2.4. Drug Application and Subsequent Quantification of Treatment Effects and Substrate Release

Cells were harvested and counted as described above. We seeded 5000–10,000 cells per well in 96-well plates (Corning Costar, #3903). The next day, the medium was replaced and the ADAM10 inhibitor GI254023X (Aobious, #3611; 3 µM) or the ADAM10/ADAM17 inhibitor GW280264X (Aobious, #3632; 3 µM) was added. This fixed concentration was recommended as a standard concentration for metalloprotease inhibition avoiding off-target-effects as reported by [13,30,31,32,33,34]. As these inhibitors were solved in DMSO, the same amount of DMSO was used as the solvent control in all experiments. Two hours later, different concentrations of cisplatin (obtained from the Clinic Pharmacy Services, UKSH, Campus Kiel) at a concentration of 1 mM dissolved in 0.9% NaCl or 0.9% NaCl used as solvent control were added. Cells were treated in three replicate wells. After 48 h of incubation, supernatants were stored at −20 °C for subsequent enzyme-linked immunosorbent assay (ELISA) quantification. Next, the Multiplex Assay ApoLive-Glo^TM (Promega, #G6411), which combines detection of viable cells and caspase3/7 activity, was performed as described in the manufacturer’s instructions (TM325). Cell viability was measured as relative fluorescence units using the following excitation sources (Ex) and emission filters (Em) (RFU, 400Ex/505Em) and caspase3/7 cleavage as relative luminescence units (RLU) using the Caspase-Glo^® 3/7 Assay System. Both assays were quantified with a microplate-reader (Infinite 200, Tecan, Männedorf, Switzerland). To facilitate comparability to 3D assays, viability and caspase are displayed separately and caspase activity was not normalized to the viability. The means of three separate experiments were plotted as a dose–response curve using four parameter logistic regressions in GraphPad Prism (GraphPad Software, Inc, San Diego, CA, USA). The half minimal (50%) inhibitory concentration (IC₅₀) (viability) or the half maximal effective concentration (EC₅₀) (caspase3/7) value was calculated and analyzed as described in the statistical section.

Cells were seeded with the same cell numbers as described above in ULA black-transparent 96-well plates (Corning, #4520), and imaged daily using NYONE^® Scientific (SYNENTEC) to monitor and analyze spheroid formation over time. As the spheroid density may vary between different cell lines, three different focal offsets were used to ensure each spheroid could be captured in focus. On day four, the diameter of spheroids before treatment was calculated using the spheroid quantification v 0.9 (1 channel) application in YT^®-Software. Afterward, treatment was performed. We carefully aspirated 150 µL medium using a multichannel pipette to leave 50 µL remaining volume, including the spheroid in the plate. We added 50 µL fresh medium. Master mixes of inhibitors or solvent control DMSO and, second, master mixes of cisplatin with NaCl were prepared in medium to reach a final volume of 220 µL/well. The ADAM10 inhibitor GI254023X (Aobious, #3611) or the ADAM10/ADAM17 inhibitor GW280264X (Aobious, #3632) was prepared with a final concentration of 3 µM or the same amount of DMSO as the control. After 2 h of incubation, cisplatin concentrations of 0.1–100 µM were added to the wells in triplicates. As a negative control, the equivalent volume of NaCl was used. The next day, CellTox^TM Green cytotoxicity (Promega #G8743) stain was added in a final dilution of 1:2000 and imaged 3 h later using NYONE^® Scientific and the following settings: brightfield (Ex: BF, Em: Green (530/43 nm)), CellTox^TM Green (Ex: Blue (475/28 nm); Em: Green (530/43 nm)). For cytotoxicity quantification, the CellTox^TM Green signal within the spheroid was analyzed using the spheroid quantification (1F) (v. 0.9) (2 channel) application in YT-Software^® using the result “Average fluorescence intensity CH1 BC [1]”. Following a second CellTox™ Green Cytotoxicity Assay measurement in NYONE^® 48 h after treatment, endpoint viability and caspase3/7 measurements were performed using two different luminescent assays. For quantification of viability, the RealTime-Glo™ MT Cell Viability Assay (Promega, #G9712) assay was applied and quantified 1 h after incubation at 37 °C using Infinite 200 (Tecan). Next, a Caspase-Glo^® 3/7 Assay System (Promega, #G8093) was used at RT, and measured 1 and 2 h after assay application because, for some spheroids, the dynamic range was higher after 2 h, probably due to penetration issues, whereas for others, 1 h was sufficient. The following luminescence filter and integration times were used: for viability, Filter 1 (BLUE2_NB–5000 ms); for caspase measurement, Filter 2 RED_NB–5000 ms) (Infinite 200, Tecan). The means of three separate experiments were plotted as a dose–response curve using four parameter logistic regressions in GraphPad Prism 9, and IC₅₀ or EC₅₀ values were calculated (GraphPad Software, Inc.). Supernatants of these experiments were stored at –20 °C for subsequent ELISA experiments.

Cell culture supernatants of 2D or 3D cell cultures 48 h after treatment were collected to investigate substrate release. The concentration of HB-EGF was measured as a surrogate for ADAM17 activity. Therefore, the Human HB-EGF DuoSet ELISA (R&D Systems, #DY259B) was used according to the manufacturer’s instructions in NUNC-IMMUNO plates (Thermo Scientific, #442404, Waltham, MA, USA). Using a microplate reader (Infinite 200, Tecan), the optical density (OD) was measured at 450 nm, and HB-EGF concentrations were calculated using MS Excel (2010). The means of three independent experiments + standard error of the mean (SEM) were determined using GraphPad Prism 9 (GraphPad Software, Inc., San Diego, CA, USA).