Methods

Cell Culture Conditions

The human alveolar rhabdomyosarcoma  cell line RH-30 (No. ACC-489, DSMZ, Braunschweig, Germany), which has a positive PAX3-FKHR fusion status , was used. Tumor cells were cultured in Dulbecco’s modified Eagle’s medium plus Ultraglutamine 1 (Lonza, Verviers, Belgium) supplemented with 10% fetal calf serum (FCS) (PAN Biotech GmbH, Aidenbach, Germany) and 1% antibiotic-antimycotic solution (Gibco, Paisley, UK). All cells were mycoplasma negative and kept under humidified conditions at 37 °C and 5% CO2 atmosphere.

Rhabdomyosarcoma xenograft model

Eighty NOD/LtSz-scid IL2Rγnullmice, with an age of 8-11 weeks and a weight of 25-30 g, were used. This immunodeficient mouse strain was initially sourced from Jackson Laboratories (Bar Harbor, ME, USA) and further obtained from own animal facility. Animals were kept under specific pathogen-free conditions, fed an autoclaved standard diet and given free access to sterilized water. The animal experiments were approved by the government ethics committee for animal studies (Regierungspräsidium Gießen, V54 – 19 c 20 15 h 01 MR 20/14, No. G38/2017). The RMS xenotransplantation procedure was described previously . Briefly, RMS tumor induction was carried out by intraperitoneal (i.p.) injection into the lower left flank of the animals of a total number of 2 x 106 RH-30 tumor cells solved in a 0.9% sterile sodium chloride solution (Ecolav® B.Braun, Melsungen, Germany).