Electrochemotherapy in 3D Ocular Melanoma Spheroids using a Customized Electrode

1. Spheroid formation Use adherently growing cancer cell lines for spheroid formation. Provide all cell culture associated steps under sterile bench conditions. Prepare the standard complete culture medium as recommended for the cell lines of interest and warm to 37 °C using a water bath. NOTE: Here, the human conjunctival cell lines CM2005.1, CRMM1, CRMM2 as well as the uveal melanoma cell lines 92.1, OMM1and OMM2 were used and cultured in Ham F-12 medium containing 10% fetal bovine serum and RPMI containing 10% fetal bovine serum, respectively. Detach the cells of interest using standard enzymatic digestion methods (e.g., trypsin-EDTA solution). To determine the number of vital cells, resuspend 10 µL of the cell suspension in 10 µL of Trypan blue solution (1:1) and count the cells using a chamber (hemocytometer or an automated cell counter) within 5 min. Cells should be <90% of confluence and in good condition. To form spheroids, seed 5 x 103 cells per well with a total volume of 200 µL of complete culture medium in 96-well ultra-low attachment plates. Incubate the plate at 37 °C in a humidified atmosphere containing 5% CO2. NOTE: For effective inhibition of cellular attachment, use ultra-low attachment plates with a covalently bound hydrophilic, non-ionic, neutrally charged hydrogel on the surface. This hydrogel inhibits immortalization of cells and force them into a suspended state to build 3D spheroids. Check the spheroid formation using a microscope. NOTE: Some cell lines do not form round 3D tumor spheroids in the first passage. Passage the tumor sphere culture before they start to develop a dark center. Dissociate the tumor masses depending on the cell line after 4-10 days by trypsinization. Afterwards, seed the single cells in a fresh 96 ultra-low attachment plate. By repeating this procedure for several passages, cells will adapt to 3D culture. 2. Electrochemotherapy of the tumor spheroids As an assay control, use untreated spheroids and spheroids treated only with EP or bleomycin alone. Adjust the settings for the electroporator (pulse counts, pulse frequency, duration time and voltage; see Table of Materials). For melanoma cells, use 8 square wave electric pulses of 750 Volts/cm strength, 100 µs pulse duration, 5 Hz repetition frequency. At day 3 of spheroid culture, prepare fresh bleomycin sulfate solution in sterile PBS at a concentration of 5 µg/mL in a 15 mL tube. Exchange the cell culture medium of the spheroids by removing 200 µL of culture medium and refilling with 100 µL of fresh complete culture medium for each well. Add 100 µL of bleomycin sulfate solution to each well (final concentration 2.5 µg/mL). Add 100 µL of fresh medium to untreated controls and spheroids only treated with EP instead of bleomycin sulfate solution. Perform the electroporation. NOTE: The electrodes shown in Figure 1 were produced in the research workshop of the University of Halle-Wittenberg. They are made of stainless steel and fit the grip of the electroporator. The electrodes were designed for use in 96 well ultra-low attachment plates. Similar stainless-steel electrodes can be easily manufactured for alternative applications too. Sterilize electrodes with 70% ethanol and allow electrodes to dry. The diameter of the electrodes is 1 mm, the gap between the two electrodes is 4 mm, and the length of each electrode is 8 mm (Figure 1a). Place the electrodes at the bottom of the well, with the spheroids between the electrodes. This allows the ideal positioning of the spheroid between the two electrodes (Figure 1b). Shake the plate to allow the spheroids to move from the bottom of the well and be placed between the electrodes. The spheroid will only be between the electrodes for a couple of seconds. Start the electroporation with preferred settings. Replace the bleomycin sulfate solution in the well by exchanging 150 µL of medium with 150 µL of fresh medium. Incubate the cells for up to 10 days at 37 °C in a humidified atmosphere containing 5% CO2. If the untreated spheroids start to develop a dark center, terminate the experiment. Exchange 150 µL of the cell culture medium every 2-3 days. Thereby, avoid spheroid disruption by positioning the pipette tip at the edge region of the well, tilting the plate and slowly pipetting. 3. Determination of spheroid size Measure the spheroid size on the day of treatment (starting point) and 3-7 days following treatment using bright field microscopy by calculating the cross sectional area of the spheroids using ImageJ Fiji11. Take an image of the single spheroids with the scale bar. Use a magnification by which the whole spheroid is visible. For example, 5x magnification was used for CM2005.1. Save the image as a JPEG. Analyze the spheroid size by calculating the cross-sectional area using ImageJ/Fiji. Install the software (https://imagej.net/Fiji/Downloads). Start ImageJ/Fiji. Import the spheroid-image: File > Open. To set the scale, choose the line button of the toolbar and mark the line of the scale bar in the image by mouse click: Analyze > Set Scale. Using a known distance, fill in the distance of the scale bar. Change the Unit of length to µm. Click the field global to assign the settings for all images with the same magnification factor. Convert the image to 8-bit: Image > Type > 8-bit. Choose Image > Adjust > Auto Threshold (Select > Do not reset range). The spheroid is marked in red or black depending on the threshold marker chosen. Adjust the sliders so that the spheroid pixels turn red, but non-spheroid pixels do not change color. Close the Threshold dialog window without clicking any of the buttons. To calculate the cross-sectional area of the spheroid, choose Analyze > Set Measurements and click Area, Limit to Threshold and Display label. Close the Set Measurements dialog window by clicking OK. To show the results, choose Analyze > Analyze Particles and change the size (µm2) = 10000-infinity to remove noise. Click the button Display results and close the window by clicking OK. In the Results, the spheroids with the label and area are shown. By repeating the analyses of several spheroids, the results are listed in the Results window. To determine the relative treatment response of the ECT treated spheroids compared to the untreated and single treated controls, calculate the percentage changes of the cross-sectional area. 4. Determination of spheroid viability Measure the spheroid viability 3-7 days following treatment using a cell viability assay suitable for 3D cell cultures according to the manufacturer instructions. The assay needs to penetrate and lyse large spheroids to allow detection of viability by quantitation of ATP, which signals the presence of metabolically active cells. NOTE: In this experiment, a luminescence-based assay as a readout method was used. The viability of the ECT-treated spheroids was compared to controls seven days following treatment.