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).