Name: Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro (Video) | JoVE Journal
Uploaded: 2014-03-18T14:30:00.000+00:00
Duration: 8 min 4 s
Description: Learn photodynamic therapy cytotoxicity in osteosarcoma cells using M-T-H-P-C photosensitizer and laser activation. This method measures cell viability via WST-1 assay to evaluate therapeutic potential.

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5 minutes

The authors would like to thank biolitec Research GmbH (Jena, Germany) for kindly providing us with the liposomal mTHPC formulation, with special thanks to Susanna Gr&#228;fe and Arno Wiehe for their help and technical expertise. We also would like to thank Kerstin Reidy, who generated a large part of the results and was coresponsible for its publication11.
This work was supported by grants from the Schweizerischer Verein Balgrist, the University of Zurich, the Krebsliga Zurich as well as by a grant from the Walter L. and Johanna Wolf Foundation, Zurich, Switzerland and a grant from EuroNanoMed ERA-NET &#8260;SNF Swiss National Science Foundation 31NM30-131004 &#8260; 1. This work was also supported by the HSM (Highly Specialized Medicine) program for Musculoskeletal Oncology of the Kanton Zurich.

1.&#160;Comparison of the Uptake of mTHPC in the Respective Low and Highly Metastatic HOS and 143B OS Cell Lines
<ol>
	<li>Prepare cell culture medium containing DMEM, Ham F12, and heat inactivated fetal calf serum in a ratio 4.5:4.5:1. 
	Note: The liposomal formulation of the PS mTHPC was dissolved in water at a final concentration of 1.5 mg/ml mTHPC.</li>
	<li>Plate 0.2 x 106 HOS and 143B cells/well in 6-well plates (triplicates for each condition) and let the cells adhere overnight. 
	Note: the number has to be adjusted to individual cell lines. A confluency of 80-90% is most suitable at the time of the experiment.</li>
	<li>On the following day, incubate the cells with a fixed concentration of mTHPC for different time periods (time dependent uptake), or with increasing concentrations of mTHPC during a fixed time period (dose dependent uptake).</li>
	<li>Incubate the cells with mTHPC for specific time points or concentrations of mTHPC. 
	Note: In this study, both the HOS and 143B cell lines were incubated with 0.6 &#181;g/ml mTHPC for 0, 2.5, 5, or 10 hr, or with 0, 0.6, 2.5, and 10 &#181;g/ml mTHPC for 5 hr. The applied time periods and dosages may vary depending on the cell line that is used.</li>
	<li>Remember to always work in the dark or in dimmed light conditions&#160;to prevent direct light exposure of the cells.</li>
	<li>After incubation of the cells at the indicated conditions, aspirate the medium and wash the cells three times with PBS. Next, detach the cells with 0.5 ml trypsin/EDTA and count the cells.</li>
	<li>After detachment, centrifuge the cells at 400 x g for 5 min, aspirate the medium and resuspend the cells in PBS to a final density of 200,000 cells/ml.</li>
	<li>Pipette 100 &#181;l of the obtained cell suspension (i.e.&#160;20,000 cells) in a 96-well plate and measure the fluorescence of these cells in a fluorescence spectrophotometer . The settings for mTHPC fluorescence measurements are: 417 nm for the excitation and 652 nm for the emission.</li>
	<li>In order to calculate the amount of internalized mTHPC per cell, normalize the relative fluorescence unit (RFU) for the cell number and cell volume as follows:</li>
	<li>Prepare a standard curve using different concentrations of mTHPC in PBS from 0-4 &#181;g/ml (use triplicate measurements). Pipette 100 &#181;l of each dilution in a 96-well plate and measure the fluorescence of the wells using the settings described under step 1.6.</li>
	<li>Divide the RFU (obtained for 20,000 cells) by the slope of the linear standard curve of mTHPC, which gives the concentration of mTHPC (in &#181;g/ml) in the well.</li>
	<li>Divide this mTHPC concentration by 10 to get the total amount of mTHPC in one well (in &#181;g, representing 100 &#181;l of cell suspension) and divide this value by the volume of 20,000 cells.</li>
	<li>Calculate the volume with the formula for a sphere: (4/3 x &#960;) x r3. Obtain the radius r by measuring the diameter of 20 trypsinized cells under a microscope, and dividing this value by 2.</li>
	<li>Finally, normalize all the values to those obtained for HOS cells after 10 hr of incubation (dose dependent uptake) or treated with 10 &#181;g/ml mTHPC (time dependent uptake), which were set to 100%.</li>
</ol>
2.&#160;Measurement of Phototoxicity of PS In Vitro
<ol>
	<li>Seed 3,000 cells/well in a 96-well plate (triplicates for each concentration of mTHPC used) and let them adhere overnight. Similarly prepare an additional 96-well plate and treat the cells as described below, but then put them aside for phase contrast imaging. 
	Note: the number has to be adjusted for each individual cell line. A confluency of 50-60% at the time of the experiment is ideal.
	<ol>
		<li>Keep in mind to add the corresponding dark toxicity control of cells for each of the mTHPC concentrations. These are cells that will be incubated in the absence or presence of selected concentrations of mTHPC, but will not be illuminated.</li>
	</ol>
	</li>
	<li>On the following day, incubate the cells with different concentrations of mTHPC (0, 0.0001, 0.001, 0.01, 0.03, 0.075, 0.15, 0.3, 0.6, 1.25, 2.5, 5,&#160;and 10 &#181;g/ml) depending on the cell lines and keep them in the dark for 5 hr.
	<ol>
		<li>Refer to Section 1.3.2 
		Note: The incubation time before illumination depends on the cell line and on the PS used, therefore it is advisable to test different concentrations and different time points in separate experiments.</li>
	</ol>
	</li>
	<li>Following incubation for the indicated time period, wash the cells twice with PBS and add new fresh medium without mTHPC.</li>
	<li>Illuminate the cells with a 652 nm diode laser, specific for the mTHPC absorption spectrum (see Figure 1).
	<ol>
		<li>Set the laser to 21.88 mW/cm2 power at a height of 13.5 cm distance from the cells, in order to get an energy dose of 5 J/cm2. Illuminate the cells for 230 sec. Do not illuminate the dark toxicity control cells (with and without mTHPC). 
		Note: Always wear glasses to protect the eyes from the laser light used for excitation of the PS.</li>
	</ol>
	</li>
	<li>After illumination, keep the cells in the dark at 37 &#186;C for 24 hr. Subsequently, add water soluble tetrazolium (WST-1) reagent (10 &#181;l/100 &#181;l of medium).</li>
	<li>Three hours after the addition of the WST-1 reagent, measure the absorbance of individual wells of the 96-well plate at 415 nm.</li>
	<li>In order to calculate the percentage of surviving cells, set the mean of the values obtained for the non mTHPC treated cells for each condition to 100% (i.e.&#160;illuminated, without mTHPC and nonilluminated, without mTHPC).</li>
</ol>
3.&#160;Estimation of Cell Number by Cell Counting
<ol>
	<li>Seed 20,000 cells/well in a 24-well plate and let them adhere overnight.</li>
	<li>Incubate the cells for 5 hr with mTHPC (0.001, 0.003, 0.15, 0.6, and 1.25 &#181;g/ml) and illuminate them as described above.</li>
	<li>Collect the medium, wash the cells once with PBS, trypsinize them and collect them by centrifugation at 400 x g for 5 min.</li>
	<li>After centrifugation, aspirate the medium and resuspend the cells in 200 &#181;l of fresh medium.</li>
	<li>Count the cells in a Neubauer chamber.</li>
</ol>

<ol><li>Juarranz, A., Jaen, P., Sanz-Rodriguez, F., Cuevas, J., Gonzalez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photodynamic+therapy+of+cancer.+Basic+principles+and+applications%5BTitle%5D)+AND+%22Clin.+Transl.+Oncol%22%5BJournal%5D)">Photodynamic therapy of cancer. Basic principles and applications.</a> Clin. Transl. Oncol. 10, 148-154 (2008).</li>
<li>Agostinis, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photodynamic+therapy+of+cancer%3A+an+update%5BTitle%5D)+AND+%22CA+Cancer%22%5BJournal%5D)">Photodynamic therapy of cancer: an update.</a> CA Cancer. 61, 250-281 (2011).</li>
<li>D'Adamo, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Appraising+the+current+role+of+chemotherapy+for+the+treatment+of+sarcoma%5BTitle%5D)+AND+%22Semin.+Oncol%22%5BJournal%5D)">Appraising the current role of chemotherapy for the treatment of sarcoma.</a> Semin. Oncol. 38 Suppl 3, 19-29 (2011).</li>
<li>Allison, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+meta-analysis+of+osteosarcoma+outcomes+in+the+modern+medical+era%5BTitle%5D)+AND+%22Sarcoma%22%5BJournal%5D)">A meta-analysis of osteosarcoma outcomes in the modern medical era.</a> Sarcoma. , (2012).</li>
<li>Anninga, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemotherapeutic+adjuvant+treatment+for+osteosarcoma%3A+where+do+we+stand%5BTitle%5D)+AND+%22Eur.+J.+Cancer%22%5BJournal%5D)">Chemotherapeutic adjuvant treatment for osteosarcoma: where do we stand.</a> Eur. J. Cancer. 47, 2431-2445 (2011).</li>
<li>Bacci, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pattern+of+relapse+in+patients+with+osteosarcoma+of+the+extremities+treated+with+neoadjuvant+chemotherapy%5BTitle%5D)+AND+%22Eur.+J.+Cancer%22%5BJournal%5D)">Pattern of relapse in patients with osteosarcoma of the extremities treated with neoadjuvant chemotherapy.</a> Eur. J. Cancer. 37, 32-38 (2001).</li>
<li>Briccoli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resection+of+recurrent+pulmonary+metastases+in+patients+with+osteosarcoma%5BTitle%5D)+AND+%22Cancer%22%5BJournal%5D)">Resection of recurrent pulmonary metastases in patients with osteosarcoma.</a> Cancer. 104, 1721-1725 (2005).</li>
<li>Milla Sanabria, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+and+indirect+photodynamic+therapy+effects+on+the+cellular+and+molecular+components+of+the+tumor+microenvironment%5BTitle%5D)+AND+%22Biochim.+Biophys.+Acta%22%5BJournal%5D)">Direct and indirect photodynamic therapy effects on the cellular and molecular components of the tumor microenvironment.</a> Biochim. Biophys. Acta. 1835, 36-45 (2013).</li>
<li>Mitra, S., Foster, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photophysical+parameters%2C+photosensitizer+retention+and+tissue+optical+properties+completely+account+for+the+higher+photodynamic+efficacy+of+meso-tetra-hydroxyphenyl-chlorin+vs+Photofrin%5BTitle%5D)+AND+%22Photochem.+Photobiol%22%5BJournal%5D)">Photophysical parameters, photosensitizer retention and tissue optical properties completely account for the higher photodynamic efficacy of meso-tetra-hydroxyphenyl-chlorin vs Photofrin.</a> Photochem. Photobiol. 81, 849-859 (2005).</li>
<li>Ma, L., Moan, J., Berg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+a+new+photosensitizer%2C+meso-tetra-hydroxyphenyl-chlorin%2C+for+use+in+photodynamic+therapy%3A+a+comparison+of+its+photobiological+properties+with+those+of+two+other+photosensitizers%5BTitle%5D)+AND+%22Int.+J.+Cancer%22%5BJournal%5D)">Evaluation of a new photosensitizer, meso-tetra-hydroxyphenyl-chlorin, for use in photodynamic therapy: a comparison of its photobiological properties with those of two other photosensitizers.</a> Int. J. Cancer. 57, 883-888 (1994).</li>
<li>Reidy, K., Campanile, C., Muff, R., Born, W., Fuchs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mTHPC-mediated+photodynamic+therapy+is+effective+in+the+metastatic+human+143B+osteosarcoma+cells%5BTitle%5D)+AND+%22Photochem.+Photobiol%22%5BJournal%5D)">mTHPC-mediated photodynamic therapy is effective in the metastatic human 143B osteosarcoma cells.</a> Photochem. Photobiol. 88, 721-727 (2012).</li>
<li>Triesscheijn, M., Ruevekamp, M., Aalders, M., Baas, P., Stewart, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+sensitivity+of+microvascular+endothelial+cells%2C+fibroblasts+and+tumor+cells+after+in+vitro+photodynamic+therapy+with+meso-tetra-hydroxyphenyl-chlorin%5BTitle%5D)+AND+%22Photochem.+Photobiol%22%5BJournal%5D)">Comparative sensitivity of microvascular endothelial cells, fibroblasts and tumor cells after in vitro photodynamic therapy with meso-tetra-hydroxyphenyl-chlorin.</a> Photochem. Photobiol. 80, 236-241 (2004).</li>
<li>Triesscheijn, M., Baas, P., Schellens, J. H., Stewart, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photodynamic+therapy+in+oncology%5BTitle%5D)+AND+%22Oncologist%22%5BJournal%5D)">Photodynamic therapy in oncology.</a> Oncologist. 11, 1034-1044 (2006).</li>
<li>Burch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+of+canine+osseous+tumors+with+photodynamic+therapy%3A+a+pilot+study%5BTitle%5D)+AND+%22Clin.+Orthop.+Relat.+Res%22%5BJournal%5D)">Treatment of canine osseous tumors with photodynamic therapy: a pilot study.</a> Clin. Orthop. Relat. Res. 467, 1028-1034 (2009).</li>
</ol>

The authors declare&#160;no competing financial interests or conflicts of interest.

To achieve optimal cytotoxicity in response to PDT, it is crucial to choose the right laser light settings and incubation times. The here described procedures are consistent and efficient to determine PS uptake and to quantify PDT induced cytotoxicity in vitro. Using the specific absorption wavelengths of the PS mTHPC, the cellular PS uptake can be determined in a direct manner, and the PS can be activated to generate cytotoxic reactive oxygen species.
Using this in vitro set...

Today&#8217;s state of the art treatment of osteosarcoma (OS), a primary bone tumor, encompasses a combination of neo adjuvant chemotherapy and surgery. This treatment regimen revealed an increase in the survival rate of patients with localized disease from approximately 20% before the use of chemotherapy, to currently between 60-70%3,4. However, in the last two decades, the overall survival of OS patients with local disease has plateaued4,5. Moreover, 30-40% of these patients relapse within 3 years after diagnosis and patients with metastatic disease continue to have a poor survival of 20-30%4,6,7. To improve the outcome of these patients, new therapeutic strategies need to be developed.
Photodynamic Therapy (PDT), a rather novel anticancer therapy, uses light of a specific wavelength for excitation of a photosensitizer (PS), which accumulates in the tumor cells after its injection into the bloodstream. Laser light excitation of the PS generates oxygen radicals in the presence of oxygen, which induce cytotoxic reaction in tumor cells and cell death. Besides this primary mechanism, two additional PDT evoked biological processes contribute to reduced tumor growth: PDT causes vasoconstriction and thrombus formation of the tumor microvasculature and, consequently local hypoxia and anoxia inside the tumor, leading to tumor infarction. Finally, PDT injured and dying tumor cells trigger a local immune response, a rather unique feature of PDT. This involves the complement system and the activation of antigen presenting dendritic cells8. Thus, conditions are created for the presentation of tumor antigens with subsequent activation of lymphoid cells, leading to tumor specific immunity.
So far, PDT has been used to treat several types of soft tissue tumors and hyperplasia&#8217;s, such as actinic keratosis, Barrett&#8217;s esophagus, endobronchial tumors, bladder cancer, basal cell carcinomas, and palliative treatment of head and neck cancer2. The treatment is known to induce local, large scale necrosis with only little side effects, and thus has the potential to selectively eradicate tumor tissue. Despite these advantages, the application of PDT remains technically more demanding than the administration of chemotherapeutic drugs. In order to achieve maximal efficacy, the PS concentration, light exposure time and total light energy transfer need to be optimized. This can be done in in vivo experiments, but, because of the relative large number of parameters that need to be optimized, it is more efficient to initially determine optimal conditions in vitro.
In the experiments described below, we tested the in vitro efficacy of PDT using the PS 5,10,15,20-tetrakis(meta-hydroxyphenyl)chlorin, abbreviated mTHPC (Figure 1A). mTHPC is the active substance in the medicinal product Foscan, which is currently used in the clinic for palliative treatment of head and neck cancer. It is one of the most potent PS, inducing massive cell damage already at low concentrations, and it was demonstrated to be superior to other PS in terms of tissue penetration9,10. Its light absorption spectrum (Figure 1B) shows two prominent peaks, one at 417 nm and a second at 652 nm, which are used for tissue localization of accumulating PS and for PDT induction, respectively.
Currently, a liposomal formulation for mTHPC is under development. Here, we describe the procedures to quantify the uptake of this liposomal formulation, and to perform PDT in two human OS cell lines; the low metastatic HOS and the high metastatic 143B cells. Some of the data presented here have been reported earlier11. The approach described here enables us to study the effect of a metastatic phenotype on PDT efficacy. 143B cells, orthotopically injected into the hind limbs of immune deficient SCID mice cause intratibial metastasizing primary tumors, a model closely mimicking the human metastasizing disease. Thus, the proposed in vitro experiments are perfectly suitable to assess the optimal PDT settings to be later used in in vivo experiments.

<table><tbody><tr><td>DMEM</td><td>PAA GmbH, Freiburg, Germany</td><td>E15-883</td><td></td></tr><tr><td>HAM F12</td><td>PAA GmbH, Freiburg, Germany</td><td>E15-817</td><td></td></tr><tr><td>Heat-inactivated fetal calf serum</td><td>GIBCO, Basel, Switzerland</td><td>10500-064</td><td></td></tr><tr><td>mTHPC</td><td>biolitec Research GmbH, Jena, Germany</td><td></td><td>As this liposomal formulation is originating from R&amp;amp;D&lt;br/&gt;no catalogue number is available at the moment. Stock: 1.5 mg/ml; provided in a 9:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG; &gt;99% purity)</td></tr><tr><td>Spectramax Gemini XS plate reader</td><td>Molecular Devices, Sunnyvale, CA</td><td>ID# 861</td><td></td></tr><tr><td>Microscope Zeiss Observer.Z1</td><td>Axio Observer, Axio Vision Release 4.6.3 SP1, Jena Germany</td><td></td><td></td></tr><tr><td>Ceralas PDT 652 nm Laser</td><td>biolitec AG, Jena, Germany</td><td>LD652nm2W400u</td><td></td></tr><tr><td>Water-soluble tetrazolium (WST) reagent</td><td>Roche Diagnostics AG, Rotkreuz, Switzerland</td><td>1644807</td><td></td></tr></tbody></table>

cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro

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.

With the here reported techniques, we investigated mTHPC-based PDT in human OS cells. First, the time  and dose dependent uptake of mTHPC was investigated in the low metastatic HOS and in the highly metastatic 143B OS cell lines. mTHPC uptake can be assessed by measuring the fluorescence of mTHPC with a fluorescence spectrophotometer (Figure 2, reproduced with permission from Reidy et al.11). Figure 2A illustrates the uptake of mTHPC in a time dependent manner. The fl...

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 ...

The overall goal of the following experiment is to show the cytotoxic efficacy of photodynamic therapy in osteosarcoma cells. This is achieved by first measuring the intracellular uptake of the photosensitizer M-T-H-P-C by osteosarcoma cells. In the second step, laser light is applied to the cells to activate the M-T-H-P-C and to produce cytotoxic oxygen radicals.Finally, cell metabolism and cell number are measured by a water soluble tetra oleum one or WST one assay. Ultimately, the WST one assay, as well as direct cell counting, can be used to measure the cytotoxicity of the photodynamic therapy on the osteosarcoma cells. The implications of this technique extend towards the therapy of osteosarcoma as it allows the measurement of photodynamic therapy efficacy in vitro and resembles the technique currently used in patients.To compare the M-T-H-P-C uptake between osteosarcoma cell lines begin by plating 0.2 times 10 to the sixth Hoss and 143 B cells per well. In triplicate in six well plates let the cells adhere overnight to an 80 to 90%co fluency, and then on the following day, incubate the cells with a fixed concentration of M-T-H-P-C for different time periods in the dark to evaluate the time dependent uptake of the photosensitizer by the cells. After the incubations, aspirate the media and wash the cells three times with PBS, then detach the cells with 0.5 milliliters of tripsin EDTA and count them.Next, spin down the cells for five minutes at 400 times G and room temperature. Aspirate the medium and resuspend the pellet in PBS to a final density of two times 10 to the fifth cells per milliliter. Pipette 100 microliters of the cell suspension into each well of a 96.Well plate and measure the fluorescence of the cells in a fluorescence spectrophotometer set to 417 nanometers excitation, and 652 nanometers emission. Then prepare a standard curve by plating 100 microliters of zero to four milligrams per milliliter concentrations of M-T-H-P-C in triplicate. In a 96 well plate and measure the fluorescence as just demonstrated to generate a standard curve for calculating the relative fluorescence unit of each cell containing well from the previously measured plate.To measure the phototoxicity of the photo sensitizer first seed 3000 cells per well in triplicate per each M-T-H-P-C concentration in a 96 well plate, including dark toxicity control cells for each PHOTOSENSITIZER concentration. Let the cells adhere overnight to a 50 to 60%co fluency the following day. Incubate the cells with a range of M-T-H-P-C concentrations in the dark for five hours following the incubation.Aspirate the medium and wash the cells twice with PBS and add fresh media without M-T-H-P-C then specific for the M-T-H-P-C absorption spectrum, wearing proper eye protection. Set a 652 nanometer diode laser to a 21.88 milliwatts per square centimeter power at a height of 13.5 centimeters. Distance from the cells to get an energy dose of five joules per square centimeter.Then illuminate the cells except for the dark toxicity control cells for 230 seconds after the illumination. Keep the cells in the dark at 37 degrees Celsius for 24 hours. Subsequently add 10 microliters of wst, one reagent per 100 microliters of medium three hours after addition of the wst one reagent measure the absorbance of the individual wells at 415 nanometers.Finally, calculate the number of surviving cells in addition to the WST one assay. Direct counting of the cells is a second method for measuring cell toxicity. To calculate the percentage of viable cells, seed two times 10 to the fourth cells per well in 2 24 well plates and let them adhere overnight.The next day incubate the cells for five hours in the dark with M-T-H-P-C and illuminate them as just demonstrated. Keep dark control cells in the dark after 24 hours, collect the supernatant, wash the cells once with PBS and trypsin eyes them finally spin down the cells and resuspend the pellet in 200 microliters of fresh medium for counting. The results illustrated in this graph show in the top panel the uptake of M-T-H-P-C in a time dependent manner with a fluorescence intensity of the cell suspension representing the intracellular levels of the M-T-H-P-C.In the bottom panel. The dose dependent M-T-H-P-C uptake can be assessed by measuring the fluorescence of M-T-H-P-C with a fluorescence spectrophotometer. This graph illustrates the uptake of a fixed concentration of M-T-H-P-C in a time dependent manner with the fluorescence intensity of the cell suspension representing the intracellular levels of the M-T-H-P-C.In this graph, the dose dependent M-T-H-P-C uptake in Haass and 143 B cells after a five hour incubation is demonstrated. The uptake in the high metastatic cell line 143 B was significantly higher than in the low metastatic parental host cell line. The dose dependent dark toxicity of the osteosarcoma cells is demonstrated.143 B cells were treated with different M-T-H-P-C dosages and compared to untreated cells and a dose dependent decrease of the metabolic activity and cell number measured by wst one assay was observed. Decreased cell viability was recognized at an M-T-H-P-C concentration equal to and higher than 2.5 micrograms per milliliter. In the bottom graph, the dose dependent toxicity of M-T-H-P-C is illustrated.The cells were treated with different M-T-H-P-C doses and subsequent illumination at five joules per square centimeter. A consequential decrease in cell metabolic activity as well as a decrease in cell number was observed. The development of this technique paved the way for researchers in the field of cancer biology to apply this methods to in vivo models of osteosarcoma as well as for other preclinical investigations.

cytotoxic-efficacy-photodynamic-therapy-osteosarcoma-cells

Research

JoVE Journal

Medicine

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.

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

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.

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

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

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

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

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.

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

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.

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

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Learn photodynamic therapy cytotoxicity in osteosarcoma cells using M-T-H-P-C photosensitizer and laser activation. This method measures cell viability via WST-1 assay to evaluate therapeutic potential.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

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