The authors thank Dr. Julia Mannheim, Walter Ehrlichmann, Ramona Stumm, Funda Cay, Daniel Bukala, Maren Harant as well as Natalie Altmeyer for the support during the experiments and data analysis. This work was supported by the Werner Siemens-Foundation, the DFG through the SFB685 (project B6) and Fort&#252;ne (2309-0-0).

<ol>
	<li>Cerri, S., 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=Intracarotid+Infusion+of+Mesenchymal+Stem+Cells+in+an+Animal+Model+of+Parkinson's+Disease,+Focusing+on+Cell+Distribution+and+Neuroprotective+and+Behavioral+Effects.">Intracarotid Infusion of Mesenchymal Stem Cells in an Animal Model of Parkinson's Disease, Focusing on Cell Distribution and Neuroprotective and Behavioral Effects.</a> Stem Cells Trans Med. 4 (9), 1073-1085 (2015).</li><li>Hasenbach, K., 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=Monitoring+the+glioma+tropism+of+bone+marrow-derived+progenitor+cells+by+2-photon+laser+scanning+microscopy+and+positron+emission+tomography.">Monitoring the glioma tropism of bone marrow-derived progenitor cells by 2-photon laser scanning microscopy and positron emission tomography.</a> Neuro Oncol. 14 (4), 471-481 (2012).</li><li>Sood, 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=Biodistribution+of+18F-FDG-Labeled+Autologous+Bone+Marrow+&#8211;+Derived+Stem+Cells+in+Patients+With+Type+2+Diabetes+Mellitus.">Biodistribution of 18F-FDG-Labeled Autologous Bone Marrow &#8211; Derived Stem Cells in Patients With Type 2 Diabetes Mellitus.</a> Clin Nucl Med. 40 (9), 697-700 (2015).</li><li>Perez-Diez, A., 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=CD4+cells+can+be+more+efficient+at+tumor+rejection+than+CD8+cells.">CD4 cells can be more efficient at tumor rejection than CD8 cells.</a> Blood. 109 (12), 5346-5354 (2007).</li><li>Muranski, P., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+of+cancer+using+CD4++T+cells.">Adoptive immunotherapy of cancer using CD4+ T cells.</a> Curr. Opin. Immunol. 21 (2), 200-208 (2009).</li><li>Braumuller, H., 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=T-helper-1-cell+cytokines+drive+cancer+into+senescence.">T-helper-1-cell cytokines drive cancer into senescence.</a> Nature. 494 (7437), 361-365 (2013).</li><li>Kochenderfer, J. 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=Eradication+of+B-lineage+cells+and+regression+of+lymphoma+in+a+patient+treated+with+autologous+T+cells+genetically+engineered+to+recognize+CD19.">Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19.</a> Blood. 116 (20), 4099-4102 (2010).</li><li>Cherry, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+Positron+Emission+Tomography+and+Applications+in+Preclinical+Drug+Development.">Fundamentals of Positron Emission Tomography and Applications in Preclinical Drug Development.</a> J. Clin. Pharmacol. 41 (5), 482-491 (2001).</li><li>Tavar&#233;, 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=An+Effective+Immuno-PET+Imaging+Method+to+Monitor+CD8-Dependent+Responses+to+Immunotherapy.">An Effective Immuno-PET Imaging Method to Monitor CD8-Dependent Responses to Immunotherapy.</a> Cancer Res. 76 (1), 73-82 (2016).</li><li>Tavar&#233;, 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=Engineered+antibody+fragments+for+immuno-PET+imaging+of+endogenous+CD8++T+cells+in+vivo.">Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 111 (3), 1108-1113 (2014).</li><li>Dobrenkov, K., 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=Monitoring+the+Efficacy+of+Adoptively+Transferred+Prostate+Cancer-Targeted+Human+T+Lymphocytes+with+PET+and+Bioluminescence+Imaging.">Monitoring the Efficacy of Adoptively Transferred Prostate Cancer-Targeted Human T Lymphocytes with PET and Bioluminescence Imaging.</a> J Nucl Med. 49 (7), 1162-1170 (2008).</li><li>Rini, J. 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=PET+with+FDG-labeled+Leukocytes+versus+Scintigraphy+with+111In-Oxine-labeled+Leukocytes+for+Detection+of+Infection.">PET with FDG-labeled Leukocytes versus Scintigraphy with 111In-Oxine-labeled Leukocytes for Detection of Infection.</a> Radiology. 238 (3), 978-987 (2006).</li><li>Ritchie, D., 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+tracking+of+macrophage+activated+killer+cells+to+sites+of+metastatic+ovarian+carcinoma.">In vivo tracking of macrophage activated killer cells to sites of metastatic ovarian carcinoma.</a> Cancer Immunol. Immunother. 56 (2), 155-163 (2006).</li><li>Adonai, 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=Ex+vivo+cell+labeling+with+64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone)+for+imaging+cell+trafficking+in+mice+with+positron-emission+tomography.">Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography.</a> Proc. Natl. Acad. Sci. U.S.A. 99 (5), 3030-3035 (2002).</li><li>Huang, J., Lee, C. C. I., Sutcliffe, J. L., Cherry, S. R., Tarantal, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiolabeling+Rhesus+Monkey+CD34++Hematopoietic+and+Mesenchymal+Stem+Cells+with+64Cu-Pyruvaldehyde-Bis(N4-Methylthiosemicarbazone)+for+MicroPET+Imaging.">Radiolabeling Rhesus Monkey CD34+ Hematopoietic and Mesenchymal Stem Cells with 64Cu-Pyruvaldehyde-Bis(N4-Methylthiosemicarbazone) for MicroPET Imaging.</a> Mol. Imaging. 7 (1), (2008).</li><li>Griessinger, C. M., 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+Tracking+of+Th1+Cells+by+PET+Reveals+Quantitative+and+Temporal+Distribution+and+Specific+Homing+in+Lymphatic+Tissue.">In Vivo Tracking of Th1 Cells by PET Reveals Quantitative and Temporal Distribution and Specific Homing in Lymphatic Tissue.</a> J Nucl Med. 55 (2), 301-307 (2014).</li><li>Griessinger, C. M., 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=64Cu+antibody-targeting+of+the+T-cell+receptor+and+subsequent+internalization+enables+in+vivo+tracking+of+lymphocytes+by+PET.">64Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET.</a> Proc. Natl. Acad. Sci. U.S.A. 112 (4), 1161-1166 (2015).</li><li>McCarthy, D. W., 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=Efficient+production+of+high+specific+activity+64Cu+using+a+biomedical+cyclotron.">Efficient production of high specific activity 64Cu using a biomedical cyclotron.</a> Nucl. Med. Biol. 24 (1), 35-43 (1997).</li><li>Kalkhof, S., Sinz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chances+and+pitfalls+of+chemical+cross-linking+with+amine-reactive+N-hydroxysuccinimide+esters.">Chances and pitfalls of chemical cross-linking with amine-reactive N-hydroxysuccinimide esters.</a> Anal. Bioanal. Chem. 392 (1), 305-312 (2008).</li><li>Bedoya, S. K., Wilson, T. D., Collins, E. L., Lau, K., Larkin Iii, ., J, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Th17+Differentiation+of+Naive+CD4+T+Lymphocytes.">Isolation and Th17 Differentiation of Naive CD4 T Lymphocytes.</a> J Vis Exp. (79), e50765 (2013).</li><li>Flaherty, S., Reynolds, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Naive+CD4++T+Cell+Isolation+and+In+vitro+Differentiation+into+T+Cell+Subsets.">Mouse Naive CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets.</a> J Vis Exp. (98), e52739 (2015).</li><li>Judenhofer, M., Wiehr, S., Kukuk, D., Fischer, K., Pichler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+363.">Chapter 363.</a> Small Animal Imaging. Basics and Practical Guide. , 363-370 (2011).</li><li>Phelps, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PET. Molecular Imaging and Its Biological Applications. , 93-101 (2004).</li><li>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=Antibodies+and+Antimatter:+The+Resurgence+of+Immuno-PET.">Antibodies and Antimatter: The Resurgence of Immuno-PET.</a> JNM. 50 (1), 2-5 (2009).</li><li>Lewis, M. 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=In+vivo+evaluation+of+pretargeted+64Cu+for+tumor+imaging+and+therapy.">In vivo evaluation of pretargeted 64Cu for tumor imaging and therapy.</a> J Nucl Med. 44 (8), 1284-1292 (2003).</li><li>Boswell, C. A., 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=Comparative+in+vivo+stability+of+copper-64-labeled+cross-bridged+and+conventional+tetraazamacrocyclic+complexes.">Comparative in vivo stability of copper-64-labeled cross-bridged and conventional tetraazamacrocyclic complexes.</a> J Med Chem. 47 (6), 1465-1474 (2004).</li><li>Ghosh, S. C., 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=Comparison+of+DOTA+and+NODAGA+as+chelators+for+(64)Cu-labeled+immunoconjugates.">Comparison of DOTA and NODAGA as chelators for (64)Cu-labeled immunoconjugates.</a> Nucl Med Biol. 42 (2), 177-183 (2015).</li><li>Johnson, T. E., Birky, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Health Physics and Radiological Health. , (2011).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Meth. 2 (12), 932-940 (2005).</li></ol>

The authors have nothing to disclose.

This protocol presents a reliable and easy method to stably radiolabel cells for in vivo tracking by PET. Utilizing this method, cOVA-TH1 cells, isolated and expanded in vitro from donor mice, could be radiolabeled with 64Cu-DOTA-KJ1-26-mAb and their homing was tracked to the pulmonary and perithymic LNs as sites of cOVA presentation in a cOVA-induced acute airway DTHR.
The modification of the mAb with the chelator requires fast and efficient working and the use of ultra-pure solutions without traces of amines. The solutions were treated with a chelating ion exchange resin to ensure proper conjugation of the DOTA-NHS active ester to amine residues of the mAb. The chelator-conjugated mAb as an intracellular radiolabel has several advantages over the use of non-specific cell labeling agents. First, the mAb provides excellent specificity allowing for the targeted labeling of a defined cell population. The mAb itself is not cytotoxic and has no detrimental effect on the viability or functionality of the cells. Next, the efflux of radioactivity from the cells was significantly reduced improving the signal-to-background ratio of the PET images17,24. By choosing a mAb with a different specificity, this method is easily transferrable to other cell populations of interest such as CD8+ TIL or CD14+ monocytes. Moreover, this cell labeling approach can be used for cell tracking studies in various animal models of inflammatory human diseases (e.g., rheumatoid arthritis and bronchial asthma) or animal models for different human cancer types. This labeling method, however, is confined to membrane-bound receptors or differentiations markers as targets since mAbs do not passively diffuse across the cell membrane. The activation-induced internalization of the target or, simply, membrane-shuttling is advantageous. A high avidity between the mAb and the target, however, might also be sufficient for in vivo imaging, possibly with a reduced signal to background ratio.
The choice of DOTA as a chelator for our experiments was based on early work using 64Cu25. However, in the meantime DOTA was shown to not be an optimal chelator for this isotope, leading to rather high activity uptake in the liver by transchelation to superoxide dismutase26. Although using the radiolabeled antibody for in vitro labeling of cells that are then adoptively transferred should lower the risk of 64Cu release and transport to the liver, modern chelators optimized for 64Cu, like 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) or 1,4,7-triazacyclononane-triacetic acid (NOTA), can be preferably used with our method to further enhance specificity of the obtained data27. Besides, other active esters can be used for 64Cu (e.g. N-chlorosuccinimide (NCS)) or other isotopes, such as 89Zr (e.g. desferrioxamine; DFO).
The choice of 64Cu as the radioisotope was based on the fact that in vivo cell migration and homing is a rather slow process compared to metabolic changes usually imaged by PET. 64Cu with a half-life time of 12.7 h enables the repeated imaging of cell migration over 48 h post injection. For 64Cu production, it is important to use high-grade solutions without trace metal ions to ensure the purity of 64Cu. Furthermore, the purity of 64Cu is of high importance for radiolabeling of the DOTA-mAb since multivalent metal ion impurities could lead to a reduced specific activity of the 64Cu solution to levels that preclude protein radiolabeling and consequently imaging. Although 64Cu produces a significant percentage of highly energetic, cytotoxic Auger electrons when decaying28, an adjusted dose of the radioisotope is well tolerated by the cells as represented by minimal effects on viability and functionality and low apoptosis induction after radiolabeling. Nevertheless, it is clearly advisable to initially titrate the activity dose used for radiolabeling for other cell types and adjust the radiolabeling protocol. Fast and easy in vitro evaluation methods have been demonstrated in the protocol, including the determination of viability by trypan blue exclusion and staining with PE-Annexin V for the determination of the apoptotic fractions of the cells. Both methods are relatively cheap and use easily accessible laboratory equipment. Combining PE-Annexin V staining with a dye for the discrimination of living and dead cells such as 7-aminoactinomycin D (7-AAD) would allow for the determination of the viability and apoptosis in one experimental step. As an alternative to assess the viability of labeled T cells, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT-) assay can be used to assess the cell metabolic activity. The evaluation of the uptake and efflux values with a dose calibrator and &#947;-counter are more advanced but still fast and cheap to achieve since nearly every laboratory working with radioactivity has the respective hardware on site. Gene sequencing or microchip-based methods would yield more detailed data. These methods, however, are not always easily accessible, need appropriate expertise and are most often linked to higher costs.
Although PET is not able to reach a cellular resolution like microscopic imaging techniques29, it provides excellent sensitivity in the picomolar range for the detection of even small numbers of cells within animals or humans8. Compared to other non-invasive imaging techniques such as optical imaging, PET is quantitative, not limited in penetration depth and provides high-quality images with three dimensional resolutions. Combining the high sensitivity of PET with the anatomical accuracy of CT allows for high precision imaging of cell migration. Moreover, in vivo data can be easily validated by several ex vivo methods including &#947;-counting for biodistribution analysis. Autoradiography of cryosections and subsequent histological staining of the cryosections can be overlaid to correlate the autoradiographic activity map with the immune cell infiltrates identified by hematoxylin and eosin staining.

Non-invasive cell tracking is a versatile tool to monitor cell function, migration and homing in vivo. Recent cell tracking studies have focused on mesenchymal1,2 or bone-marrow derived stem cells3 in the context of regenerative medicine, autologous peripheral white blood cells in inflammation or T lymphocytes in adoptive cell therapies against cancer3,4. The elucidation of the sites of action and the underlying biological principles of cell-based therapies is of tremendous importance. CD8+ cytotoxic T lymphocytes, genetically engineered chimeric antigen receptor (CAR) T cells or tumor-infiltrating lymphocytes (TILs) were widely considered as the gold standard. However, tumor-associated antigen-specific TH1 cells have proven to be an effective alternative treatment option4,5,6,7.
As key players in inflammation, organ-specific autoimmune diseases (e.g., rheumatoid arthritis or bronchial asthma), and cells of high interest in cancer immunotherapy, it is important to characterize the temporal distribution and homing patterns of TH1 cells. Noninvasive in vivo imaging by PET presents a quantitative, highly sensitive method8 to examine cell migration patterns, in vivo homing, and the sites of T cell action and responses during inflammation, allergies, infections or tumor rejection9,10,11.
Clinically, 111In-oxine is used for leukocyte scintigraphy for the discrimination of inflammation and infection12, while 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is commonly used for cell tracking studies by PET3,13. One major disadvantage of this PET tracer, however, is the short half-life of the radionuclide 18F at 109.7 min and the low intracellular stability that impedes imaging at later time points post adoptive cell transfer. For longer term in vivo cell tracking studies by PET, although unstable in the cells, 64Cu-PTSM is frequently used to nonspecifically label cells14,15 with minimized detrimental effects on T cell viability and function16.
This protocol describes a method to further reduce disadvantageous effects on cell viability and function using a T cell receptor (TCR)-specific radiolabeled mAb. First, the production of the radioisotope 64Cu, the conjugation of the mAb KJ1-26 with the chelator DOTA, and the subsequent 64Cu-radiolabeling are shown. In a second step, the isolation and expansion of cOVA-TH1 cells of DO11.10 donor mice and the radiolabeling with 64Cu-loaded DOTA-conjugated mAb KJ1-26 (64Cu-DOTA-KJ1-26) are described in detail. The assessment of uptake values and efflux of radioactivity with a dose calibrator and by &#947;-counting, respectively, as well as the evaluation of the effects of 64Cu-radiolabeling on cell viability by trypan blue exclusion and functionality with IFN-&#947; ELISA are presented. For non-invasive in vivo cell tracking, the elicitation of a mouse model of cOVA-induced acute airway DTHR and image acquisition by PET/CT after adoptive cell transfer are described.
Moreover, this labeling approach can be transferred to different disease models, murine T cells with different TCRs or general cells of interest with membrane-bound receptors or expression markers underlying continuous membrane shuttling17.

<table><tbody><tr><td>HCl, Suprapur</td><td>Merck, Darmstadt, Germany</td><td>1.00318</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Methanol, Suprapur</td><td>Merck, Darmstadt, Germany</td><td>1.06007</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Isopropanol, Suprapur</td><td>Merck, Darmstadt, Germany</td><td>1.0104</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Pt/Ir (90/10) plate</td><td>&amp;Ouml;gussa</td><td>Custom made</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>PEEK chamber</td><td>WKL</td><td>Custom made</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>&lt;sup&gt;64&lt;/sup&gt;Ni</td><td>Chemotrade</td><td></td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Polygram SIL G/UV 254 plate</td><td>Macherey-Nagel</td><td>805021</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Ion exchange column</td><td>BioRad</td><td>AG1-X8</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Solid state target system for PETtrace</td><td>WKL</td><td>costum made</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>&lt;sup&gt;64&lt;/sup&gt;Cu work-up module</td><td>WKL</td><td>costum made</td><td>&lt;sup&gt;64&lt;/sup&gt;Cu production</td></tr><tr><td>Dose calibrator</td><td>Capintec</td><td>CRC-25R</td><td></td></tr><tr><td>PETtrace cyclotron</td><td>General Electric Medical Systems</td><td></td><td></td></tr><tr><td>DOTA-NHS</td><td>Macrocyclics</td><td>B-280</td><td>DOTA-conjugation</td></tr><tr><td>Anti-cOVA-TCR antibody (KJ1-26)</td><td></td><td>Isolated from hybridoma cell culture</td><td>DOTA-conjugation</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>71633</td><td>DOTA-conjugation</td></tr><tr><td>H&lt;sup&gt;+&lt;/sup&gt; Chelex 100</td><td>Sigma-Aldrich</td><td>C7901</td><td>DOTA-conjugation</td></tr><tr><td>Amicon Ultra-15 filter unit</td><td>Merck Millipore</td><td>UFC910008</td><td>DOTA-conjugation</td></tr><tr><td>Rotipuran ultrapure water</td><td>Carl Roth</td><td>HN68.3</td><td>DOTA-conjugation</td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>32301</td><td>DOTA-conjugation</td></tr><tr><td>PBS</td><td>University Tuebingen</td><td></td><td>DOTA-conjugation</td></tr><tr><td>Micro Bio-spin P-6 column</td><td>Bio-Rad Laboratories</td><td>7326221</td><td>DOTA-conjugation</td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>71497</td><td>DOTA-conjugation</td></tr><tr><td>Cyclone Plus PhosphorImager&amp;nbsp;</td><td>Perkin-Elmer</td><td>L2250116</td><td>DOTA-conjugation</td></tr><tr><td>DMEM</td><td>Merck Millipore</td><td>102568</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>FCS</td><td>Merck Millipore</td><td>S0115/1004B</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>Sodium pyruvate</td><td>Merck Millipore</td><td>L0473</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>MEM-amino acids</td><td>Merck Millipore</td><td>K0293</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>HEPES&amp;nbsp;</td><td>Merck Millipore</td><td>L 1613</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Penicillin/Streptomycin</td><td>Merck Millipore</td><td>A2212</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>0.05 mM 2-&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td>ingredient for T cell medium&amp;nbsp;</td></tr><tr><td>DO11.10 mice</td><td></td><td>in-house breeding</td><td>TH1 cell culture</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190144</td><td>TH1 cell culture</td></tr><tr><td>Cell strainer 40 &amp;micro;m&amp;nbsp;</td><td>Corning</td><td>352340</td><td>TH1 cell culture</td></tr><tr><td>ACK Lysing Buffer</td><td>Lonza</td><td>10-548E</td><td>TH1 cell culture</td></tr><tr><td>CD4 MicroBeads, mouse</td><td>Miltenyi Biotech</td><td>130-097-145</td><td>TH1 cell culture</td></tr><tr><td>QuadroMACS separator</td><td>Miltenyi Biotech</td><td>130-090-976</td><td>TH1 cell culture</td></tr><tr><td>LS column</td><td>Miltenyi Biotech</td><td>130-042-401</td><td>TH1 cell culture</td></tr><tr><td>anti-CD4 antibody (Gk1.5)</td><td></td><td>Isolated from hybridoma cell culture</td><td>TH1 cell culture</td></tr><tr><td>anti-CD8 antibody (5367.2)</td><td></td><td>Isolated from hybridoma cell culture</td><td>TH1 cell culture</td></tr><tr><td>Anti-rat antibody (MAR18.5)</td><td></td><td>Isolated from hybridoma cell culture</td><td>TH1 cell culture</td></tr><tr><td>Rabbit complement MA</td><td>tebu-Bio</td><td>CL3221</td><td>TH1 cell culture</td></tr><tr><td>Anti-IL-4 antibody (11B11)</td><td></td><td>Isolated from hybridoma cell culture</td><td>TH1 cell culture</td></tr><tr><td>cOVA 323-339-peptide&amp;nbsp;</td><td>EMC-micro-collections</td><td>Custom order</td><td>TH1 cell culture</td></tr><tr><td>CPG1668-oligonucleotides</td><td>Eurofins MWG Operon</td><td>Custom order</td><td>TH1 cell culture</td></tr><tr><td>IL-2</td><td>Novartis</td><td>65483-116-07</td><td>TH1 cell culture</td></tr><tr><td>96-well plates</td><td>Greiner&amp;nbsp;</td><td>655180</td><td>TH1 cell culture</td></tr><tr><td>24-well plates</td><td>Greiner&amp;nbsp;</td><td>662160</td><td>TH1 cell culture</td></tr><tr><td>cell culture flask</td><td>Greiner&amp;nbsp;</td><td>660175</td><td>TH1 cell culture</td></tr><tr><td>48-well plates</td><td>Greiner&amp;nbsp;</td><td>677 180</td><td>cell labeling</td></tr><tr><td>Gammacell 1000</td><td>Best Theratronics</td><td>&lt;em&gt;via&lt;/em&gt; inquiry&amp;nbsp;</td><td></td></tr><tr><td>Gulmay RT225</td><td>Gulmay</td><td>&lt;em&gt;via&lt;/em&gt; inquiry&amp;nbsp;</td><td></td></tr><tr><td>Trypan blue</td><td>Merck Millipore</td><td>L6323</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Mouse IFN-&amp;gamma; ELISA</td><td>BD Biosciences</td><td>558258</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>PE Annexin V Apoptosis Detection Kit&amp;nbsp;</td><td>BD Biosciences</td><td>559763</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Tube 5 mL</td><td>Sarstedt</td><td>55.476</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Round-bottom tubes&amp;nbsp;</td><td>BD Biosciences</td><td>352008</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Wizard &amp;gamma;-counter</td><td>Perkin-Elmer</td><td>2480-0010</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>ELISA Reader MultiscanEX</td><td>Thermo Fisher Scientific</td><td>51118177</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>Microscope</td><td>Leica</td><td>&lt;em&gt;via&lt;/em&gt; inquiry&amp;nbsp;</td><td>&lt;em&gt;in vitro&lt;/em&gt; evaluation</td></tr><tr><td>BD LSRII&amp;nbsp;</td><td>BD Biosciences</td><td>&lt;em&gt;via&lt;/em&gt; inquiry&amp;nbsp;</td><td>&lt;em&gt;in vitro&lt;/em&gt;evaluation</td></tr><tr><td>BALB/c mice</td><td>Charles River</td><td>028</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Aluminum gel</td><td>Serva Electrophoresis</td><td>12261.01</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Xylazine</td><td>Bayer HealthCare</td><td>Ordered &lt;em&gt;via&lt;/em&gt; University hospital</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Ketamine</td><td>Ratiopharm</td><td>Ordered &lt;em&gt;via&lt;/em&gt; University hospital</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Isoflurane</td><td>CP-Pharma</td><td>Ordered &lt;em&gt;via&lt;/em&gt; University hospital</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>30 G needle</td><td>BD Biosciences</td><td>304000</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Syringe</td><td>BD Biosciences</td><td>11612491</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Capillaries 10 &amp;micro;L</td><td>VWR</td><td>612-2439</td><td></td></tr><tr><td>Inveon PET scanner</td><td>Siemens Healthineers</td><td>no longer available</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking, alternative companies: Bruker, Mediso&amp;nbsp;</td></tr><tr><td>Inveon SPECT/CT scanner</td><td>Siemens Healthineers</td><td>no longer available</td><td>&lt;em&gt;in vivo&lt;/em&gt; cell trafficking</td></tr><tr><td>Inveon Research Workplace</td><td>Siemens Healthineers</td><td></td><td>image analysis, alternative software: Pmod</td></tr></tbody></table>

murine lymphocyte labeling by 64cu-antibody receptor targeting for in vivo cell trafficking by pet/ct

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Figure 1 summarizes the labeling of cOVA-TH1 cells with the 64Cu-DOTA-KJ1-26-mAb and the experimental design for the in vitro and in vivo studies covered in this protocol.
<img alt="Figure 1" src="/files/ftp_upload/55270/55270fig1.jpg" /> 
Figure 1: 64Cu-DOTA-KJ1-26-mAb Labeling Process &#38; Experimental Design. (A) Schematic representation of radioactive cell labeling with 64Cu-DOTA-KJ1-26-mAb. The 64Cu-DOTA-KJ1-26-mAb binds to cOVA-TCR receptors on the cell surface of the TH1 cells and is internalized with the receptor within 24 h. cOVA-TCRs are re-expressed 24 h after radiolabeling. (B) CD4+ T cells were isolated, expanded and radiolabeled with 64Cu-DOTA-KJ1-26-mAb in vitro. Uptake and efflux values were determined by a dose calibrator and &#947;-counting. Effects of the radioactive antibody on cell viability were assessed with trypan blue staining, on functionality with IFN-&#947; ELISA, and on induction of apoptosis with Phycoerythrin (PE)-Annexin V staining. (C) cOVA-airway DHTR was induced in female BALB/c mice. 107 TH1 cell were adoptively transferred into cOVA-airway DHTR-diseased animals directly after radiolabeling. PET/CT images were acquired 3, 24 and 48 h post adoptive cell transfer. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55270/55270fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The successful intracellular labeling of the TH1 cells with the 64Cu-DOTA-KJ1-26-mAb was confirmed by immunohistochemistry (Figure 2A). The 64Cu-DOTA-KJ1-26 antibody (green) was co-localized with CD3 at the cell membrane 3 h after radiolabeling, while the signal of the mAb at the cell membrane was only faint at 24 h (yellow). At 48 h after radiolabeling, the 64Cu-DOTA-KJ1-26-TCR complex was internalized via endocytosis with a strong signal in the cytoplasm of the TH1 cells. The applied radioactive dose of 0.7 MBq reduced viability of the TH1 cells by only 8% while higher doses of 1.4 MBq and 2.1 MBq 64Cu-DOTA-KJ1-26-mAb had more pronounced effects. Compared to radiolabeling with 64Cu-PTSM, however, no significant advantage of 64Cu-DOTA-KJ1-26-mAb was observed (Figure 2B). 64Cu-antibody radiolabeled cells showed little loss in functionality as shown by IFN-&#947; secretion (Figure 2C), reduced efflux of radioactivity (Figure 2D), and reduced induction of apoptosis (Figure 2E) compared to 64Cu-PTSM-labeled TH1 cells.
<img alt="Figure 2" src="/files/ftp_upload/55270/55270fig2.jpg" /> 
Figure 2: In Vitro Evaluation of Radioactive TH1 Cell Labeling with 64Cu-DOTA-KJ1-26-mAb. (A) Confocal microscopy proves the intracellular uptake of 64Cu-DOTA-KJ1-26-mAbs (green) in cOVA-TH1 cells (cell membrane; red) within 24 h after initial labeling (This figure has been modified from reference17). (B) Titration of radioactive labeling doses of 64Cu-DOTA-KJ1-26-mAb or 64Cu-PTSM revealed an equal influence on the viability as detected by trypan blue exclusion 24 h post labeling. The usage of 0.7 MBq for cell labeling induced the lowest impairments of the viability (mean &#177; SD in percent; this figure has been modified from references16,17. (C) Titration of radioactive labeling doses of 64Cu-DOTA-KJ1-26-mAb or 64Cu-PTSM revealed a stronger impairment of the functionality after 64Cu-PTSM labeling, as detected by IFN-&#947; ELISA 24 h post labeling. An increasing dose of 64Cu-DOTA-KJ1-26-mAb for cell labeling had no influences on the functionality (mean &#177; SD in percent; statistics: Student&#39;s t-test; * p &#60;0.05, ** p &#60;0.01, *** p &#60;0.001; this figure has been modified from references16,17). (D) Efflux measurements of radiolabeled cells (&#947;-counting) showed a higher labeling stability of 64Cu-DOTA-KJ1-26-mAb compared to 64Cu-PTSM 5 h and 24 h post labeling (mean &#177; SD in percent; statistics: Student&#39;s t-test; *** p&#60; 0.001; this figure has been modified from17). (E) Respective flow cytometry (PE-Annexin V) diagrams indicate a higher apoptosis induction 24 h after 64Cu-PTSM labeling compared to 64Cu-DOTA-KJ1-26-mAb-labeling (This figure has been modified from reference17). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55270/55270fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After adoptive transfer of 107 64Cu-DOTA-KJ1-26-mAb-labeled cOVA-TH1 cells into cOVA-DTHR-diseased animals and untreated controls, high resolution static PET and anatomical CT images were acquired. For image analysis, images were fused in the coronal, axial and sagittal view with the help of glass capillaries containing a small amount of 64Cu-DOTA-KJ1-26-mAb (Figure 3A). VOIs were drawn on the pulmonary and perithymic LNs on the decay-corrected and normalized PET images to calculate the percentage injected dose per cm3 (Figure 3B).
<img alt="Figure 3" src="/files/ftp_upload/55270/55270fig3.jpg" /> 
Figure 3: PET/CT-co-registration and Analysis. (A) PET and CT images were co-registered in image analysis software. If automated registration by the software failed, the images were fused by overlaying the PET and CT signals of glass capillaries (markers), filled with radioactivity and fixed under the animal bed. (B) VOIs are placed on the corrected and normalized PET signals of the perityhmic and pulmonary LNs. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55270/55270fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
cOVA-TH1 cell migration was tracked to the pulmonary and perithymic LN as cOVA-presentation sites in airway DTHR. In vivo PET signals derived from 64Cu-DOTA-KJ1-26-mAb-labeled cOVA-TH1 cells were considerably higher than signals from 64Cu-PTSM-labeled cells (Figure 4A). cOVA-TH1 cell uptake values in the pulmonary and perithymic LNs were significantly increased in DTHR-diseased animals compared to untreated control littermates (Figure 4B).
<img alt="Figure 4" src="/files/ftp_upload/55270/55270fig4.jpg" /> 
Figure 4: cOVA-TH1 Cell Tracking in the Airway DTHR Model by In Vivo PET/CT. (A) Respective PET/CT images of adoptively transferred cOVA-TH1 cells, which were previously labeled with 64Cu-DOTA-KJ1-26-mAb or 64Cu-PTSM, into cOVA-DTHR-diseased or untreated mice. The transferred cOVA-TH1 cells homed to the pulmonary and perithymic LNs. (B) Quantitative analysis of the homing sites of the cOVA-TH1 cells proved a higher homing to inflamed tissues (mean &#177; SD; statistics: Student&#39;s t-test; * p &#60;0.05, ** p &#60;0.01). The labeling with 64Cu-DOTA-KJ1-26-mAb proved a higher sensitivity (increased %ID/cm3) compared to 64Cu-PTSM in the different homing sites (mean &#177; SD, statistics: Student&#39;s t-test; # p &#60;0.05, ## p &#60;0.01, ### p &#60;0.001). This figure has been modified from reference16,17. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55270/55270fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT at JoVE.com

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

murine-lymphocyte-labeling-64cu-antibody-receptor-targeting-for-vivo

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.8K Views.  Eberhard Karls University Tübingen.  Na de bereiding van een 64 Cu-gemodificeerd monoklonaal antilichaam dat aan een transgene muizen T-celreceptor, T-cellen radiogelabeld in vivo geanalyseerd op levensvatbaarheid functionaliteit labeling stabiliteit en apoptose en adoptief overgebracht in muizen met een luchtweg vertraagde hypersensitiviteit reactie voor niet-invasieve beeldvorming met positron emission tomography / computed tomography (PET / CT). 

Video: Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

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

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 ...

Cancer Research

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Bioengineering

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological deficits and disability as the disease progresses. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. Currently, there is no way to accurately select patients for specific anti-B cell therapies or to non-invasively quantify the effects of these treatments on B cell load in the CNS and peripheral organs. Positron emission tomography (PET) imaging has enormous potential to provide highly specific, quantitative information regarding the in vivo spatiotemporal distribution and burden of B cells in living subjects.
This paper reports methods to synthesize and employ a PET tracer specific for human CD19+ B cells in a well-established B cell-driven mouse model of MS, experimental autoimmune encephalomyelitis (EAE), which is induced with human recombinant myelin oligodendrocyte glycoprotein 1-125. Described here are optimized techniques to detect and quantify CD19+ B cells in the brain and spinal cord using in vivo PET imaging. Additionally, this paper reports streamlined methods for ex vivo gamma counting of disease-relevant organs, including bone marrow, spinal cord, and spleen, together with high-resolution autoradiography of CD19 tracer binding in CNS tissues.

Imaging CD19+ B Cells in an Experimental Autoimmune Encephalomyelitis Mouse Model using Positron Emission Tomography

This paper details methodology for radiolabeling a human-specific anti-CD19 monoclonal antibody and how to use it to quantify B cells in the central nervous system and peripheral tissues of a mouse model of multiple sclerosis using in vivo PET imaging, ex vivo gamma counting, and autoradiography approaches.

Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in neurological ...

Neuroscience

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Biology

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.remotexs.ntu.edu.sg/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 ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Labeling Murine T Helper Lymphocytes using Radiolabeled Monoclonal Antibodies

Source: Hoffmann, S. H. L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55270">Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the intracellular radiolabeling of T helper cells using radioactive isotope-conjugated monoclonal antibodies for non-invasive positron emission tomography imaging.

Source: Hoffmann, S. H. L., et al., Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT. J. Vis. Exp. ...

Immune Systems and Components

Assays and Functional Profiling

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

Dialyze 0.5 milliliters of the antibody using a dialysis cassette in 1 liter of conjugation buffer. After four hours, transfer the dialysis cassette to fresh buffer, and dialyze overnight. To set up a conjugation reaction, add 0.03 micrograms of IRDye 800CW per 1 milligram of antibody in a total volume of 500 microliters.
Incubate the mixture for two hours at 20 degrees Celsius. Then, purify the labeled conjugates by extensive dialysis against PBS. Estimate the degree of labeling by measuring the absorbance of the dye at 780 nanometers, and absorbance of the protein at 280 nanometers.
To develop mouse xenografts, gently, lift the skin of the animal and separate it from the underlying muscle layer. Slowly inject 100 microliters of cell suspension under the skin with a 26-gauge needle. Wait for a few seconds before taking the needle out so that basement matrix medium can form a semi-solid gel-like structure along with cells under the skin, which will prevent the mixture from coming out of the injection site.
To perform in vivo imaging, inject the dye-labeled antibody via the tail vein. After anesthetizing the mouse, check for the lack of response to pedal reflexes, and dilate the vein by applying warm water. Use a 1 CC insulin syringe with a 26-gauge needle to inject 25 micrograms of the labeled antibody in a volume of 100 microliters. As a negative control, label and inject the non-specific IgG isotype antibody which does not target cancer cells.
Perform in vivo imaging 8, 24, and 48 hours after antibody injections. In the imaging software, click Initialize. Confirm that the stage temperature is 37 degrees Celsius. In the control panel, set up fluorescence imaging through the imaging wizard, and set the excitation to 773 nanometers, and emission to 792 nanometers.
Transfer the anesthetized mouse into the imaging chamber and position it on the imaging field using the nose cone. When ready, click Acquire on the control panel for the image acquisition, and click Auto Expose. The generated image is the overlay of the fluorescence on the photographic image, with optical fluorescence intensity displayed in units of counts or photons.

Murine Lymphocyte Labeling by ⁶⁴Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

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