Name: near infrared photoimmunotherapy for mouse models of pleural dissemination
Uploaded: 2021-02-09T12:30:00
Description: Watch this Scientific Journal Video about Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination at JoVE.com

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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 con...

<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. 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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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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)&#160;</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 ...

This method can be used to evaluate the therapeutic effects of NIR-PIT on thoracic tumors in a clinically relevant tumor model. The NIR-PIT procedures using the pleural disseminated cancer model are easy to understand and to perform. To set up a mouse dissemination model, use polystyrene foam to make a stopper, and disinfect it with 70%ethanol.Attach the needle to the stopper with the tip extending five millimeters out of the stopper. Bend the tip of a 30-gauge needle with clean forceps or against a hard object cleaned with 70%ethanol, and fill a syringe with one times 10 to the six target tumor cells in 100 microliters of PBS. Confirm a lack of response to pedal reflex in an anesthetized, 19 to 21 gram, eight to 12 week old, female, homozygote, athymic nude mouse.Insert the needle into the chest through the intercostal space, moving the needle up and down to avoid contacting the ribs. When the tip has passed through the intercostal space, position the syringe so that it is pressed against the mouse and inject the entire volume of target cells. After the injection, roll the mouse two to three times to spread the cells throughout the thoracic cavity, and return the mouse to its cage with monitoring until full recovery.24 hours after cell injection and every day thereafter, inject the anesthetized tumor cell-injected mice with 200 microliters of 15 milligrams per milliliter of D-luciferin. After 10 minutes, place the mice into a bioluminescence imager, and open the Acquisition Control Panel in the imager software. Select Luminescent, Photograph, and Overlay.Set the Exposure Time to Auto, the Binning to Small, the F/Stop to one for Luminescent and to eight for Photograph, and the Field of View to C.Click Acquire to image the bioluminescence. Set the Display Format to Radians, and select the circle from the Region Of Interest Tools in the Tool Palette panel. Click Measure Regions Of Interest to measure the surface bioluminescent intensity, and use Configure Measurement to select the values relevant to the experiment.Export this data table as a csv file. Then use the total flux values for the bioluminescent intensity quantification in the file. Before performing near-infrared phototherapy of the tumor-injected mice, use a power meter to measure the light dose of a 690-nanometer wavelength laser, and adjust the output to 100 milliwatts per square centimeter.24 hours before the treatment, intravenously injected 100 micrograms of antibody photosensitizer conjugate in 50 to 200 microliters of PBS via the tail vein of the tumor-injected animal. On the day of the phototherapy treatment, place the anesthetized conjugate-injected, tumor-laden mouse in the supine position, and shield the non-target sites with aluminum foil. When all of the shields have been placed, use a 100-joules-per-square-centimeter laser to irradiate the thoracic cavity with near-infrared light for about 30 seconds.When the irradiation is complete and the mouse has awoken, return the animal to its cage, and measure the bioluminescence daily as demonstrated. In this representative analysis, the conjugation of anti-podoplanin antibody with IR700 was confirmed by SDS-PAGE analysis. After tumor cell injection, bioluminescence imaging and diffuse luminescence imaging tomography should be performed to determine which mice express sufficient luciferase activity in the chest cavity for further study.At day five after injection, anti-podoplanin antibody IR700-injected mice demonstrate high IR700 fluorescence and luciferase activity in thoracic tumors, indicating that intravenously injected IR700-conjugated antibody reaches disseminated pleural tumor sites. Notably, pleural disseminated mice treated with near-infrared photoimmunotherapy demonstrate a decreased luciferase activity, while the relative light units in the control group exhibit a gradual increase in intensity. Since the required NIR irradiation energy depends on the cell line and antibodies, be sure to check the conditions in advance in vitro.

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