All neuroblastoma cell lines were purchased from Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ). The cells were cultivated for not more than 40 passages at 37 °C in a humidified 5 % CO2 atmosphere. The cell line Kelly was cultured in RPMI 1640 (Gibco®, Cat. No. 11875093) supplemented with 15 % fetal bovine serum (FBS, Biochrom), 1% penicillin/streptomycin (Biochrom, Cat. No. A2212) and 2 mM L-glutamine (Gibco®, Cat. No. 25030081). The cell line IMR-32 was cultured in RPMI 1640 with supplemented with 20% FBS, 1% penicillin/streptomycin and 1% non-essential amino acids (MEM NEAA, Gibco®, Cat. No. 11140050). The cell line SK-N-BE(2) was cultured in EMEM (Lonza, Cat. No. 12-125F)/Ham´s F12 (Biochrom, Cat. No. F0815) 1:1 supplemented with 10% FBS, 1% penicillin/streptomycin and 2 mM L-glutamine. All cell lines were tested periodically for mycoplasma contamination.

AZD3147 (Cat. No. 5615), PP242 (Cat. No. 4257), Torin-1 (Cat. No. 4247), Torin-2 (Cat. No. 4248) and SN-38 (active metabolite of Irinotecan, Cat. No. 2684) were purchased from Tocris Bioscience. Rapamycin (Cat. No. R-5000) and Dasatinib (Cat. No. D-3307) were purchased from LC Laboratories. Temozolomide (TMZ) was purchased from BioVision (Cat. No. 2226-10). All drugs were dissolved in DMSO (Sigma, Cat. No. D2650) following the manufacturer´s instructions and stored at -20 °C. Cells were treated with indicated concentrations and for indicated time-periods. As control, cells were treated with appropriate DMSO concentrations. The applied drug concentrations for single or combination treatment are listed in Table Table33 and Table Table4,4, respectively. The treatment protocol has been performed as shown in Figure Figure1212.

In vitro protocol for the multimodal RIST, respectively TIST treatment. Pre-treatment with the drug combination Dasatinib + Rapamycin for RIST respectively Dasatinib + Torin-2 for TIST for 72 h. Thereafter the medium was changed and 24h later the drug combination of conventional chemotherapeutics Irinotecan (SN-38) + Temozolomide (TMZ) was applied for additional 72 h.

Drug concentrations for single treatment

Drug concentrations for combination treatment

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