Irinotecan

All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee, School of Basic Medical Sciences, Shandong University. All mice were housed in a pathogen-free animal facility. To evaluate the cancer cell colonization in lungs, 1 × 10⁶ cells in 100 μL PBS were injected into six-week-old male BALB/c nude mice (Beijing Vital River Laboratory Animal Technology, Cat# 401, Beijing, China) through the tail veins. To evaluate liver metastasis after splenic injection, 2 × 10⁶ cells in 100 μL PBS were injected into the distal spleen of seven-week-old female nude mice. 2 × 10⁶ cells in 50 μL PBS were injected into cecum of seven-week-old female nude mice to generate orthotopic tumors according to the published protocol [24 (link)].
For the in vivo chemoresistance model, 1 × 10⁶ cells in 100 μL PBS were subcutaneously injected into the flank of six-week-old male BALB/c nude mice. The tumors were measured every 3 days and tumor volumes were calculated by the formula: volume = width² × length/2. When the tumors became palpable (50–100 mm³), irinotecan (Cat# HY-16562, MedChemExpress, Shanghai, China) was administered by intraperitoneal injection at 40 mg/kg twice a week. After six injections, the mice were sacrificed and the tumors were collected and imaged.

Selumetinib, carboplatin, irinotecan, and temsirolimus were purchased from MedChem Express. JQ1 was kindly provided by Dr. James Bradner (Dana-Farber Cancer Institute, Boston, MA, USA). Drugs were resuspended in N-Methyl-2-pyrrolidone (NMP) to create a stock solution (Sigma Aldrich) and diluted in PTD buffer (30 % PEG-400; 5 % Tween 80; 65 % Dextrose water, D5W, Sigma Aldrich) before drug dosing.