Cisplatin

XTT viability assays were performed as described previously [26 (link)]. Briefly, 1–3 × 10³ cells were plated onto 96‐well plates before treatment with cis‐diamminedichloroplatinum‐II (Cisplatin; Accord Healthcare, London, UK) for up to 96 h. Regarding the effect of conditioned media on Cisplatin sensitivity, GCT cells were pretreated with CM from TM cells for 24 h before Cisplatin treatment. Every day viability was screened by adding 50 μL 2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐5‐[(phenylamino)carbonyl]‐2H‐tetrazolium (XTT; 1 mg· mL⁻¹;neoLab Migge GmbH, Heidelberg, Germany) and 0.5 μL N‐methyl dibenzopyrazine methyl sulfate (PMS; 1.25 mm; Sigma‐Aldrich) and measuring absorbance 4 h later in a UV/VIS spectrometer (450 nm vs. 650 nm, iMark Microplate Absorbance Reader, BioRad, Feldkirchen, Germany). Each time point/concentration was measured in quadruplicates. LD₅₀ doses were calculated using graphpad prism software version 8 (GraphPad Software, San Diego, CA, USA).

The human UC cell lines (UCCs) RT-112 and T-24 were obtained from the DSMZ (ACC 418, ACC 376), whereas 253J and J82 cell lines were kindly provided by J. Fogh (New York, USA). Parental UCCs and their long-term cisplatin-treated sublines were grown in DMEM GlutaMAX-I (Gibco, Darmstadt, Germany) containing 10% heat-inactivated fetal bovine serum. Long-term cisplatin-treated sublines (LTTs) were established by continuously escalating cisplatin (Accord Healthcare, Muenchen, Germany) dosages over several months until stable resistant cell lines could be maintained at doses of 50 μM (RT-112-LTT), 3.3 μM (J82-LTT), 6.6 μM (253J-LTT), and 23 μM (T-24-LTT) as described previously^{5 (link)}.