The authors have no acknowledgements.

1. Preparation of Cells
<ol>
	<li>Plate 20,000 cells in 2 mL of culture medium into each well of a 6-well plate using sterile technique in a biosafety cabinet.</li>
	<li>Place 6-well plates in a humidified incubator (37 &#176;C, 5% CO2) for 24 h to allow the cells to adhere to plates.</li>
</ol>
2. Preparation of Photosensitizer for Treatment of Cells
<ol>
	<li>Prepare 0.5, 1, and 2 mM 5-ALA solutions in culture medium. If fetal bovine serum (FBS) is an ingredient of culture medium, prepare and treat cells with 5-ALA in culture medium solution with 0.1% FBS for incubations.11,12 In this case, SCC-13 cells do not require FBS, so 5-ALA was added directly to keratinocyte serum-free medium supplemented with bovine pituitary extract and epidermal growth factor.</li>
	<li>Aspirate the culture medium and wash the cells with at least 2 mL of phosphate buffered saline (PBS).</li>
	<li>Aspirate PBS and add 2 mL of 0, 0.5, 1, or 2 mM 5-ALA solutions into separate plates or wells.</li>
	<li>Incubate the cells with 0, 0.5, 1, or 2 mM 5-ALA on a 36 to 37 &#176;C heating block for 30 min. Protect the cells from light exposure during incubation using aluminum foil.</li>
	<li>Aspirate the 0, 0.5, 1, and 2 mM 5-ALA solutions and wash the cells with 2 mL of PBS.</li>
	<li>Aspirate PBS and add 2 mL of culture medium for blue light irradiation.</li>
</ol>
3. Blue Light Phototherapy
<ol>
	<li>Before irradiating the cells, turn on the blue light device (e.g., light-emitting diode, fluorescent, or halogen light) and run for one cycle (1,000 s) to warm up the device. The blue light device should have an output wavelength of 417 +/- 5 nm. Allowing the device to run for one cycle before treatments ensures that the blue light wavelength and irradiance are consistent throughout the photoactivation phase.</li>
	<li>Use a photometer to measure irradiance at the cell surface. The blue light irradiance at the cell surface should be 10 mW/cm2. The distance between the light and the cells may need to be adjusted to achieve an irradiance of 10 mW/cm2 depending on the intensity of the light source.</li>
	<li>Place 0, 0.5, 1, and 2 mM ALA treatment groups on a black surface under the blue light source. Irradiate the cells with blue light for 1,000 s (16 min and 40 s) for a total fluence of 10 J/cm2. Following blue light photoactivation, the cells are ready for analysis.</li>
</ol>
4. Collection and Staining
<ol>
	<li>Prepare flow buffer according to manufacturer guidelines (see Table of Materials).</li>
	<li>Aspirate culture medium and wash cells with 2 mL of PBS.</li>
	<li>Add 1 mL of 0.25% trypsin-EDTA and allow cells to detach for approximately 3 to 5 min. The cells may be examined under the microscope to confirm cell detachment.</li>
	<li>Add 1 mL of culture medium (or PBS) with 10% fetal bovine serum to deactivate the trypsin.</li>
	<li>Collect cell suspension into labeled 5 mL flow tubes.</li>
	<li>Spin 5 mL flow tube in a centrifuge at 201 x g for 5 min. Aspirate solution without removing cell pellet.</li>
	<li>Add 200 &#181;L of flow buffer with conjugated annexin-V antibody (1 &#181;L of annexin-v per 39 &#181;L of flow buffer) to each sample.</li>
	<li>Resuspend the cells and place in the incubator (37 &#176;C, 5% CO2) for 20 min to allow annexin-V binding.</li>
	<li>Add 3 &#181;L of 7-AAD to each sample. Incubate for 5 min at room temperature.</li>
</ol>
5. Flow Cytometric Analysis
<ol>
	<li>Follow flow cytometer manufacturer guidelines for set-up (see Table of Materials).</li>
	<li>Collect and analyze samples with flow cytometry.13</li>
	<li>Perform statistical comparison of treatment and control groups using analysis of variance (ANOVA)14. Compare the means of the treatment groups to the mean of the control with a Dunnett&#39;s test for post hoc analysis.</li>
</ol>

<ol>
	<li>Morton, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=European+Dermatology+Forum+guidelines+on+topical+photodynamic+therapy.">European Dermatology Forum guidelines on topical photodynamic therapy.</a> European Journal of Dermatology. 25 (4), 296-311 (2015).</li><li>Ozog, D. 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=Photodynamic+therapy:+A+clinical+consensus+guide.">Photodynamic therapy: A clinical consensus guide.</a> Dermatologic Surgery: Official Publication for American Society for Dermatologic Surgery. 42 (7), 804-827 (2016).</li><li>Abrahamse, H., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+photosensitizers+for+photodynamic+therapy.">New photosensitizers for photodynamic therapy.</a> Biochemical Journal. 473 (4), 347-364 (2016).</li><li>Josefsen, L. B., Boyle, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+and+the+development+of+metal-based+photosensitisers.">Photodynamic therapy and the development of metal-based photosensitisers.</a> Metal-Based Drugs. 2008, 276109 (2008).</li><li>Ohgari, 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=Mechanisms+involved+in+&#948;-aminolevulinic+acid+(ALA)-induced+photosensitivity+of+tumor+cells:+Relation+of+ferrochelatase+and+uptake+of+ALA+to+the+accumulation+of+protoporphyrin.">Mechanisms involved in &#948;-aminolevulinic acid (ALA)-induced photosensitivity of tumor cells: Relation of ferrochelatase and uptake of ALA to the accumulation of protoporphyrin.</a> Biochemical pharmacology. 71 (1-2), 42-49 (2005).</li><li>Kessel, D., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+photodamage+and+PDT-induced+apoptosis.">Mitochondrial photodamage and PDT-induced apoptosis.</a> Journal of Photochemistry and Photobiology B: Biology. 42 (2), 89-95 (1998).</li><li>Goldberg, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+in+skin+rejuvenation.">Photodynamic therapy in skin rejuvenation.</a> Clinics in Dermatology. 26 (6), 608-613 (2008).</li><li>Sakamoto, F. H., Lopes, J. D., Anderson, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+acne+vulgaris:+A+critical+review+from+basics+to+clinical+practice:+Part+I.+Acne+vulgaris:+When+and+why+consider+photodynamic+therapy.">Photodynamic therapy for acne vulgaris: A critical review from basics to clinical practice: Part I. Acne vulgaris: When and why consider photodynamic therapy.</a> Journal of the American Academy of Dermatology. 63 (2), 183-193 (2010).</li><li>Boen, M., Brownell, J., Patel, P., Tsoukas, M. 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Annexin V and Propidium Iodide Labeling.</a> JoVE. , (2018).</li><li>Mamalis, A., Koo, E., Sckisel, G., Siegel, D., Jagdeo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependent+impact+of+thermal+aminolaevulinic+acid+photodynamic+therapy+on+apoptosis+and+reactive+oxygen+species+generation+in+human+dermal+fibroblasts.">Temperature-dependent impact of thermal aminolaevulinic acid photodynamic therapy on apoptosis and reactive oxygen species generation in human dermal fibroblasts.</a> British Journal of Dermatology. 175 (3), 512-519 (2016).</li><li>Gilaberte, 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=Cellular+intrinsic+factors+involved+in+the+resistance+of+squamous+cell+carcinoma+to+photodynamic+therapy.">Cellular intrinsic factors involved in the resistance of squamous cell carcinoma to photodynamic therapy.</a> Journal of Investigative Dermatology. 134 (9), 2428-2437 (2014).</li><li>Yoon, H. 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=Nanophotosensitizers+engineered+to+generate+a+tunable+mix+of+reactive+oxygen+species,+for+optimizing+photodynamic+therapy,+using+a+microfluidic+device.">Nanophotosensitizers engineered to generate a tunable mix of reactive oxygen species, for optimizing photodynamic therapy, using a microfluidic device.</a> Chemistry of Materials. 26 (4), 1592-1600 (2014).</li><li>Chen, Y. -. C., Lou, X., Zhang, Z., Ingram, P., Yoon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cancer+cell+sphere+formation+for+characterizing+the+efficacy+of+photo+dynamic+therapy+in+3D+cell+cultures.">High-throughput cancer cell sphere formation for characterizing the efficacy of photo dynamic therapy in 3D cell cultures.</a> Scientific Reports. 5, 12175 (2015).</li><li>Campos, C., Inada, N., Kurachi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+PDT+on+breast+cancer+spheroids.">Low-dose PDT on breast cancer spheroids.</a> Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XXVII. , (2018).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+temperature+on+5-aminolevulinic+acid-based+photodynamic+reaction+in+keratinocytes+in+vitro.">The influence of temperature on 5-aminolevulinic acid-based photodynamic reaction in keratinocytes in vitro.</a> Photodermatology, Photoimmunology &amp; Photomedicine. 26 (2), 83-88 (2010).</li><li>Juzeniene, A., Juzenas, P., Kaalhus, O., Iani, V., Moan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effect+on+Accumulation+of+protoporphyrin+IX+after+topical+application+of+5-aminolevulinic+acid+and+its+methylester+and+hexylester+derivatives+in+normal+mouse+skin.">Temperature effect on Accumulation of protoporphyrin IX after topical application of 5-aminolevulinic acid and its methylester and hexylester derivatives in normal mouse skin.</a> Photochemistry and Photobiology. 76 (4), 452-456 (2002).</li><li>Novak, B., Heesen, L., Schary, N., Lubbert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+different+illumination+parameters+on+protoporphyrin+IX+induced+cell+death+in+squamous+cell+carcinoma+cells.">The influence of different illumination parameters on protoporphyrin IX induced cell death in squamous cell carcinoma cells.</a> Photodiagnosis and Photodynamic Therapy. 21, 385-392 (2018).</li></ol>

DUSA/Sun Pharmaceutical provided 5-ALA and the BLU-U light device. This manuscript was supported by Dr. Jagdeo&#39;s residual funding. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

There was a significant increase in SCC-13 apoptosis following 30 min incubations of 0.5, 1, and 2 mM 5-ALA followed by 1,000 s of blue light. These results are consistent with our previously published research demonstrating that 5-ALA incubations of 30 min followed by blue light activation leads to a dose-dependent increase in the percentage of fibroblast cells undergoing apoptosis12,14.
This experimental approach to studying PDT has advantages and limitations. The described methods conform to clinical practice as a commercially available PS and light source were used. Researchers may customize the methods with different cell types, PSs, light sources, and irradiation parameters.However, there are limitations to this approach as this protocol is designed for adherent cells cultured in a monolayer. In clinical practice, differences in tissue architecture or disease pathology (i.e., hyperkeratosis or fibrosis) may decrease PS absorption or light penetration15. As a result, the treatment doses and experimental findings may not directly correspond to clinical practice. Spheroid tumor models and microfluidic chip have been studied as methods to more closely replicate tumor microenvironments16,17,18. Spheroids have inner and outer cellular niches that may differentially incorporate the photosensitizers and represent heterogeneous tumor architecture. Other researchers have used microfluidic chips to screen treatment conditions and vascular delivery of photosensitizers. However, microfluidic chips may be costly to develop or difficult to implement for researchers unfamiliar with the technique. Furthermore, spheroids and microfluidic chips may not reflect the clinical treatment of skin cancers in which photosensitizers are directly applied to the tumor surface without vascular dissemination. Researchers may need to evaluate the pros and cons of monolayer culture, spheroid tumor models, and microfluidic chips for studying PDT in different disease systems. As there are no immune cells or extracellular matrix, it is not possible to determine how PDT affects complex cell-cell interactions. Researchers may need to confirm in vitro experimental results using animal models and clinical trials. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments.
Temperature is known to alter the efficacy of PDT19.One study demonstrated that cell death, 5-ALA uptake, PP-IX formation, and cytokine release may be enhanced by incubating cells with 5-ALA at temperatures up to 44 &#176;C19. Incubation temperatures above 44 &#176;C may lead to thermal induced cell death, and incubation temperatures below 20 &#176;C may not lead to significant 5-ALA uptake and accumulation19. We recommend that researchers perform PS incubations on a temperature-adjustable heating block at a constant temperature to control for potentially confounding thermal effects. Additionally, it is important to incubate the photosensitizer for a sufficient amount of time to allow for the conversion of 5-ALA into PP-IX. In this protocol, SCC-13 cells were incubated with 5-ALA for 30 min, which successfully induced cell apoptosis. We have previously demonstrated that a 10 min incubation of 5-ALA did not significantly increase apoptosis in fibroblast compared to untreated control fibroblasts11,14. PP-IX starts accumulating in mouse skin after 5-ALA is incubating for six minutes at 37 &#176;C. Therefore, after ten minutes of 5-ALA incubation, there may not be sufficient accumulation of PP-IX to induce cell apoptosis20. We recommend a minimum incubation period of 20 to 30 min for 5-ALA based on our previous studies11,14. Researchers may need to optimize incubation period based on the laboratory setting, photosensitizer of interest, cell type, and clinical indication. Antibody titration and optimization experiments may be performed for best results using manufacturers guidelines. Other assays of interest may be performed following blue light photoactivation including dihydroethidium flow cytometric quantification of ROS.14
Variation in the amount of light delivered per unit of surface area (i.e., irradiance) and total irradiation dose (i.e., fluence) during the photoactivation phase may affect ROS formation and cell apoptosis21. The relationship between fluence, irradiance, and time can be described by the following equation:
Fluence (J/cm2) = Irradiance (W/cm2) x time (s)
As fluence is dependent on time, lengthening or shortening the photoactivation phases may change treatment efficacy. The irradiance is proportional to distance squared between the light source and the target tissue. As a result, if the light is too far away, there may not be sufficient light energy delivered to the targeted tissue to excite the PS. Alternatively, the irradiance may be increased by light reflecting off the surrounding surfaces. Therefore, we recommend performing light irradiations with the cell culture plates placed on a black surface to prevent light reflection. Researchers may acquire commercially available or FDA-approved blue light-emitting diodes and fluorescent devices to use in PDT experiments, but these devices may have different power outputs and light field uniformity. A wavelength-specific photometer should be used to measure the irradiance at the cell surface and uniformity of the light field before every experiment for consistent results. A commercially available diffuser may be used to enhance field uniformity, if necessary.
In summary, we have described an in vitro approach to investigating PDT by detailing theory, experimental methods, limitations, potential pitfalls, and recommendations for optimization. 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.

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 (FDA) has approved PDT for the treatment of actinic keratosis (AK), and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris1,2. Emerging non-dermatological uses for PDT include treatment of gastrointestinal, prostate, and gynecological cancers3. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scarring2.
In the first step of PDT, a PS is applied and allowed to accumulate intracellularly2. In the United States, topical 5-aminolevulinic acid (5-ALA) is commonly used to treat AKs. During the incubation phase, 5-ALA is converted into protoporphyrin IX (PP-IX) via the heme biosynthesis pathway in incorporated cells3. Cancer and pre-cancer cells have decreased ferrochelatase activity, which converts protoporphyrin IX (PP-IX), into the end product, heme. As a result, cancers cells accumulate PP-IX compared to normal cells4,5. This accumulation of PP-IX in cancer and pre-cancer cells relative to the surrounding tissue allows for PDT to be a targeted approach with minimal adverse events. Light irradiation leads to reactive oxygen species (ROS) generation when oxygen accepts excited electrons from PP-IX. PP-IX has a peak absorption in the ultraviolet spectrum with smaller peaks throughout the visible light spectrum, including blue and red light2. ROS formation may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover6,7,8,9,10.
PDT was developed in the 1970s and is an evolving therapeutic modality3. The FDA-approved protocol for the treatment of AKs recommends 5-ALA skin incubation for 14&#8211;18 h, but 1 to 2 h incubations are commonly used in clinical practice2. In vitro, we have shown that shorter 15 min 5-ALA incubations may increase fibroblast cell apoptosis compared to untreated fibroblasts11,12. Additionally, novel chlorin, phthalocyanine, and nanoparticle-based PSs are being studied to improve PDT efficacy3. Herein, we present an in vitro method to study PDT in an adherent squamous cell carcinoma cells. In this protocol, SCC-13 cells are incubated with 0, 0.5, 1.0, and 2 mM 5-ALA for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure is apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D (7-AAD) flow cytometry. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with other cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Preparation of additional control groups may be necessary, including unstained, single fluorophore stained, negative, and positive controls for flow cytometry. A comprehensive review of theory, protocols, experimental design, and gating for apoptosis/necrosis flow cytometry can be found elsewhere13.

<table><tbody><tr><td>Keratinocyte serum free medium&amp;nbsp;</td><td>Thermofisher</td><td>17005042</td><td>Culture medium for SSC-13 cells</td></tr><tr><td>DMEM low glucose glutamax</td><td>Thermofisher</td><td>10567022</td><td>Possible culture medium for other cell types</td></tr><tr><td>6-well culture plates</td><td>Thermofisher</td><td>720083</td><td></td></tr><tr><td>PBS</td><td>Thermofisher</td><td>14190250</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Thermofisher</td><td>25200056</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermofisher</td><td>15250061</td><td>For cell counting and plating&amp;nbsp;</td></tr><tr><td>BLU-U light&amp;nbsp;</td><td>DUSA</td><td>Request from manufacturer</td><td>Other blue LED, flourescent, or halogen lights may used&amp;nbsp;</td></tr><tr><td>5-ALA</td><td>DUSA</td><td>Request from manufacturer</td><td></td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>C17032</td><td></td></tr><tr><td>FlowCellect Annexin Red Kit</td><td>Millipore Sigma&amp;nbsp;</td><td>FCCH100108&amp;nbsp;</td><td>Propidium iodide may replace 7-AAD. Comes with annexin-V, 7-AAD, and flow buffer</td></tr><tr><td>FACSCanto II</td><td>BD</td><td>Request from manufacturer</td><td>Any flow cytometer may be used&amp;nbsp;</td></tr><tr><td>Counting Chamber</td><td>Hausser</td><td>3120</td><td>For cell counting and plating&amp;nbsp;</td></tr><tr><td>FlowJo</td><td>FlowJo</td><td>Request from manufacturer</td><td>Flow cytometry analysis software</td></tr></tbody></table>

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.

After SCC-13 cells were incubated for 30 min with 0, 0.5, 1, and 2 mM 5-ALA and irradiated with 1,000 s of blue light, there was a dose-dependent increase in cell apoptosis. Total apoptosis (mean &#177; standard error of mean) was 3.94 &#177; 0.34, 7.90 &#177; 0.52, 23.86 &#177; 0.52, and 38.33 &#177; 1.81 after 30 min of incubation with 0, 0.5, 1, and 2 mM 5-ALA, respectively, and 1000 seconds of blue light (Figure 1). We compared the mean percentage of apoptotic cell among 0, 0.5, 1, and 2 mM 5-ALA incubated groups using ANOVA. Cells treated with 1 and 2 mM 5-ALA had a significant increase in cell apoptosis compared to 0 mM 5-ALA treated cells. Figure 2A shows representative flow cytometric gating of forward scatter (FSC) versus side scatter (SSC) for SCC-13 cells incubated with 0, 0.5, 1, and 2 mM 5-ALA. The circle gating encloses the SCC-13 cell population. For this experiment, 7500 events were collected and the SCC-13 cell population gate captured at least 90% of events. Figure 2B shows a representative plot of the 7-AAD on the x-axis and annexin-V on the y-axis. The cell populations are gated into four quadrants (Q1 to Q4) to discriminate apoptotic cells. Q1 (high annexin-v, low 7-AAD) represents cells undergoing early apoptosis. Q2 (high annexin-v, high 7-AAD) represent cells undergoing late apoptosis or necrosis. Q3 (low annexin-v, high 7-AAD) represents cells undergoing necrosis. Q4 (low annexin-v, low 7-AAD) represents cells not undergoing apoptosis or necrosis. To calculate the percentage of cells undergoing apoptosis, we added the percentage of cells in Q1 and Q2. Inconsistency in 5-ALA incubation period length, incubation temperature, or photoactivation irradiation dose may alter PDT efficacy. Figure 3 demonstrates a representative annexin-V and 7-AAD flow cytometric plot when the incubation temperature is varied (i.e., 27, 36, or 42 &#176;C). There was a temperature dependent increase in apoptosis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58190/58190fig1.jpg" /> 
Figure 1: Dose-dependent increase in apoptosis after thirty-minute incubation of 5-ALA. 5-ALA incubated at 36 &#176;C for thirty minutes followed by 1,000 s of blue light in SCC-13 cells. Bars represent average percent annexin-V positive cells in each treatment and control group. Experiments were performed in technical triplicate. Statistical significance was determined by ANOVA, with p &#60;0.05 denoted by an asterisk (*). Error bars represent the mean &#177; standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/58190/58190fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58190/58190fig2.jpg" /> 
Figure 2: Representative Flow Cytometry plots. (A) Representative forward scatter vs. side scatter cytometry plots in Arbitrary Units (AU) for x- and y-axis for 0, 0.5, 1, and 2 mM 5-ALA incubated cells. For this experiment, 7500 events were collected and the SCC-13 cell population gate captured 90% of events. (B) Representative annexin-V vs. 7-AAD flow cytometry plots in AU for x- and y-axis for 0, 0.5, 1, and 2 mM 5-ALA incubated cells. Q1: early apoptotic cells, Q2: late apoptotic/necrotic cells, Q3: necrotic cells, and Q4: viable cells. <a href="https://www.jove.com/files/ftp_upload/58190/58190fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58190/58190fig3.jpg" /> 
Figure 3: Incubating 5-ALA at different temperatures may alter PDT efficacy.&#160;Representative annexin-V vs. 7-AAD flow cytometry plots in AU for x- and y-axis for SCC-13 cells incubated with 0.5 mM 5-ALA at 27, 36, and 42 &#176;C. Q1: early apoptotic cells, Q2: late apoptotic/necrotic cells, Q3: necrotic cells, and Q4: viable cells. <a href="https://www.jove.com/files/ftp_upload/58190/58190fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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.

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 ...

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Medicine

8.6K Views.  University of California, Davis. 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.

Video: An In Vitro Approach to Photodynamic Therapy

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for Photodynamic Therapy (PDT) is reported. The nanoparticles are formed by hydrophobic interaction of the semiconducting polymer MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) with the fullerene PCBM (phenyl-C61-butyric acid methyl ester) in the presence of a non-compatible solvent. MEH-PPV has a high extinction coefficient that leads to high rates of triplet formation, and efficient charge and energy transfer to the fullerene PCBM. The latter processes enhance the efficiency of the PDT system through fullerene assisted triplet and radical formation, and ultrafast deactivation of MEH-PPV excited stated. The results reported here show that this nanoparticle PDT sensitizing system is highly effective and shows unexpected specificity to cancer cell lines.

This protocol describes a method for the fabrication of conducting polymer nanoparticles blended with fullerene. These nanoparticles were investigated for their potential use as a next generation photosensitizers for Photodynamic Therapy (PDT).

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next ...

Chemistry

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT. 
After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive ...

Cancer Research

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

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

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 ...

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 ...

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells

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>&#160;J. Vis. Exp.&#160;(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. In this protocol video, PDT-based method is used to evaluate the phototoxic potential of a potential photosensitizer in lung cancer cells.

Source: Chang, J. E., et al. Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.&#160;J. Vis. Exp.&#160;(2016).
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Lung Cancer

In vitro study

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 ...

An In Vitro Approach to Photodynamic Therapy

Summary

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Chapters in this video

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

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

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

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells

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