and Conformational Epitopes.</a> PLoS ONE. 10 (3), (2015).</li><li>Woof, J. M., Burton, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+antibody-Fc+receptor+interactions+illuminated+by+crystal+structures.">Human antibody-Fc receptor interactions illuminated by crystal structures.</a> Nature Reviews Immunology. 4 (2), 89-99 (2004).</li><li>Brooks, 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=Translational+genomics:+The+challenge+of+developing+cancer+biomarkers.">Translational genomics: The challenge of developing cancer biomarkers.</a> Genome Research. 22 (2), 183-187 (2012).</li><li>Tabrizi, M., Bornstein, G. G., Suria, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+Mechanisms+of+Therapeutic+Monoclonal+Antibodies+in+Health+and+Disease.">Biodistribution Mechanisms of Therapeutic Monoclonal Antibodies in Health and Disease.</a> The AAPS Journal. 12 (1), 33-43 (2009).</li><li>Duraiyan, J., Govindarajan, R., Kaliyappan, K., Palanisamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+immunohistochemistry.">Applications of immunohistochemistry.</a> Journal of Pharmacy &amp; Bioallied Sciences. 4 (Suppl 2), S307-S309 (2012).</li><li>Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+cancer+with+positron+emission+tomography.">Molecular imaging of cancer with positron emission tomography.</a> Nature Reviews. Cancer. 2 (9), 683-693 (2002).</li><li>Freise, A. C., Wu, A. M. <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+with+Antibodies+and+Engineered+Fragments.">In vivo Imaging with Antibodies and Engineered Fragments.</a> Molecular Immunology. 67 (200), 142-152 (2015).</li><li>Cilliers, C., Menezes, B., Nessler, I., Linderman, J., Thurber, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Tumor+Penetration+and+Single-Cell+Targeting+of+Antibody-Drug+Conjugates+Increases+Anticancer+Efficacy+and+Host+Survival.">Improved Tumor Penetration and Single-Cell Targeting of Antibody-Drug Conjugates Increases Anticancer Efficacy and Host Survival.</a> Cancer Research. 78 (3), 758-768 (2018).</li><li>Wilson, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectroscopic+Photoacoustic+Molecular+Imaging+of+Breast+Cancer+using+a+B7-H3-targeted+ICG+Contrast+Agent.">Spectroscopic Photoacoustic Molecular Imaging of Breast Cancer using a B7-H3-targeted ICG Contrast Agent.</a> Theranostics. 7 (6), 1463-1476 (2017).</li><li>Wischhusen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+molecular+imaging+as+a+non-invasive+companion+diagnostic+for+netrin-1+interference+therapy+in+breast+cancer.">Ultrasound molecular imaging as a non-invasive companion diagnostic for netrin-1 interference therapy in breast cancer.</a> Theranostics. 8 (18), 5126-5142 (2018).</li><li>Guy, C. T., Cardiff, R. D., Muller, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+tumors+by+expression+of+polyomavirus+middle+T+oncogene:+a+transgenic+mouse+model+for+metastatic+disease.">Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.</a> Molecular and Cellular Biology. 12 (3), 954-961 (1992).</li><li>Hober, S., Nord, K., Linhult, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+A+chromatography+for+antibody+purification.">Protein A chromatography for antibody purification.</a> Journal of Chromatography B. 848 (1), 40-47 (2007).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> Journal of Visualized Experiments JoVE. (65), (2012).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Bachawal, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Earlier+detection+of+breast+cancer+with+ultrasound+molecular+imaging+in+a+transgenic+mouse+model.">Earlier detection of breast cancer with ultrasound molecular imaging in a transgenic mouse model.</a> Cancer Research. 73, 1689-1698 (2013).</li><li>Fitamant, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrin-1+expression+confers+a+selective+advantage+for+tumor+cell+survival+in+metastatic+breast+cancer.">Netrin-1 expression confers a selective advantage for tumor cell survival in metastatic breast cancer.</a> Proceedings of the National Academy of Sciences. 105 (12), 4850-4855 (2008).</li><li>Kennedy, T. E., Serafini, T., de la Torre, J. R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord.">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78 (3), 425-435 (1994).</li><li>Ryman, J. T., Meibohm, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+of+Monoclonal+Antibodies.">Pharmacokinetics of Monoclonal Antibodies.</a> CPT: Pharmacometrics &amp; Systems Pharmacology. 6 (9), 576-588 (2017).</li><li>Scalia, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+Masking+During+Fixation+and+Embedding,+Dissected.">Antigen Masking During Fixation and Embedding, Dissected.</a> Journal of Histochemistry and Cytochemistry. 65 (1), 5-20 (2017).</li><li>Robertson, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+labeled+tomato+lectin+for+imaging+vasculature+structures.">Use of labeled tomato lectin for imaging vasculature structures.</a> Histochemistry and Cell Biology. 143 (2), 225-234 (2015).</li><li>Chen, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+reprograms+lymphatic+vessels+to+blood+vessels.">Blood flow reprograms lymphatic vessels to blood vessels.</a> The Journal of Clinical Investigation. 122 (6), 2006-2017 (2012).</li><li>Anderson, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+staining+for+discrimination+of+vascular+and+tissue+leukocytes.">Intravascular staining for discrimination of vascular and tissue leukocytes.</a> Nature Protocols. 9 (1), 209-222 (2014).</li><li>de Boer, E., et al. <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+Fluorescence+Immunohistochemistry:+Localization+of+Fluorescently+Labeled+Cetuximab+in+Squamous+Cell+Carcinomas.">In Vivo Fluorescence Immunohistochemistry: Localization of Fluorescently Labeled Cetuximab in Squamous Cell Carcinomas.</a> Scientific Reports. 5, (2015).</li><li>Jenkins, R. W., Barbie, D. A., Flaherty, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+resistance+to+immune+checkpoint+inhibitors.">Mechanisms of resistance to immune checkpoint inhibitors.</a> British Journal of Cancer. 118 (1), 9-16 (2018).</li><li>Rexer, B. N., Arteaga, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+acquired+resistance+to+HER2-targeted+therapies+in+HER2+gene-amplified+breast+cancer:+mechanisms+and+clinical+implications.">Intrinsic and acquired resistance to HER2-targeted therapies in HER2 gene-amplified breast cancer: mechanisms and clinical implications.</a> Critical Reviews in Oncogenesis. 17 (1), 1-16 (2012).</li></ol>

The authors have nothing to disclose.

This method has several critical steps and requires potential modifications to ensure successful implementation. First, the dosage and timing of the antibody/antibody conjugate intravenous injection must be tailored to the specific application. Generally, dosages should be used that are consistent with how the antibody conjugate will typically be used, i.e., matching dosages of the therapeutic antibody or antibody-based contrast agent. Also, the timing of the collection of target tissues should be carefully considered. A...

Monoclonal antibodies (mAb) are large glycoproteins (approximately 150 kDa) of the immunoglobulin superfamily that are secreted by B cells and have a primary function in the immune system to identify and either inhibit the biological function of, or mark for destruction, bacterial or viral pathogens, and can recognize abnormal protein expression on cancer cells1. Antibodies can have an extremely high affinity to their specific epitopes down to femtomolar concentrations making them highly promising tools in biomedicine2. With the development of hybridoma technology by Milstein and K&#246;hler (awarded the Nobel Prize in 1984), the production of mAbs became possible3. Later, human mAbs were generated using the phage display technology or transgenic mouse strains and revolutionized their use as novel research tools and therapeutics4,5.
Cancer is a worldwide health issue and a major cause of death creating the need for novel approaches for prevention, detection, and therapy6. To date, mAbs have allowed extrication of the role of genes and their proteins in tumorigenesis and when directed against cancer biomarkers, can enable tumor detection and characterization for patient stratification. For cancer therapy, bispecific mAbs, antibody-drug conjugates, and smaller antibody fragments are being developed as therapeutics, and for the targeted drug delivery to enhance therapeutic efficacy7. Additionally, antibodies serve for the biomarker targeting of contrast agents for molecular imaging modalities such as fluorescence-guided surgery, photoacoustic (PA) imaging, ultrasound (US) molecular imaging, and clinically used positron emission tomography (PET) or single photon emission computed tomography (SPECT)8. Finally, antibodies can also be used as theranostic agents enabling stratification of patients and response monitoring for targeted therapies9. Therefore, novel mAbs are beginning to play a critical role in cancer detection, diagnosis, and treatment.
Despite critical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be rendered ineffective due to the complexity of the tumor environment. Antibody interactions are dependent on the type of epitope, i.e., whether it is linear or conformational10. In addition to the recognition of antigens, antibodies need to overcome natural barriers such as vessel walls, basal membranes, and the tumor stroma to reach target cells expressing the antigen. Antibodies interact with the tissue not only through the variable fragment antigen binding (Fab) domain but also through the constant crystalline fragment (Fc) which further leads to off-site interactions11. Targeting is also complicated by the heterogeneous expression of tumor markers throughout the tumor bulk and heterogeneity in tumor vascularization and the lymphatics system12,13. In addition, the tumor microenvironment is composed of cancer-associated fibroblasts which support tumor cells, tumor immune cells that suppress anti-tumor immune reactions, and the tumor endothelium which supports the transport of oxygen and nutrients, all of which interfere with the penetration, distribution, and availability of antibody-based therapeutics or diagnostics. Overall, these considerations can limit therapeutic or diagnostic efficacy, reduce treatment response, and may result in tumor resistance.
Therefore, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate within the tumor microenvironment. Currently, in preclinical studies, marker expression in tumor research models is analyzed ex vivo by immunofluorescence (IF) staining of tumor sections14. Standard IF staining is performed with primary marker-specific antibodies which are then highlighted by secondary fluorescently labeled antibodies on ex vivo tumor tissue slices that have been isolated from the animal. This technique highlights the static location of the marker at the time of tissue fixation and does not provide insight into how the antibody-based therapeutics or diagnostics might distribute or interact in physiological conditions. Molecular imaging by PET, SPECT, US, and PA can provide information about the antibody-conjugated contrast agent distribution in living preclinical models8,15. As these imaging modalities are non-invasive, longitudinal studies can be performed and time-sensitive data can be collected with a minimal number of animals per group. However, these non-invasive molecular imaging approaches are not sensitive enough and do not have enough resolution for the localization of antibody distribution at the cellular level. Additionally, the physical and biological characteristics of the primary antibody may be drastically changed by the conjugation of a contrast agent16.
In order to take the in vivo physiological and pathological conditions into consideration of how antibody-based therapeutics and diagnostics interact within the tumor environment and to obtain high-resolution cellular and even sub-cellular distribution profiles of non-conjugated antibodies, we propose an IF approach, deemed In Vivo Immunofluorescence Localization (IVIL), in which the antigen-specific antibody is intravenously injected in vivo. The antibody-based therapeutic or diagnostic, acting as a primary antibody, circulates in functional blood vessels and binds to its target protein in the highly accurate, living tumor environment. After isolation of in vivo-labeled tumors with the primary antibody, a secondary antibody is used to localize accumulated and retained antibody conjugates. This approach is similar to a previously described IF histology approach injecting fluorescently labeled antibodies17. Though here, the use of non-conjugated antibodies avoids a potential change in biodistribution characteristics induced by antibody modification. Furthermore, ex vivo application of fluorescent secondary antibody avoids a possible loss of fluorescence signal during tissue collection and processing and provides amplification of fluorescence signal intensity. Our labeling approach reflects in vivo biodistribution of antibody-based drugs and targeted agents and can provide important insights for the development of novel diagnostic and therapeutic agents.
Here, we describe two applications of the IVIL method as applied in previous studies investigating the biodistribution and accessibility of antibody-based contrast agents for molecular imaging approaches for breast cancer detection. First, the biodistribution of an antibody-near infrared dye conjugate (anti-B7-H3 antibody bound to the near infrared fluorescence dye, indocyanine green, B7-H3-ICG) and the isotype control agent (Iso-ICG) for&#160;fluorescence and photoacoustic molecular imaging is explored18. This application&#39;s method is described in the protocol. Next, the biodistribution results of a conformationally sensitive antibody to netrin-1, typically not detectable with traditional IF imaging, used with ultrasound molecular imaging, is quantified and presented in the representative results19. At the conclusion of this protocol paper, readers should feel comfortable adopting the IVIL method for their own antibody-based research applications.

<table><tbody><tr><td>&lt;strong&gt;Animal Model&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FVB/N-Tg(MMTV-PyMT)634Mul/J</td><td>The Jackson Laboratory</td><td>002374</td><td>Females, 4-6 weeks of age</td></tr><tr><td>&lt;strong&gt;Animal Handling Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>27G Catheter</td><td>VisualSonics</td><td>Please call to order</td><td>Vevo MicroMarker Tail Vein Access Cannulation Kit</td></tr><tr><td>Alcohol Wipes</td><td>Fisher Scientific</td><td>22-246073</td><td></td></tr><tr><td>Gauze Sponges (4&quot; x 4&quot; 16 Ply)</td><td>Cardinal Health</td><td>2913</td><td></td></tr><tr><td>Heat Lamp</td><td>Morganville Scientific</td><td>&amp;nbsp;HL0100</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein Animal Health</td><td>29404</td><td></td></tr><tr><td>Ophthalmic Ointment</td><td>Fisher Scientific</td><td>NC0490117</td><td></td></tr><tr><td>Surgical Tape</td><td>3M</td><td>1530-1</td><td></td></tr><tr><td>&lt;strong&gt;Tissue Collection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disposable Base Molds</td><td>Fisher Scientific</td><td>22-363-556</td><td></td></tr><tr><td>Optimal Cutting Temperature (OCT) Medium</td><td>Fisher Scientific</td><td>23-730-571</td><td></td></tr><tr><td>Surgical London Forceps</td><td>Fine Science Tools</td><td>11080-02</td><td></td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AlexaFluor-488 goat anti-rat IgG</td><td>Life Technologies</td><td>A-11006</td><td></td></tr><tr><td>AlexaFluor-546 goat anti-rabbit IgG</td><td>Life Technologies</td><td>A-11010</td><td></td></tr><tr><td>AlexaFluor-594 goat anti-human IgG</td><td>Life Technologies</td><td>A11014</td><td></td></tr><tr><td>Human IgG Isotype Control</td><td>Novus Biologicals</td><td>NBP1-97043</td><td></td></tr><tr><td>Humanized anti-netrin-1 antibody&amp;nbsp;</td><td>Netris Pharma</td><td></td><td>contact@netrispharma.com</td></tr><tr><td>Rabbit anti-Mouse CD276 (B7-H3)</td><td>Abcam</td><td>ab134161</td><td>EPNCIR122 Clone</td></tr><tr><td>Rat anti-Mouse CD31</td><td>BD Biosciences</td><td>550274</td><td>MEC 13.3 Clone</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153-50G</td><td></td></tr><tr><td>Clear Nail Polish</td><td></td><td></td><td>Any local drug store</td></tr><tr><td>Indocyanine Green - NHS</td><td>Intrace Medical</td><td>ICG-NHS ester</td><td></td></tr><tr><td>Mounting Medium</td><td>ThermoFisher Scientific</td><td>TA-006-FM</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Fisher Scientific</td><td>ICN19135680</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Fisher Scientific</td><td>AAJ19943K2</td><td></td></tr><tr><td>Sterile Phosphate Buffered Saline (PBS)</td><td>ThermoFisher Scientific</td><td>14190250</td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adhesion Glass Slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Desalting Columns</td><td>Fisher Scientific</td><td>45-000-148</td><td></td></tr><tr><td>Glass Cover Slips</td><td>Fisher Scientific</td><td>12-544G</td><td></td></tr><tr><td>Hydrophobic Barrier Pen</td><td>Ted Pella</td><td>22311</td><td></td></tr><tr><td>Microcentrifuge Tubes</td><td>Fisher Scientific</td><td>05-402-25</td><td></td></tr><tr><td>Slide Staining Tray</td><td>VWR</td><td>87000-136</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FIJI</td><td>LOCI, UW-Madison.</td><td>Version 4.0</td><td>https://fiji.sc/</td></tr></tbody></table>

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 IVIL method was used here to examine the in vivo biodistribution and tissue interaction of B7-H3-ICG and Iso-ICG, by allowing the agents, after intravenous injection into a living animal, to interact with the target tissue for 96 h, and then once the tissues are harvested, to act as the primary antibodies during ex vivo immunostaining. The IVIL method was also compared to the standard ex vivo IF staining of the tissues for the B7-H3 marker. Normal murine mammary glands do not express ...

Watch this Scientific Journal Video about In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research at JoVE.com

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

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 ...

in-vivo-immunofluorescence-localization-for-assessment-therapeutic

Research

JoVE Journal

Cancer Research

8.9K Views.  INSERM U1052-CNRS UMR5286, Centre Léon Bérard. Dit protocol voor in vivo immunofluorescentielokalisatie, of IVIL, beoordeelt de in vivo biodistributie van diagnostische en therapeutische antilichamen of op antilichamen gebaseerde middelen voor kankeronderzoek, detectie en behandeling. In tegenstelling tot conventionele immunofluorescente kleuring waar cellulaire expressie van antigenen ex vivo wordt onthuld, verduidelijkt de IVIL-methode hoe antilichamen en antilichaamgebaseerde agentia zich verspreiden in het levende dier. Deze video hel...

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

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 ...

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Medicine

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

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

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

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

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

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

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

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

Source: Shivange, G. et al., <a href="https://app.jove.com/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 ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

An In Vivo PET Imaging Technique to Detect Tumors in a Murine Model Using Radiolabeled Antibodies

Source: Zeglis, B. M., et al. <a href="https://app.jove.com/v/52521">The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies.</a> J. Vis. Exp. (2015).
This video demonstrates a technique involving a radioimmunoconjugate for the in vivo imaging of a tumor in a mouse model. Upon injection of the radioimmunoconjugate, the antibodies of the conjugate bind to a specific membrane antigen on the cancer cells. The radiotracer of the conjugate emits positrons that interact with neighboring electrons to produce energy, which is detected by a positron emission tomography (PET) scanner to create an image of the tumor.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. In ...

A Technique to Assess the In Vivo Localization of Tumor-Specific Antibodies

Source: Shivange, G., et al. <a href="https://app.jove.com/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody.</a> J. Vis. Exp. (2020).
This video demonstrates a technique to study the in vivo localization of tumor-specific antibodies in a mouse tumor xenograft model. Upon subcutaneously injecting cancer cells into mice for tumor formation, fluorescently-labeled antibodies are injected that bind to tumor-specific receptors, enabling in vivo assessment of antibody localization via imaging.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Immunodiagnostics

Imaging and Visualization Techniques

An In Vivo Immunofluorescence Localization Method to Study Antibody Biodistribution

Source: Wischhusen, J. C., et al. <a href="https://app.jove.com/v/59810">In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research.</a> J. Vis. Exp. (2019).
This video demonstrates an in vivo immunofluorescence localization (IVIL) technique to study the biodistribution of tumor-targeting antibodies in a murine model of breast cancer. Upon the intravenous injection of a tumor-specific antibody in the mouse, the tumor is surgically isolated, and the localization of the antibody within the tumor tissue is confirmed via ex vivo immunostaining.

An In Vivo Technique to Track Bispecific Antibody-Induced T Cell Trafficking to Tumors

After anesthetizing the mouse using a 26-gauge needle, inject 20 million T cells in 100 microliters of media retro-orbitally. Administer 1,000 units of recombinant IL-2 subcutaneously to support T cell survival in vivo. Then, administer the bispecific antibody retro-orbitally or intraperitoneally.
For in vivo imaging, administer 100 microliters of three milligrams of D-luciferin by retro-orbital injection with a 26-gauge needle in an anesthetized mouse. To capture images, place the mouse in the light-tight chamber of the imager, such that the flank bearing the xenograft is facing up toward the camera. Using the acquisition control panel, select luminescent, photograph, and overlay. Set the exposure time to auto, the binning to medium, and F-stop to one, and acquire images.
After the first image, set the exposure time to match that automatically calculated for the first image so that subsequent images may be compared directly. Take another set of images with the mouse supine to evaluate T cell presence in the lungs.

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

Samenvatting

Meer video's verkennen