Icg 001

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Laboratory Animal Centre of Peking University Shenzhen Graduate School (permit YW; the permit from Tsinghua Shenzhen International Graduate School is “Ethical Development no. 37 (year 2019)”. Six-week-old male BALB/c athymic nude mice were grouped by simple randomization using a random number table method. The principle of sample size followed the “The ARRIVE Guidelines” [35 (link)]. Mice were subcutaneously injected with Capan-1-ctrl/TFF3 (1 × 10⁷ cells) or SW1990-shctrl/shTFF3 (5 × 10⁶ cells). For drug treatment, the mice were subcutaneously injected with SW1990 (5 × 10⁶ cells). Six xenograft-bearing mice were each randomized into the vehicle, single-drug, double-drug, or triple-drug groups, and were treated with intraperitoneal injections of vehicle (1% DMSO/10% PEG400 in normal saline), 20 mg/kg AMPC, 10 mg/kg ICG-001 (Selleck Chemicals, USA), or 5 mg/kg gemcitabine (Eli Lily, USA). Xenograft volume was calculated as previously described [33 (link)]. Immunohistochemical analysis of xenograft histology sections was performed as previously described [30 (link), 36 (link)].

Next, we evaluated the effect of WBC pathway inhibition in vitro. For these experiments, we used the HPV-positive HNSCC cell line SCC154. The Cal27, an HPV-negative HNSCC, cell line served as a control. They were both acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were regularly tested for mycoplasma contamination. Dulbecco’s modified eagle’s medium (DMEM), Penicillin/Streptomycin (P/S) and fetal calf serum (FBS) were obtained from Gibco (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were kept in a humidified environment at 37 °C and 5% CO₂ (Hera Cell 240, Heraeus Holding GmbH, Hanau, Germany) and cultivated in DMEM supplemented with 10% FBS and 1% P/S. Cells were split once per week by trypsinization. The inhibitor ICG-001 was acquired from Selleckchem (Selleckchem S2662, Houston, TX, USA). All in vitro experiments were performed at least three times. Mean values ± standard deviations (SD) were calculated and used for further analysis and graphical representation. Displayed graphical presentation show representative images.