Aprepitant

Naproxen (Tocris, 10 mg/kg), codeine (St. Martins Pharmacy, 7 or 30 mg/kg), sumatriptan (Tocris, 10 mg/kg), and gabapentin (Sigma, 30 mg/kg) were dissolved in physiological saline (Braun) and administered via a single i.p. injection. Phenytoin (Sigma, 15 mg/kg) and aprepitant (MedChemExpress, 20 mg/kg) were dissolved in PEG-400 (Sigma) and delivered via i.p. injections. Drug injection schedules are detailed in Figure 1A: Naproxen, sumatriptan, and aprepitant were administered once on the last testing day. Codeine was administered at the last testing day twice (1 h before testing and when testing was concluded–before the night cycle). Phenytoin was administered once daily over a 15 day period, starting with the first injection of oxaliplatin. gabapentin was injected once per day over a period of 5 consecutive days starting 2 days after CCI-surgery.

Human NSCLC cell lines NCI-H1975, NCI-H1944, NCI-H226, A549, HCC827 were purchased from American Type culture collection (ATCC, Manassas, VA, USA) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (ThermoFisher, Waltham, USA) and 1% penicillin/streptomycin solution (Sangon, Shanghai, China). The cells were cultured in a humidified incubator at 37 °C with 5% CO₂. EGFR genotyping was performed in each cell line according to the instruction of the manufacture (Yihe, Shanghai, China). Aprepitant was obtained from MedChemExpress (NJ, USA). Osimertinib and gefitinib were from Selleck Chemicals (Houston, TX, USA). All chemicals were dissolved in dimethyl sulfoxide at the concentration of 100 mM and aliquoted and stored at −20 °C until use. Human hemokinin-1 (hHK-1) peptide (TGKASQFFGLM-NH₂) and substance P (SP) (RPKPQQFFGLM-NH2) were synthesized by Fmoc solid-phase synthesis system as described before [28 (link)]. The molecular weight of the peptide was confirmed by ESI-TOF mass spectrometry. The purity of peptide was quantified to be >95% using reversed-phase HPLC by a C18 column as the solid phase and a H₂O:acetonitrile gradient as the liquid phase.

A stock solution of aprepitant (MedChemExpress, United States) at the concentration of 100 mM, was attained through dissolving the compound in sterile dimethyl sulfoxide (DMSO) (Sigma-Aldrich, United States). Then it was diluted to different concentrations (6.25–100 μM), so that the final concentration of DMSO in the culture medium did not exceed 0.1% in all the treatments.