Irinotecan

For the tumor drug treatment assay, 2 × 10⁶ cells per mouse were subcutaneously injected into the right dorsal flanks of female BALB/c athymic nude mice (4–6 weeks of age, 18–20 g), which were obtained from the Animal Center of Southern Medical University, Guangzhou, China). The tumor-bearing mice were observed until the tumor volume reached approximately 150 mm³, and the mice were randomly grouped. Mice in the two groups were intraperitoneally injected with 20 mg/kg irinotecan (Selleck, S2217) or DMSO three times per week. The tumor diameters were measured twice a week. Approximately 50 days later, the tumors were excised and placed in 10% neutral buffered formalin for 24 h. For flow cytometry, the indicated cells were seeded into plates with the medium as mentioned above, and 5 μg/ml irinotecan (Selleck, S2217) or PBS was added when the cells adhered to the plates. After 48 h, the cells were detected by flow cytometry according to the manufacturer’s instructions. For tumor sphere formation analysis, a mixture culture medium was prepared as mentioned above and an additional 5 μg/ml irinotecan (Selleck, S2217) or PBS was added. Then, the cells were cultured in these medium in an incubator at 37 °C and 5% CO² with saturated humidity for 12–14 days.

Doxorubicin hydrochloride (#D1515), vincristine, and insulin growth factor 2 (IGF2, #I2526) were purchased from Merck. Irinotecan (#S2217), MK-2206, capivasertib (AZD5363), alpelisib (HY-15244), nilotinib (S1033), pazopanib (S3012), and ponatinib (S1490) were purchased from Selleckchem. Everolimus (RAD001, SRP020750e) was purchased from Sequoia Research Products, etoposide was purchased from Sandoz and AVE 1642 from Immunogen. D-188514, an ifosfamide analog that does not require metabolic activation, was purchased from Niomech. The PI3K/mTOR dual inhibitor NVP-BEZ235 was kindly provided by Novartis. Trabectedin (ET-743) was kindly provided by PharmaMar. Working dilutions of all drugs were prepared immediately before use.

Chemotherapy exposure was performed in a dose-response curve with 5-fluorouracil (5FU, Sigma)), oxaliplatin (OX, Selleck Chemicals), a 1:1 combination of 5FU and OX (OXF) or irinotecan (IR, Selleck Chemicals). Stocks were made in DMSO and stored at −20°C until use.