Sirolimus

Sirolimus was purchased from MedChem Express, LLC (Princeton, NJ). The drug was dissolved in dimethyl sulfoxide (DMSO) at 50 μg/μL (55 mM) and stored in at −20 °C. This concentrated stock was diluted to a final concentration of 0.5 μg/μL in dH₂O immediately before use. Ketamine (50 mg/mL), xylazine (suspended in methanol at 10 mg/mL), and remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Ketamine-xylazine solution consisted of 80 μL (4 mg) Ketamine plus 20 μL (0.2 mg) xylazine and stored at 4 °C for a maximum of 2 weeks. The solution was diluted 1:10 with dH₂O immediately prior to use. Mice were injected intraperitoneally with 10 μL/g (40 μg/g Ketamine +2 μg/g xylazine) before inoculation and other surgical procedures. Adequate sedation was confirmed before intranasal inoculations by the absence of footpad reflexes. Mice were held in a supine position (at a 45° angle) with the back supported by the palm and the neck skin fold by the thumb and index finger. The inoculum was slowly released from a 10-μL micropipette as two small drops covering the two nostrils. The mice were allowed to inhale the volume without forming bubbles. They were then maintained in the same position until they regained consciousness and their rapid breathing returned to normal.

PSOs were cultured in 96-well plates, with 2-3 PSOs per well. Eight wells constituted each test group, and each test group was treated with various chemotherapeutic drugs or targeted drugs, such as paclitaxel (A71384, Innochem, China), sirolimus (53123-88-9, MedChemExpress, USA), oxaliplatin (A27295, Innochem, China) and capecitabine (C124969, Aladdin, China). The drugs were administered at the respective clinical-relevant concentrations, as indicated in the figure legends, for a period of 5-7 days. Subsequently, Live/Dead cell double staining kit (KGAF001, KeygenBiotech, China) was used to stain the PSOs, per the manufacturer’s specifications. After staining, the PSOs were visualized using a fluorescence microscope (BDS400, Cnoptec, China) with 500-550nm excitation light. The green and red fluorescence emissions were isolated and analyzed separately. The mean fluorescence intensities of each group were then quantified using ImageJ software (version v1.53h, NIH).