This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

Note: The following protocol describes the necessary steps to eliminate specific cells using NIR-PIT. Controls and other details about NIR-PIT and cell viability can be found elsewhere18.
1. Conjugation of IR700 to Monoclonal Antibodies (mAb)
<ol>
	<li>Prepare mAb of interest at 2-5 mg/ml in 0.1 M Na2HPO4 (pH 8.6) solution.</li>
	<li>Mix 6.8 nmol of mAb with 30.8 nmol of 10 mM IR700 in 0.1 M Na2HPO4 solution (pH 8.6) in a microcentrifuge tube and incubate at RT for 1 hr, covered with aluminum foil.</li>
	<li>Wash the PD-10 column (see Table of Materials/Equipment) with 15 ml PBS, twice. Load the sample from step 1.2.</li>
	<li>Purify the mixture via the PD-10 column by PBS elution according to manufacturer&#39;s instructions. 
	Note: Here, the elution was along the band color of IR700. For a 300 &#181;l sample, PBS elution fraction is typically between 2.5 ml to 4.4 ml.</li>
	<li>Determine the protein concentration with Coomassie staining by measuring the absorption at 595 nm with a spectrophotometer9. Determine the concentration of IR700 with absorption at 689 nm to confirm the number of fluorophore molecules conjugated to each mAb molecule9. 
	Note: It is important to determine an optimal conjugation number of IR700 molecules in 1 mAb with a spectrophotometer. Generally, around 3 IR700 molecules in 1 mAb molecule is optimal for both in vitro and in vivo work. HPLC and SDS-PAGE methods can be used to confirm whether the mAb and IR700 are bound or not.</li>
	<li>Store at 4 &#176;C after determining the protein concentration.</li>
</ol>
2. Preparation of Mixed 3D Cell Culture (Mixed Spheroid)
<ol>
	<li>Apply sterile water (around 1 ml) into the plate reservoir section of hanging drop plates.</li>
	<li>Prepare various ratios of the cell types of interest &#8212; A431-luc-GFP cells and 3T3-RFP cells &#8212; with each sample containing 5,000 cells total, suspended in 50 &#181;l of culture media. 
	Note: Prepare 1:100, 10: 100, 25:100, 50:100, etc. ratios of cells types of interest in culture media depending on the cell-type.</li>
	<li>Incubate the mix for 5-7 days in the 96 well hanging drop plate in a humidified incubator at 37 &#176;C and 5% carbon dioxide. Change the culture media every 2 days. Note: To make the 3D spheroid precisely, handle the plate gently, as drops containing cells fall off easily before forming 3D shapes.</li>
	<li>Observe the morphology and size of the spheroids using an inverted brightfield microscope at 10X - 40X magnification. Note: Although it depends on cell types and the size of hanging-drop, ensure that the diameter of the spheroid is around 400-600 &#181;m after 7 days of incubation in the 96 well hanging drop plate.</li>
</ol>
3. In Vitro NIR-PIT for Mixed 3D Cell Culture
<ol>
	<li>Change the media of the hanging drop plates to 10 &#181;g/ml antibody-photoabsorber conjugate (APC) containing media, and incubate for 6 hr in a humidified incubator at 37 &#176;C and 5% carbon dioxide.</li>
	<li>After 6 hr incubation, wash the spheroid twice with fresh culture media (phenol red free). Gently transfer the spheroid to a glass-bottomed 50 mm dish with 100 &#181;l fresh phenol red free culture media using a sterile 200 &#181;l pipette tip with the tip cut off. Place one spheroid in each dish.</li>
	<li>Observe the spheroid with an inverted brightfield microscope to detect the change of morphology. To observe the optical reporters (e.g., GFP and RFP) use fluorescence microscope with the following filter settings: GFP&#160;&#8212; 469 nm excitation filter, and 525 nm emission filter; RFP&#160;&#8212; 559 nm excitation filter, and 630 nm emission filter.</li>
	<li>Place the light-emitting diode (LED) above the glass-bottomed dish for irradiation. 
	Note: Spheroids can be exposed to NIR light either on the microscope or in the laminar hood.
	<ol>
		<li>Measure the power density of the NIR-light with an optical power meter9. According to this measurement, irradiate NIR-light via light-emitting diodes (LEDs) which emit light at wavelengths of 670 to 710 nm at 2 J/cm2. 
		Note: LED light can penetrate maximum depth of approximately 5 inches. Cytotoxic effects of NIR-PIT is dependent only on given energy regardless of power density and duration of exposure23.</li>
	</ol>
	</li>
	<li>After irradiation, transfer the spheroid into a new hanging drop plate with 50 &#181;l of fresh culture media and incubate for 1 day in a humidified incubator at 37 &#176;C and 5% carbon dioxide.</li>
	<li>Gently transfer the spheroid to a glass-bottomed dish with 100 &#181;l of fresh culture media (phenol red free) using a sterile 200 &#181;l pipette tip with the tip cut off. Observe the spheroid with a fluorescence microscope 1 day after NIR-PIT using filter settings described in step 3.3.
	<ol>
		<li>Detect dead cells by adding propidium iodide to the media at a final concentration of 2 &#181;g/ml, or by loss of cytoplasmic GFP-fluorescence using a fluorescence microscope (Figure 2B)14,18,22.</li>
	</ol>
	</li>
	<li>Repeat steps 3.1-3.6 if target cells are not completely eliminated.</li>
</ol>

<ol>
	<li>Robinton, D. A., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promise+of+induced+pluripotent+stem+cells+in+research+and+therapy.">The promise of induced pluripotent stem cells in research and therapy.</a> Nature. 481 (7381), 295-305 (2012).</li><li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+past,+present,+and+future.">Induced pluripotent stem cells: past, present, and future.</a> Cell stem cell. 10 (6), 678-684 (2012).</li><li>Birchall, M. A., Seifalian, 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=Tissue+engineering's+green+shoots+of+disruptive+innovation.">Tissue engineering's green shoots of disruptive innovation.</a> Lancet. 6736 (14), 11-12 (2014).</li><li>Ben-David, U., Benvenisty, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumorigenicity+of+human+embryonic+and+induced+pluripotent+stem+cells.">The tumorigenicity of human embryonic and induced pluripotent stem cells.</a> Nat. Rev. Cancer. 11 (4), 268-277 (2011).</li><li>Hanna, J. H., Saha, K., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotency+and+cellular+reprogramming:+facts,+hypotheses,+unresolved+issues.">Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues.</a> Cell. 143 (4), 508-525 (2010).</li><li>Lee, M. -. O., Moon, S. 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=Inhibition+of+pluripotent+stem+cell-derived+teratoma+formation+by+small+molecules.">Inhibition of pluripotent stem cell-derived teratoma formation by small molecules.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (35), 3281-3290 (2013).</li><li>Miura, K., Okada, 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=Variation+in+the+safety+of+induced+pluripotent+stem+cell+lines.">Variation in the safety of induced pluripotent stem cell lines.</a> Nat. Biotechnol. 27 (8), 743-745 (2009).</li><li>Tang, C., Lee, A. 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=An+antibody+against+SSEA-5+glycan+on+human+pluripotent+stem+cells+enables+removal+of+teratoma-forming+cells.">An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells.</a> Nat. Biotechnol. 29 (9), 829-834 (2011).</li><li>Mitsunaga, M., Ogawa, M., Kosaka, N., Rosenblum, L. T., 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=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> Nat. Med. 17 (12), 1685-1691 (2011).</li><li>Mitsunaga, M., Nakajima, T., Sano, K., Kramer-Marek, G., 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=Immediate+in+vivo+target-specific+cancer+cell+death+after+near+infrared+photoimmunotherapy.">Immediate in vivo target-specific cancer cell death after near infrared photoimmunotherapy.</a> BMC Cancer. 12 (1), 345 (2012).</li><li>Nakajima, T., Sano, K., 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=Real-time+monitoring+of+in+vivo+acute+necrotic+cancer+cell+death+induced+by+near+infrared+photoimmunotherapy+using+fluorescence+lifetime+imaging.">Real-time monitoring of in vivo acute necrotic cancer cell death induced by near infrared photoimmunotherapy using fluorescence lifetime imaging.</a> Cancer Res. 72 (18), 4622-4628 (2012).</li><li>Sano, K., Mitsunaga, M., Nakajima, 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=Acute+cytotoxic+effects+of+photoimmunotherapy+assessed+by+18F-FDG+PET.">Acute cytotoxic effects of photoimmunotherapy assessed by 18F-FDG PET.</a> J. Nucl. Med. 54 (5), 770-775 (2013).</li><li>Sato, K., Watanabe, 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=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> Mol. Oncol. 8 (3), 620-632 (2014).</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 Lett. 365 (1), 112-121 (2015).</li><li>Sato, K., Hanaoka, H., Watanabe, R., Nakajima, 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+Disseminated+Peritoneal+Ovarian+Cancer.">Near Infrared Photoimmunotherapy in the Treatment of Disseminated Peritoneal Ovarian Cancer.</a> Mol. Cancer Ther. 14 (8), 141-150 (2014).</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 (11), 113276 (2014).</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 (7), 698-709 (2015).</li><li>Sato, K., Nakajima, 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=Selective+cell+elimination+in+vitro+and+in+vivo+from+tissues+and+tumors+using+antibodies+conjugated+with+a+near+infrared+phthalocyanine.">Selective cell elimination in vitro and in vivo from tissues and tumors using antibodies conjugated with a near infrared phthalocyanine.</a> RSC Adv. 5, 25105-25114 (2015).</li><li>Watanabe, R., Hanaoka, 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=Photoimmunotherapy+Targeting+Prostate-Specific+Membrane+Antigen:+Are+Antibody+Fragments+as+Effective+as+Antibodies.">Photoimmunotherapy Targeting Prostate-Specific Membrane Antigen: Are Antibody Fragments as Effective as Antibodies.</a> J. Nucl. Med. 56 (1), 140-144 (2014).</li><li>Nakajima, T., Sano, 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=Improving+the+efficacy+of+Photoimmunotherapy+(PIT)+using+a+cocktail+of+antibody+conjugates+in+a+multiple+antigen+tumor+model.">Improving the efficacy of Photoimmunotherapy (PIT) using a cocktail of antibody conjugates in a multiple antigen tumor model.</a> Theranostics. 3 (6), 357-365 (2013).</li><li>Shirasu, N., Yamada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+antitumor+effect+of+CEA-targeted+photoimmunotherapy.">Potent and specific antitumor effect of CEA-targeted photoimmunotherapy.</a> Int J Cancer. 135 (11), 1-14 (2014).</li><li>Sato, K., Nagaya, T., Nakamura, Y., Harada, 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+prevents+lung+cancer+metastases+in+a+murine+model.">Near infrared photoimmunotherapy prevents lung cancer metastases in a murine model.</a> Oncotarget. 6 (23), 19747-19758 (2015).</li><li>Nakajima, T., 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=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 (1), 389 (2014).</li><li>Klimanskaya, I., Rosenthal, N., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derive+and+conquer:+sourcing+and+differentiating+stem+cells+for+therapeutic+applications.">Derive and conquer: sourcing and differentiating stem cells for therapeutic applications.</a> Nat. Rev. Drug Discov. 7 (2), 131-142 (2008).</li><li>Burmester, G. R., Feist, E., D&#246;rner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+cell+and+cytokine+targets+in+rheumatoid+arthritis.">Emerging cell and cytokine targets in rheumatoid arthritis.</a> Nat. Rev. Rheumatol. 10 (2), 77-88 (2014).</li><li>Pardoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blockade+of+immune+checkpoints+in+cancer+immunotherapy.">The blockade of immune checkpoints in cancer immunotherapy.</a> Nat. Rev. Cancer. 12 (4), 252-264 (2012).</li></ol>

The authors have nothing to disclose.

We demonstrate a method of specific cell elimination from a mixed 3D cell culture without damage to non-target cells by using NIR-PIT. So far, there is no practical method of cell elimination once the tissue is established or after transplantation. Thus, NIR-PIT is a promising method to accomplish this. This technique could also be utilized in vivo18,22, since APCs show similar pharmacokinetics as mAb itself. The target cell type can be adapted with various APC. Various antibodies or antibody fragments, including anti-EGFR, anti-PSMA, anti-mesothelin, and anti-CEA were already used as APCs15-21. Additionally, changing the region of NIR-irradiation can minimize the region treated. Here, with quantification of luciferase activity and fluorescence on target cells, complete elimination of specific cells was confirmed.
NIR-PIT is a light based therapy, thus a critical point is the distance between the NIR-light source and the target, since light energy and therefore cell necrosis decrease according to an inverse-square law (step 3.4). To make the 3D spheroid precisely, the plate should be treated and incubated as gently as possible. The drops containing cells easily are destroyed or fall off by a little shock before forming 3D shapes.
NIR-PIT has several unique advantages. First, NIR-PIT APCs demonstrate similar intravenous pharmacokinetics to parental non-conjugated antibodies, due to the hydrophilic characteristics of IR700, that lead to highly specific binding to target cells and minimal non-target accumulation. Thus, even after transplantation of the tissue, NIR-PIT can treat target cells via intravenous injection of the APC followed by exposure of the region of interest to NIR. Second, NIR-light can penetrate much deeper into tissue than UV or visible light, therefore, NIR-PIT could treat not only the surface but also the entire thickness of the transplanted tissue by exposure to NIR at the surface. Finally, NIR-PIT can be repeatedly used to eliminate target cells without limitation. This method is useful when target cells express sufficient copies of target molecules on the cell surface18.
However, there are a few limitations to this technique. Firstly, the permeability of the APC is limited since it is a large molecule. To overcome this problem, repeated NIR-PIT treatment or antibody fragments can be exploited18,19,22. Another limitation is light penetration. Although the NIR-light can properly penetrate in vitro culture, translation to in vivo treatment would require adapted approaches such as digestive endoscopy, bronchoscopy, or direct thoracoscopy with fiberoptics to move the light source closer to the target region.
Further adaptations of NIR-PIT will allow for the use of this method in both local and specific cell elimination in many fields, such as immunomodulation or tumor microenvironments24-26.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, and there is an urgent need for a cell elimination method in the tissue engineering field. Nowadays in the field of regenerative medicine, tissue cultures using embryonic stem cells (ES), pluripotent stem cells (PSCs), or induced pluripotent stem cell (iPS) are promising materials1-3.
Although this tissue regeneration is promising, contamination with unwanted cells is a major concern. Moreover, there is a safety concern of tumorigenicity after transplantation4,5. Although many studies have focused on these issues to eliminate specific cells, especially in regenerative medicine6-8, no practical method has been developed.
Near infrared photoimmunotherapy (NIR-PIT) is a treatment based on an antibody-photoabsorber conjugate (APC). An APC consists of a cell-specific monoclonal antibody (mAb) and a photoabsorber, IR700. IR700 is a hydrophilic silica-phthalocyanine derivative and does not induce phototoxicity by itself9. IR700 is covalently conjugated to the antibody via amide residues on the side chain of lysine molecules. The APC binds target molecules on the cell membrane and then induces nearly immediate cell necrosis after exposure to NIR light at 690 nm. During the exposure to NIR-light, the cellular membrane ruptures leading to cell death9-14. NIR-PIT has proven to be effective with multiple antibodies or antibody fragments, including anti-EGFR, anti-HER2, anti-PSMA, anti-CD25, anti-mesothelin, anti-GPC3, and anti-CEA15-21. Therefore, NIR-PIT can be used against a wide variety of target molecules. Moreover, NIR-PIT is a well-controlled treatment that allows selective treatment of specific regions by restricting the NIR-light irradiation18,22.
Here, we present a method of specific cell elimination using NIR-PIT from mixed 3D cultures.

<table><tbody><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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>SIGMA-ALDRICH (St. Louis, MO, USA)</td><td>S9763</td><td></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></td></tr><tr><td>Coomassie (bradford) Plus protein assay</td><td>Thermo Fisher Scientific Inc (Waltham, MA, USA)</td><td>PI-23200</td><td></td></tr><tr><td>Perfecta3D 96-Well hanging Drop Plates</td><td>3D Biomatrix Inc (Ann Arbor, MI, USA)</td><td>HDP1096-8</td><td></td></tr><tr><td>Optical power meter</td><td>Thorlabs (Newton, NJ, USA)</td><td>PM100</td><td></td></tr><tr><td>LED: L690-66-60</td><td>Marubeni America Co. (Santa Clara, CA, USA)</td><td>L690-66-60</td><td></td></tr><tr><td>Vectibix (panitumumab)</td><td>Amgen (Thousand Oaks, CA, USA)</td><td></td><td></td></tr><tr><td>35&amp;nbsp;mm glass bottom dish, dish size 35&amp;nbsp;mm, well size 10&amp;nbsp;mm</td><td>Cellvis (Mountain View, CA, USA)</td><td>D35-10-0-N</td><td></td></tr></tbody></table>

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.

To optically monitor the effect of NIR-PIT, the A431 cell line, which overexpresses EGFR, was genetically modified to also express GFP and luciferase (A431-luc-GFP). As a non-target of NIR-PIT, the Balb/3T3 cell line was optically modified to express RFP (3T3-RFP). The APC, panitumumab-IR700 (pan-IR700), was synthesized. Mixed spheroids, which were composed of various ratios of cells (A431-luc-GFP and 3T3-RFP) were fabricated according to this protocol (Figure 1). Repeated NIR-PIT was performed with the incubation of pan-IR700 (see regimen in Figure 2A). Target cell elimination from this mixed 3D culture was achieved and monitored with fluorescence and luciferase activities (Figure 2B, C). On the other hand, non-target cells continued to grow.
<img alt="Figure 1" src="/files/ftp_upload/53633/53633fig1.jpg" /> 
Figure 1. Representative images of various ratios of mixed spheroids. Representative images of mixed spheroids various ratios of A431-luc-GFP cells and 3T3-RFP cells at day 7 are shown. Scale bar = 200 &#181;m. This figure has been modified from16. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53633/53633fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53633/53633fig2.jpg" /> 
Figure 2. Target cell elimination in 3D cell spheroids (mixed spheroids of A431-luc-GFP and 3T3-RFP cells). (A) NIR-PIT (2 J/cm2) regimen is shown. (B) Repeated NIR-PIT completely eliminated target cells (A431-luc-GFP) with no harm to non-target cells (3T3-RFP), in a mixed 3D spheroid. Bar = 200 &#181;m. (C) Quantification of luciferase activities (RLU ratio) demonstrated complete elimination of target cells (n = 10 spheroids in each group). This figure has been modified from18. Data are expressed as means &#177; s.e.m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53633/53633fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

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

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 ...

selective-cell-elimination-from-mixed-3d-culture-using-near-infrared

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.8K Views.  National Cancer Institute. 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). 

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

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Cancer Research

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates.
As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 &#177; 0.6 J/cm2). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug&#39;s cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

Here, we describe a novel, simple, and low-cost device to successfully perform in vitro photodynamic therapy (PDT) assays using two-dimensional HeLa cell culture and verteporfin as a photosensitizer.

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built ...

Biochemistry

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

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Medicine

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.

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 ...

Analyzing Petite Colony Formation and Drug Resistance in Candida glabrata

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial photodynamic therapy (aPDT) targets multiple fungal structures, unlike antibiotics/antifungals, potentially thwarting resistance. Traditional methods for inducing petite colonies rely on ethidium bromide or fluconazole, which can influence drug susceptibility and stress responses. This study investigated the application of green light (peak 520 nm) and rose bengal (RB) photosensitizer to combat a drug-resistant Candida glabrata isolate. The findings revealed that aPDT treatment significantly inhibited cell growth (&#8805;99.9% reduction) and effectively induced petite colony formation, as evidenced by reduced size and loss of mitochondrial redox indicator staining. This study provides initial evidence that aPDT can induce petite colonies in a multidrug-resistant C. glabrata strain in vitro, offering a potentially transformative approach for combating resistant fungal infections.

Author Spotlight: Photodynamic Therapy as a Novel Approach to Induce Petite Colonies in Drug&#45;Resistant Candida for Antifungal Research

The significance of petite colonies in Candida spp. drug resistance has not been fully explored. Antimicrobial photodynamic therapy (aPDT) offers a promising strategy against drug-resistant fungal infections. This study demonstrates that rose bengal-mediated aPDT effectively deactivates Candida glabrata and induces petite colonies, presenting a unique procedure.

Facing a 40% mortality rate in candidemia patients, drug-resistant Candida and their petite mutants remain a major treatment challenge. Antimicrobial ...

Biology

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

Source: Yasui, H., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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 ...

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

Summary

Explore More Videos

Chapters in this video

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

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

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

An In Vitro Approach to Photodynamic Therapy

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

Induction of Petite Colonies in Candida glabrate via Rose Bengal-Mediated Photodynamic Therapy

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

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