Sarcoma cells are seeded in 96-well E-plates with integral sensor electrode arrays. After 24 hrs, the cells are treated with different RG7112 concentrations and cell proliferation is monitored noninvasively, in real-time and label-free using the xCELLigence Real-Time Cell Analyzer for up to 72 hours. The 96-well E-plates used with the xCELLigence system have gold electrodes on their bottom surface, serving as sensors of alternating current modified by the number of adherent cells and cell status (including cell viability, morphology, and adhesion). A dimensionless parameter termed cell index (CI) is derived as a relative change in measured electrical impedance to represent cell status. CI normalized to the time of medium switch/treatment is presented in xCELLigence recordings.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

For studies with hormone-dependent LNCaP xenografts, castrated male Balb/c nude are implanted with 12.5 mg sustained-release testosterone pellets 5 days before subcutaneous inoculation with 1×10⁷ cells suspended in 0.2 mL of Matrigel:PBS. Mice are randomized into treatment groups (n=10 per group) when mean tumor volume reaches approximately 150 to 400 mm³. Mice receive either vehicle (1% Klucel LF/0.1% Tween 80) or RG7112, administered as an oral suspension at the dose indicated (25-200 mg/kg). For assessment of androgen ablation treatment in combination with RG7112 in LNCaP xenograft-bearing mice, testosterone pellets are removed under ketamine/xylazine anesthesia. Tumor volume is monitored by caliper measurement and body weights are recorded 2 to 3 times weekly.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Obrador-Hevia A, et al. RG7112, a small-molecule inhibitor of MDM2, enhances trabectedin response in soft tissue sarcomas. Cancer Invest. 2015;33(9):440-50.

  [2]. Tovar C, et al. MDM2 small-molecule antagonist RG7112 activates p53 signaling and regresses human tumors in preclinical cancer models. Cancer Res. 2013 Apr 15;73(8):2587-97.

  [3]. Verreault M, et al. Preclinical Efficacy of the MDM2 Inhibitor RG7112 in MDM2-Amplified and TP53 Wild-type Glioblastomas. Clin Cancer Res. 2016 Mar 1;22(5):1185-96.

  [4]. Makii C, et al. MDM2 is a potential therapeutic target and prognostic factor for ovarian clear cell carcinomas with wild type TP53. Oncotarget. 2016 Nov 15;7(46):75328-75338.

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Room temperature in continental US; may vary elsewhere