225 The role of the MR 7 Real-time quantitative polymerase chain reaction (RTQ-PCR) RNA was isolated using TRIzol reagent (Thermo Fisher Scientific). Real-time quantitative polymerase chain reaction (RTQ-PCR) was performed as previously described.12 Expression levels were calculated relative to the expression of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) household gene. For normalized expression levels, the expression of non-doxycycline induced cells in the absence of steroid treatment was set to one. Primers used were GAPDH Fw primer 5′-GTCGGAGTCAACGGATT-3′, GAPDH Rev primer 5′-AAGCTTCCCGTTCTCAG-3′, NR3C1 Fw primer 5’-TGTTTTGCTCCTGATCTGA-3’, NR3C1 Rev primer 5’-TCGGGGAATTCAATACTCA-3’, NR3C2 Fw primer 5’-GAGCTGGCAGAGGTTCTA-3’, NR3C2 Rev primer 5’- CTGGTCGCTGATGATCTC-3’, BIM Fw primer: 5’-CGCCCAGAGATATGGAT-3’, BIM Rev primer: 5’-CGCAAAGAACCTGTCAAT-3’, GILZ Fw primer 5′-TGGCCATAGACAACAAGAT-3′, GILZ Rev primer 5′-TTGCCAGGGTCTTCAA-3′, FKBP5 Fw primer 5′-GAATGGTGAGGAAACGC-3′, FKPB5 Rev primer 5′-ATGCCTCCATCTTCAAATAA-3′. Antagonist approach To be able to distinguish between the cytotoxic effect of the GR and MR in primary patient samples, we assessed three MR antagonists (spironolactone, eplerenone and RU-28318). First, we performed a cytotoxicity (MTT) assay with fixed prednisolone concentrations (0.5879 µM for the NRC31 clone, 0.0245 µM for the NR3C2 clone) and increasing concentrations of antagonists. After establishing three effective antagonist doses, we performed an MTT assay with these doses and ascending prednisolone concentrations. Thereafter the best antagonist and optimal dose was determined and subsequently added in our MTT assays with different steroids in ascending concentrations. RU-28318 was the most effective MR antagonist and therefore used in the subsequent experiments (4 µM). Patient and PDX samples As proof of concept, we used one ALL patient-derived xenograft model (PDX) as well as two primary patient samples and treated cells with different steroid concentrations in combination with RU-28318. Since cells with an ETV6-RUNX1 gene fusion have a relatively high expression of both GR and MR,8 the used samples all harbored this gene fusion. Viability readout was performed by amino staining using FACS (viability staining: LIVE/DEAD Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitation (Cytoflex_S, Beckman Coulter)). Amino staining was performed in accordance with manufacture guidelines. Patient cells were first fixated using human telomerase reverse transcriptase mesenchymal stem cells (hTERT MSCs). Because of loss of cells after fixation, the percentage of live cells was used in the final viability calculations. Area under the curve (AUC) values were calculated to compare the in vitro cytotoxicity of prednisolone, dexamethasone and hydrocortisone in combination with RU-28318 (Prism software Version 9.3.0 from GraphPad).
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