None

All&#160;in vivo&#160;experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animal resources of Nagoya University Animal Care and Use Committee (approval #2017-29438, #2018-30096, #2019-31234, #2020-20104). Six-week-old homozygote athymic nude mice were purchased and maintained at the Animal Center of Nagoya University. When performing the procedure in mice, they were anesthetized with isoflurane (introduction: 4-5%, maintenance 2-3%); the paw was pressed with tweezers to confirm the depth of anesthesia.
1.&#160;Conjugation of IR700 with mAb
<ol>
	<li>Incubate mAb (1 mg, 6.8 nmol) with IR700 NHS ester (66.8 mg, 34.2 nmol, 5 mmol/L in DMSO) in 0.1 mol/L Na2HPO4&#160;(pH 8.6) at 15-25 &#176;C for 1 h.</li>
	<li>Purify the mixture using a column (e.g., Sephadex). Prepare and wash the column with PBS. Then, apply the mixture onto the column and collect the drop, which contains the purified IR700-conjugated antibody. This IR700-conjugated antibody is referred to as the antibody photosensitizer conjugate (APC).</li>
	<li>Measure the protein and IR700 concentration in the APC.
	<ol>
		<li>Prepare calibration curves for protein and IR700 using a spectrophotometer.</li>
		<li>Mix standard concentrations of albumin with a protein assay kit following the kit protocol (see&#160;Table of Materials, Coomassie Brilliant Blue (CBB) protein staining). Measure the absorbance of albumin at 595 nm wavelength, and plot the calibration curve (a linear approximation formula) for the protein using the following equation: y = ax + b (x: concentration, y: absorbance).</li>
		<li>Obtain calibration curves for IR700 with absorption at 690 nm using the same procedure. The standard concentration of IR700 is recommended at 0.1-5 &#181;M (0.1954-9.77 &#181;g/mL).</li>
		<li>Measure the protein concentration and IR700 concentration in the APC using a calibration curve [x = (y-b)/a (x: concentration, y: absorbance)].</li>
		<li>Determine the number of IR700 dyes bound per mAb with the results of the molar concentration. 
		NOTE: It is important to determine the optimal conjugation number of IR700 molecules per mAb molecule. Generally, approximately three IR700 molecules bound on a single mAb molecule would be effective both&#160;in vitro&#160;and&#160;in vivo. Many IR700 bound per antibody (e.g., six) makes it easier to be trapped in the liver during&#160;in vivo&#160;experiments. The ratio of antibody bound to IR700 was in the range of 1:2-1:4. The proportion of IR700 was reduced, if necessary.</li>
	</ol>
	</li>
	<li>Perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a confirmation for the formation of an APC. Image the gel at 700 nm using a fluorescent imager, and stain the protein in the gel using a protein staining kit following the kit protocol (see&#160;Table of Materials, CBB protein staining).</li>
</ol>
2.&#160;Generation of a pleural dissemination model
<ol>
	<li>Prepare luciferase-expressing target cells and suspend 1.0 &#215; 106&#160;target cells in 100 &#181;L of phosphate-buffered saline (PBS) 
	NOTE: Intrathoracic cancer cells such as lung cancer and MPM are suitable as target cells. Luciferase-expressing cells were prepared via luciferase gene transfection, and high expression of luciferase was confirmed after &#62; 10 cell passages. Cells were cultured in a medium supplemented with 10% fetal bovine serum and penicillin (100 IU/mL) and streptomycin (100 mg/mL). The number of cells was adjusted according to the tumor growth rate and the time course of treatment (1.0. &#215; 105-6.0 &#215; 106&#160;cells/body weight).</li>
	<li>Prepare 8-12-week-old female homozygote athymic nude mice, with a preferable body weight of 19-21 g.</li>
	<li>Anesthetize mice during the procedure with isoflurane (introduction: 4-5%, maintenance 2-3%); press the tail with tweezers to confirm that there is no reaction.</li>
	<li>Make a stopper with polystyrene foam and attach the stopper to the 30 G needle so that the tip remains at 5 mm to prevent lung injury. Bend the needle&#160;tip with clean forceps or by pressing it against a hard object cleaned with 70% EtOH&#160;to avoid pneumothorax (Figure 1). 
	CAUTION: Be careful not to pierce oneself. Use forceps to bend the needle. Do not hold the stopper when attaching it to the needle. It is safer to stick the stopper before filling the cells into the syringe.</li>
	<li>Fill a syringe (1 mL) with target cells, and attach a 30G needle with a stopper.</li>
	<li>Disinfect the chest of the animal with 70% EtOH before the procedure.</li>
	<li>Pierce a needle into the chest of the mouse through the intercostal space. Owing to the resistance while hitting against the ribs at that time, the needle tip moved up and down. After passing through the intercostal space, press the syringe against the mouse and inject 100 &#181;L of target cells (Figure 2). 
	NOTE: The mouse breaths deeply when the needle properly enters the chest cavity. With the bending of the needle tip, pneumothorax and inappropriate injection of cells into the lung could be avoided. 
	NOTE: Right chest wall puncture is recommended to avoid the risk of cardiac wall puncture.</li>
	<li>Roll the mouse 2-3 times to spread the cells throughout the thoracic cavity.</li>
	<li>Return the mouse to a clean and warm&#160;cage and monitor until ambulatory. After the procedure, the mouse will wake up from anesthesia and behave normally.</li>
</ol>
3.&#160;Measurement of bioluminescence
NOTE: The software used for data acquisition is listed in the&#160;Table of Materials.
<ol>
	<li>To confirm the generation of the pleural dissemination model, evaluate the bioluminescence images every day after injecting the cells into the thoracic cavity.</li>
	<li>Anesthetize mice (step 2.3) and inject intraperitoneally with D-luciferin (15 mg/mL, 200 &#181;L).</li>
	<li>Ensure that the mouse is breathing normally before and after imaging. Use a heater if necessary to prevent hypothermia during anesthesia.&#160;</li>
	<li>Ten minutes after the injection, set the mouse in the bioluminescence imaging (BLI) measuring equipment. For image acquisition, open the&#160;Acquisition&#160;Control Panel&#160;of the software. Select&#160;Luminescent,&#160;Photograph, and&#160;Overlay&#160;(Figure 3).</li>
	<li>Set exposure time as&#160;Auto. Set Binning as small.</li>
	<li>Set f/stop as&#160;1&#160;for luminescent and&#160;8&#160;for photograph; f/stop controls the amount of light received by the charged-coupled device detector.</li>
	<li>Set the Field of View as&#160;C.</li>
	<li>Once the mouse sample is ready for imaging, click&#160;Acquire&#160;for imaging acquisition. Mice with sufficient luciferase activity were selected for further studies. 
	NOTE: A suitable pleural dissemination model shows strong luminescence on the diffused site in the chest when viewed from the ventral side. If the BLI images are not diffused in the thorax, and only at the injection site, the tumor may be transplanted subcutaneously.</li>
	<li>After displaying the image, set the display format to&#160;Radiance. Open the&#160;Tool&#160;Palette&#160;panel (Figure 4A).</li>
	<li>Select&#160;ROI Tools. We recommend using the&#160;Circle&#160;to range the bioluminescent area on images.</li>
	<li>Click&#160;Measure ROIs&#160;to measure the surface bioluminescent intensity (Figure 4B).</li>
	<li>Use&#160;Configure Measurement&#160;on the left corner of the ROI measurement panel to select the values/information needed. Export this data table as a .csv file (Figure 4C).</li>
	<li>Use the values of&#160;Total Flux (p/s)&#160;as the bioluminescent intensity quantification in the .csv file.</li>
</ol>
4.&#160;Diffuse luminescence imaging tomography (DLIT)
NOTE: The software used for data acquisition is listed in the&#160;Table of Materials.
<ol>
	<li>Turn on the&#160;X-ray Armed&#160;button.</li>
	<li>Anesthetize the mice (step 2.3) and then inject D-luciferin (15 mg/mL, 200 &#181;L) intraperitoneally into the mice. To shoot DLIT continuously from 3.2 to 3.7, skip this step.</li>
	<li>Ten minutes after injection, set the mouse in the BLI equipment.</li>
	<li>Open the&#160;Acquisition Control Panel&#160;of the software. Select&#160;Luminescent,&#160;Photograph,&#160;CT,&#160;Standard-One Mouse, and&#160;Overlay. Other settings were the same as in 3.4-3.6 (Figure 5A).</li>
	<li>Select the&#160;Imaging Wizard&#160;on the&#160;Acquisition Control Panel.</li>
	<li>Select&#160;Bioluminescence&#160;and then&#160;DLIT&#160;(Figure 5B).</li>
	<li>Select&#160;Firefly&#160;as the wavelength to measure (Figure 5C).</li>
	<li>Set the Imaging Subject as&#160;Mouse, Exposure parameters as&#160;Auto Settings, Field of View as&#160;C-13.4 cm, and Subject Height as&#160;1.5 cm&#160;(Figure 5D).</li>
	<li>Push the&#160;X-rays will be produced when energized. Acquire.</li>
	<li>Open the CT sequential image data.</li>
	<li>Open&#160;Surface Topography&#160;on the Tool Palette. Select&#160;Show&#160;(Figure 6A).</li>
	<li>Adjust the threshold as the purple display shows only the body surface (Figure 6B). Then, select the Subject&#160;Nude Mouse&#160;and click the&#160;Generate Surface. Make sure that the outline of the mouse is accurately drawn (Figure 6C).</li>
	<li>Open the&#160;Tool Palette,&#160;DLIT 3D Reconstruction&#160;Properties&#160;tab. Select Tissue Properties as&#160;Mouse Tissue&#160;and Source Spectrum as&#160;Firefly&#160;(Figure 6D). Next, open the&#160;Analyze&#160;tab and confirm the data for each selected wavelength data. Finally, click the&#160;Reconstruct&#160;button (Figure 6E).</li>
	<li>Confirm the presence of BLI in the chest cavity in the configured DLIT image.</li>
</ol>
5.&#160;NIR-PIT for&#160;in vivo&#160;pleural dissemination model
<ol>
	<li>Measure the light dose of 690 nm wavelength (NIR) laser with a power meter, and adjust the output to 100 mW/cm2. 
	NOTE: The laser light is coherent with a precise coil size; thus, the light energy hardly changes regardless of the distance within 50 cm. If there are many adverse events, such as burns, reduce the output within the range of 40 mW/cm2.</li>
	<li>Clean the tail with 70% EtOH and intravenously inject APC (100 &#181;g)&#160;via&#160;the tail vein 24 h before NIR irradiation. Apply pressure to the injection site to control bleeding. 
	NOTE: Adjust the volume of APC to 50-200 &#181;L for injection.</li>
	<li>Anesthetize the mice (step 2.3), and lay them on their back. To avoid NIR irradiation to the non-target site, shield other sites with aluminum foil (Figure 7A). Irradiate with NIR light with a laser of 100 J/cm2; if the tumor is disseminated back to the belly, the NIR-light irradiation dose could be divided in multiple directions (Figure 7B). 
	NOTE: Adjust the dose at 30-150 J/cm2&#160;depending on the&#160;in vitro&#160;results and adverse events such as burns.</li>
	<li>When the NIR irradiation is complete and the mouse is awake, return it to the cage.</li>
	<li>Observe the BLI and measure the ROI over time (every day) (See 3.2-3.12).</li>
	<li>For&#160;ex vivo&#160;imaging, euthanize mice with carbon dioxide 24 h after the APC injection, immediately before the NIR irradiation.
	<ol>
		<li>To observe the inside of the chest of the mouse, remove the thorax and cut the ribs and sternum. Capture the fluorescence image (700 nm) alongside the control without APC administration. Then, apply D-luciferin (150 &#181;g/mL) over the exposed thorax, and the BLI was taken (refer 3.2-3.7).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Xu, X., Lu, H., Lee, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+Infrared+Light+Triggered+Photo/Immuno-Therapy+Toward+Cancers.">Near Infrared Light Triggered Photo/Immuno-Therapy Toward Cancers.</a> Frontiers in Bioengineering and Biotechnology. 8, (2020).</li><li>Mitsunaga, 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=Cancer+cell-selective+in+vivo+near+infrared+photoimmunotherapy+targeting+specific+membrane+molecules.">Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules.</a> Nature Medicine. 17, 1685-1691 (2011).</li><li>Kobayashi, H., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-Infrared+Photoimmunotherapy+of+Cancer.">Near-Infrared Photoimmunotherapy of Cancer.</a> Accounts of Chemical Research. 52, 2332-2339 (2019).</li><li>Oseroff, A. R., Ohuoha, D., Hasan, T., Bommer, J. C., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-targeted+photolysis:+Selective+photodestruction+of+human+T-cell+leukemia+cells+using+monoclonal+antibody-chlorin+e6+conjugates.">Antibody-targeted photolysis: Selective photodestruction of human T-cell leukemia cells using monoclonal antibody-chlorin e6 conjugates.</a> Proceedings of the National Academy of Sciences of the United States of America. 83, 8744-8748 (1986).</li><li>Mew, D., Wat, C. K., Towers, G. H., Levy, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoimmunotherapy:+treatment+of+animal+tumors+with+tumor-specific+monoclonal+antibody-hematoporphyrin+conjugates.">Photoimmunotherapy: treatment of animal tumors with tumor-specific monoclonal antibody-hematoporphyrin conjugates.</a> Journal of Immunology. 130, 1473-1477 (1983).</li><li>Vrouenraets, M. B., 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=Development+of+meta-tetrahydroxyphenylchlorin-monoclonal+antibody+conjugates+for+photoimmunotherapy.">Development of meta-tetrahydroxyphenylchlorin-monoclonal antibody conjugates for photoimmunotherapy.</a> Cancer Research. 59, 1505-1513 (1999).</li><li>Goff, B. 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=Photoimmunotherapy+and+biodistribution+with+an+OC125-chlorin+immunoconjugate+in+an+in+vivo+murine+ovarian+cancer+model.">Photoimmunotherapy and biodistribution with an OC125-chlorin immunoconjugate in an in vivo murine ovarian cancer model.</a> British Journal of Cancer. 70, 474-480 (1994).</li><li>Sato, 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=Photoinduced+Ligand+Release+from+a+Silicon+Phthalocyanine+Dye+Conjugated+with+Monoclonal+Antibodies:+A+Mechanism+of+Cancer+Cell+Cytotoxicity+after+Near-Infrared+Photoimmunotherapy.">Photoinduced Ligand Release from a Silicon Phthalocyanine Dye Conjugated with Monoclonal Antibodies: A Mechanism of Cancer Cell Cytotoxicity after Near-Infrared Photoimmunotherapy.</a> ACS Central Science. 4, 1559-1569 (2018).</li><li>Sato, K., Nagaya, T., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+infrared+photoimmunotherapy+in+the+treatment+of+pleural+disseminated+NSCLC:+Preclinical+experience.">Near infrared photoimmunotherapy in the treatment of pleural disseminated NSCLC: Preclinical experience.</a> Theranostics. 5, 698-709 (2015).</li><li>Nishinaga, 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=Targeted+Phototherapy+for+Malignant+Pleural+Mesothelioma:+Near-Infrared+Photoimmunotherapy+Targeting+Podoplanin.">Targeted Phototherapy for Malignant Pleural Mesothelioma: Near-Infrared Photoimmunotherapy Targeting Podoplanin.</a> Cells. 9, 1019 (2020).</li><li>Sato, 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=Near+infrared+photoimmunotherapy+prevents+lung+cancer+metastases+in+a+murine+model.">Near infrared photoimmunotherapy prevents lung cancer metastases in a murine model.</a> Oncotarget. 6, 19747-19758 (2015).</li><li>Sato, K., Nagaya, T., Mitsunaga, M., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+infrared+photoimmunotherapy+for+lung+metastases.">Near infrared photoimmunotherapy for lung metastases.</a> Cancer Letters. 365, 112-121 (2015).</li><li>Isobe, 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=Near+infrared+photoimmunotherapy+targeting+DLL3+for+small+cell+lung+cancer.">Near infrared photoimmunotherapy targeting DLL3 for small cell lung cancer.</a> EBioMedicine. 52, 102632 (2020).</li><li>Nakamura, 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=Near+infrared+photoimmunotherapy+in+a+transgenic+mouse+model+of+spontaneous+epidermal+growth+factor+receptor+(EGFR)-expressing+lung+cancer.">Near infrared photoimmunotherapy in a transgenic mouse model of spontaneous epidermal growth factor receptor (EGFR)-expressing lung cancer.</a> Molecular Cancer Therapeutics. 16, 408-414 (2017).</li><li>Nagaya, 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=Near+infrared+photoimmunotherapy+with+avelumab,+an+anti-programmed+death-ligand+1+(PD-L1)+antibody.">Near infrared photoimmunotherapy with avelumab, an anti-programmed death-ligand 1 (PD-L1) antibody.</a> Oncotarget. 8, 8807-8817 (2017).</li><li>Sato, 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=Spatially+selective+depletion+of+tumor-associated+regulatory+T+cells+with+near-infrared+photoimmunotherapy.">Spatially selective depletion of tumor-associated regulatory T cells with near-infrared photoimmunotherapy.</a> Science Translational Medicine. 8, (2016).</li><li>Sato, 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=Comparative+effectiveness+of+light+emitting+diodes+(LEDs)+and+lasers+in+near+infrared+photoimmunotherapy.">Comparative effectiveness of light emitting diodes (LEDs) and lasers in near infrared photoimmunotherapy.</a> Oncotarget. 7, 14324-14335 (2016).</li><li>Sato, K., Choyke, P. L., Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoimmunotherapy+of+Gastric+Cancer+Peritoneal+Carcinomatosis+in+a+Mouse+Model.">Photoimmunotherapy of Gastric Cancer Peritoneal Carcinomatosis in a Mouse Model.</a> PLoS One. 9, 113276 (2014).</li><li>McLemore, T. L., 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+intrapulmonary,+percutaneous+intrathoracic,+and+subcutaneous+models+for+the+propagation+of+human+pulmonary+and+nonpulmonary+cancer+cell+lines+in+athymic+nude+mice.">Comparison of intrapulmonary, percutaneous intrathoracic, and subcutaneous models for the propagation of human pulmonary and nonpulmonary cancer cell lines in athymic nude mice.</a> Cancer Research. 48, 2880-2886 (1988).</li><li>Manzotti, C., Audisio, R. A., Pratesi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+orthotopic+implantation+for+human+tumors+as+model+systems:+relevance+to+metastasis+and+invasion.">Importance of orthotopic implantation for human tumors as model systems: relevance to metastasis and invasion.</a> Clinical &amp; Experimental Metastasis. 11, 5-14 (1993).</li><li>Lwin, T. M., Hoffman, R. M., Bouvet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+patient-derived+orthotopic+mouse+models+and+genetic+reporters+for+developing+fluorescence-guided+surgery.">Advantages of patient-derived orthotopic mouse models and genetic reporters for developing fluorescence-guided surgery.</a> Journal of Surgical Oncology. 118, 253-264 (2018).</li><li>Sordat, B. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> From Ectopic to Orthotopic Tumor Grafting Sites: Evidence for a Critical Role of the Host Tissue Microenvironment for the Actual Expression of the Malignant Phenotype. , 43-53 (2017).</li><li>Sato, 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=Photoimmunotherapy:+comparative+effectiveness+of+two+monoclonal+antibodies+targeting+the+epidermal+growth+factor+receptor.">Photoimmunotherapy: comparative effectiveness of two monoclonal antibodies targeting the epidermal growth factor receptor.</a> Molecular Oncology. 8, 620-632 (2014).</li><li>Nakajima, 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=The+effects+of+conjugate+and+light+dose+on+photo-immunotherapy+induced+cytotoxicity.">The effects of conjugate and light dose on photo-immunotherapy induced cytotoxicity.</a> BMC Cancer. 14, 389 (2014).</li><li>Nagaya, 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=Near+infrared+photoimmunotherapy+of+B-cell+lymphoma.">Near infrared photoimmunotherapy of B-cell lymphoma.</a> Molecular Oncology. 10, 1404-1414 (2016).</li><li>Sato, 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=Near+infrared+photoimmunotherapy+in+the+treatment+of+disseminated+peritoneal+ovarian+cancer.">Near infrared photoimmunotherapy in the treatment of disseminated peritoneal ovarian cancer.</a> Molecular Cancer Therapeutics. 14, 141-150 (2015).</li><li>Colin, D. J., Bejuy, O., Germain, S., Triponez, F., Serre-Beinier, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+and+monitoring+by+pet/ct+of+an+orthotopic+model+of+human+pleural+mesothelioma+in+athymic+mice.">Implantation and monitoring by pet/ct of an orthotopic model of human pleural mesothelioma in athymic mice.</a> Journal of Visualized Experiments. 2019, (2019).</li><li>Opitz, I., 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=Local+recurrence+model+of+malignant+pleural+mesothelioma+for+investigation+of+intrapleural+treatment.">Local recurrence model of malignant pleural mesothelioma for investigation of intrapleural treatment.</a> European Journal of Cardio-Thoracic Surgery. 31, 772-778 (2007).</li><li>Bunn, P. A., Kelly, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+chemotherapeutic+agents+prolong+survival+and+improve+quality+of+life+in+non-small+cell+lung+cancer:+a+review+of+the+literature+and+future+directions.">New chemotherapeutic agents prolong survival and improve quality of life in non-small cell lung cancer: a review of the literature and future directions.</a> Clinical Cancer Research. 4, 1087-1100 (1998).</li><li>Astoul, P., Wang, X., Hoffman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-like+nude-mouse+and+scid-mouse+models+of+human+lung+and+pleural+cancer+(review).">Patient-like nude-mouse and scid-mouse models of human lung and pleural cancer (review).</a> International Journal of Oncology. 3, 713-718 (1993).</li><li>Yamaguchi, H., Pantarat, N., Suzuki, T., Evdokiou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+photoimmunotherapy+using+a+small+protein+mimetic+for+HER2-overexpressing+breast+cancer.">Near-infrared photoimmunotherapy using a small protein mimetic for HER2-overexpressing breast cancer.</a> International Journal of Molecular Sciences. 20, (2019).</li><li>Jing, 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=Imaging+and+selective+elimination+of+glioblastoma+stem+cells+with+theranostic+Near-Infrared-Labeled+CD133-Specific+antibodies.">Imaging and selective elimination of glioblastoma stem cells with theranostic Near-Infrared-Labeled CD133-Specific antibodies.</a> Theranostics. 6, 862-874 (2016).</li><li>Burley, T. 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=Near-infrared+photoimmunotherapy+targeting+EGFR-Shedding+new+light+on+glioblastoma+treatment.">Near-infrared photoimmunotherapy targeting EGFR-Shedding new light on glioblastoma treatment.</a> International Journal of Cancer. 142, 2363-2374 (2018).</li><li>Nagaya, 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=Near+infrared+photoimmunotherapy+using+a+fiber+optic+diffuser+for+treating+peritoneal+gastric+cancer+dissemination.">Near infrared photoimmunotherapy using a fiber optic diffuser for treating peritoneal gastric cancer dissemination.</a> Gastric Cancer. 22, 463-472 (2019).</li><li>Nagaya, 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=Endoscopic+near+infrared+photoimmunotherapy+using+a+fiber+optic+diffuser+for+peritoneal+dissemination+of+gastric+cancer.">Endoscopic near infrared photoimmunotherapy using a fiber optic diffuser for peritoneal dissemination of gastric cancer.</a> Cancer Science. 109, 1902-1908 (2018).</li><li>Harada, 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=Near-infrared+photoimmunotherapy+with+galactosyl+serum+albumin+in+a+model+of+diffuse+peritoneal+disseminated+ovarian+cancer.">Near-infrared photoimmunotherapy with galactosyl serum albumin in a model of diffuse peritoneal disseminated ovarian cancer.</a> Oncotarget. 7, 79408-79416 (2016).</li><li>Journals, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JNCI+Journal+of+the+National+Cancer+Institute+Way+to+Better+DNA.">JNCI Journal of the National Cancer Institute Way to Better DNA.</a> Annals of Internal Medicine. 37, 1-9 (2008).</li></ol>

The authors declare that they have no competing financial interests.

In this study, we demonstrated a method for measuring the therapeutic effect of NIR-PIT on the pleural dissemination model of MPM. Highly selective cell killing was performed with NIR-PIT; thus, the normal tissue was hardly damaged23,24,25. With this type of selective cell killing, NIR-PIT was demonstrated to be safe in disseminated models9,26<...

Cancer remains one of the leading causes of mortality despite decades of research. One reason is that radiation therapy and chemotherapy are highly invasive techniques, which may limit their therapeutic benefits. Cellular- or molecular-targeted therapies, which are less invasive techniques, are receiving increased attention. Photoimmunotherapy is a treatment method that synergistically enhances the therapeutic effect by combining immunotherapy and phototherapy. Immunotherapy enhances tumor immunity by increasing the immunogenicity of the tumor microenvironment and reducing immunoregulatory suppression, resulting in the destruction of tumors in the body. Phototherapy destroys primary tumors with a combination of photosensitizers and light rays, and tumor-specific antigens released from the tumor cells enhance tumor immunity. Tumors can be selectively treated using photosensitizers as they are specific and selective for the target cells. The modality of phototherapy includes photodynamic therapy (PDT), photothermal therapy (PTT), and photochemistry-based therapies1.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed method of antitumor phototherapy that combines photochemical-based therapy and immunotherapy1,2. NIR-PIT is a molecularly targeted therapy that targets specific cell surface molecules through the conjugation of a near-infrared silicon phthalocyanine dye, IRdye 700DX (IR700), to a monoclonal antibody (mAb). The cell membrane of the target cell is destroyed upon irradiation with NIR light (690 nm)3.
The concept of using targeted light therapy by combining conventional photosensitizers and antibodies or targeted PDT is over three decades old4,5. Previous studies have attempted to target conventional PDT agents by conjugating them to antibodies. However, there was limited success because these conjugates were trapped in the liver, owing to the hydrophobicity of the photosensitizers6,7. Moreover, the mechanism of NIR-PIT is completely different from that of conventional PDT. Conventional photosensitizers generate oxidative stress that results from an energy conversion that absorbs light energy, dislocates to an excited state, transitions to the ground state, and causes apoptosis. However, NIR-PIT causes rapid necrosis by directly destroying the cell membrane by aggregating photosensitizers on the membrane through a photochemical reaction8. NIR-PIT is superior to conventional targeted PDT in many ways. Conventional photosensitizers have low extinction coefficients, requiring the attachment of large numbers of photosensitizers to a single antibody molecule, potentially reducing binding affinity. Most conventional photosensitizers are hydrophobic, making it difficult to bind the photosensitizers to antibodies without compromising their immunoreactivity or&#160;in vivo&#160;target accumulation. Conventional photosensitizers typically absorb light in the visible range, reducing tissue penetration.
Several studies on NIR-PIT targeting intrathoracic tumors such as lung cancer and malignant pleural mesothelioma (MPM) cells have been reported9,10,11,12,13,14,15,16,17. However, only a few reports have described the efficacy of NIR-PIT in pleural disseminated MPM or lung cancer models9,10,11,12. Subcutaneous tumor xenograft models are thought to be standard tumor models and are currently widely used to evaluate the antitumor effects of new therapies18. However, the subcutaneous tumor microenvironment is not permissive for the development of an appropriate tissue structure or a condition that properly recapitulates a true malignant phenotype19,20,21,22. Ideally, orthotopic disease models should be established for a more precise evaluation of the antitumor effects.
Here, we demonstrate a method of efficacy evaluation in a mouse model of pleural disseminated lung cancer, which was treated using NIR-PIT. A pleural dissemination mouse model is generated by injecting tumor cells into the thoracic cavity and confirmed using luciferase luminescence. The mouse was treated with an intravenous injection of mAb conjugated with IR700 and NIR irradiation to the chest. The therapeutic effect was evaluated using luciferase luminescence.

<table><tbody><tr><td>0.25w/v% Trypsin-1mmol/l EDTA 4Na Solution with Phenol Red</td><td>Wako</td><td>209-016941</td><td>for cell culture</td></tr><tr><td>1mL syringe</td><td>TERUMO</td><td>SS-01T</td><td>for mice experiment</td></tr><tr><td>30G needle</td><td>Nipro</td><td>1907613</td><td>for mice experiment</td></tr><tr><td>BALB/cSlc-nu/nu</td><td>Japan SLC</td><td></td><td></td></tr><tr><td>Collidal Blue Staining Kit</td><td>Invitrogen</td><td>LC6025</td><td>use for gel protein staining</td></tr><tr><td>Coomassie (bradford) Plus protein assay</td><td>Thermo Fisher Scientific Inc (Waltham, MA, USA)</td><td>PI-23200</td><td>for measuring the APC concentration</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Wako</td><td>043-07216</td><td>use for conjugation of IR700</td></tr><tr><td>D-Luciferin (potassium salt)</td><td>Cayman Chemical</td><td>14681</td><td>for bioluminescence imaging and DLIT</td></tr><tr><td>GraphPad Prism7</td><td>GraphPad software</td><td></td><td>for statistical analysis</td></tr><tr><td>Image Studio</td><td>Li-Cor Biosciences</td><td></td><td>for analyzing 700 nm fluorescent image</td></tr><tr><td>IRDye 700DX Ester Infrared Dye</td><td>LI-COR Bioscience (Lincoln, NE, USA)</td><td>929-70011</td><td></td></tr><tr><td>isoflurane</td><td>Wako</td><td>095-06573</td><td>for mice anesthesia</td></tr><tr><td>IVIS Spectrum CT</td><td>PerkinElmer</td><td></td><td>for capturing bioluminescent image and DLIT</td></tr><tr><td>Living Image</td><td>PerkinElmer</td><td></td><td>for analyzing bioluminescent image and DLIT</td></tr><tr><td>Na2HPO4</td><td>SIGMA-ALDRICH (St. Louis, MO, USA)</td><td>S9763</td><td>use for conjugation of IR700</td></tr><tr><td>NIR Laser</td><td>Changchun New Industries Optoelectronics Technology</td><td>MRL-III-690R</td><td>for NIR irradiation</td></tr><tr><td>Novex WedgeWell 4 to 20%, Tris-Glycine, 1.0 mm, Mini Protein Gel, 12 well</td><td>Invitrogen</td><td>XP04202BOX</td><td>use for SDS-PAGE</td></tr><tr><td>NuPAGE LDS Sample Buffer (x4)</td><td>Invitrogen</td><td>NP0007</td><td>use for SDS-PAGE</td></tr><tr><td>Optical power meter</td><td>Thorlabs (Newton, NJ, USA)</td><td>PM100</td><td>for measuring the output of the NIR laser&amp;nbsp;</td></tr><tr><td>PBS(-)</td><td>Wako</td><td>166-23555</td><td></td></tr><tr><td>Pearl Trilogy imaging system</td><td>Li-Cor Biosciences</td><td></td><td>for capturing 700 nm fluorecent image</td></tr><tr><td>Penicilin-Streptomycin Solution (x100)</td><td>Wako</td><td>168-23191</td><td>for cell culture</td></tr><tr><td>Puromycin Dihydrochloride</td><td>ThermoFisher</td><td>A1113803</td><td>for luciferase transfection</td></tr><tr><td>RediFect Red-Fluc-Puromycin Lentiviral Prticles</td><td>PerkinElmer</td><td>CLS960002</td><td>for luciferase transfection</td></tr><tr><td>RPMI-1640 with L-glutamine and Phenol Red</td><td>Wako</td><td>189-02025</td><td>for cell culture</td></tr><tr><td>Sephadex G25 column (PD-10)&amp;nbsp;</td><td>GE Healthcare (Piscataway, NJ, USA)</td><td>17-0851-01</td><td>use for conjugation of IR700</td></tr><tr><td>UV-1900i</td><td>Shimadzu</td><td></td><td>for measuring the APC concentration</td></tr></tbody></table>

near infrared photoimmunotherapy for mouse models of pleural dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Anti-podoplanin antibody NZ-1 was conjugated with IR700 to generate NZ-1-IR700. We confirmed the binding of NZ-1 and IR700 on an SDS-PAGE (Figure 8). Luciferase-expressing H2373 (H2373-luc) was prepared by transfecting malignant mesothelioma cells (H2373) with a luciferase gene10.
We anesthetized 8-12-week-old female homozygote athymic nude mice and injected 1 &#215; 105&#160;H2373-luc cells into the thoracic cavity. The day of i...

Watch this Scientific Journal Video about Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination at JoVE.com

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...

near-infrared-photoimmunotherapy-for-mouse-models-pleural

Research

JoVE Journal

Medicine

3.2K Views.  Nagoya University Graduate School of Medicine. Deze methode kan worden gebruikt om de therapeutische effecten van NIR-PIT op thoracale tumoren te evalueren in een klinisch relevant tumormodel. De NIR-PIT procedures met behulp van het pleura gedissemineerde kankermodel zijn gemakkelijk te begrijpen en uit te voeren. Om een muisverspreidingsmodel op te zetten, gebruikt u polystyreenschuim om een stop te maken en desinfecteert u deze met 70% ethanol.Bevestig de naald aan de stop met de punt die vijf millimeter uit de stop reikt. Buig ...

Video: Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

Biology

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Bioengineering

Examining Tumor Cell Dissemination from Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Author Spotlight: Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

Cancer Research

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

Chemistry

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Immunology and Infection

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

A Near-Infrared Photoimmunotherapy Technique to Treat Lung Cancer in a Murine Model

Source: Yasui, H., et al. <a href="https://app.jove.com/v/61593">Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination.</a> J. Vis. Exp. (2021)
This video demonstrates a near-infrared photoimmunotherapy technique to treat lung cancer in an engineered mouse model exhibiting luciferase-expressing tumor cells. Upon injecting an antibody-photosensitizer conjugate (APC) that binds to specific antigens on the tumor cells, the mouse is exposed to near-infrared radiation, inducing photochemical changes in the APC and leading to necrotic death of the bound tumor cells. The effectiveness of the treatment is monitored by injecting luciferin, a luciferase substrate, and measuring the bioluminescence over time, correlating with tumor growth.

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

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

Near-Infrared Photoimmunotherapy for Targeted Cell Elimination in Mixed 3D Cultures

Source: Sato, K. et al., <a href="https://app.jove.com/v/53633">Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates a technique for selectively removing specific cells from a mixed 3D cell culture through near-infrared photoimmunotherapy. In this method, targeted cells are accurately identified using an antibody conjugated with an NIR photoabsorber. Subsequent irradiation with infrared light induces cell death, effectively eliminating the targeted cells from the 3D culture.

NOTE: The following protocol describes the necessary steps to eliminate specific cells using Near infrared photoimmunotherapy (NIR-PIT). Controls and ...

Cellular Immunotherapy

In Vivo Antitumor Efficacy Analysis of PDT: A Technique to Determine the Phototoxic Potential of a Photosensitizer in Tumor Bearing Mice

Source: Chang, J. E., et al. <a href="https://www.jove.com/v/54865/anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted">Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.</a> J. Vis. Exp. (2016).
Photodynamic therapy (PDT) is a non-invasive and non-surgical method for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light. This video describes the in vivo anti-tumor efficacy analysis using photosensitizer containing nanoparticles in a tumor-bearing mouse, to determine the phototoxic potential of the photosensitizer.

Source: Chang, J. E., et al. Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles. J. Vis. Exp. (2016).
Photodynamic ...

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

Samenvatting

Meer video's verkennen