*4.10. Animal Studies*

All animal experiment procedures were approved by the Kyung Hee University Institutional Animal Care and Use Committee (KHSASP-20-250). Five-week-old male BALB/C nu/nu mice were obtained from Raonbio (Seoul, Republic of Korea). The mice were maintained for 1 week under controlled temperature (23 ± 3 ◦C) and humidity (55 ± 15%) in a 12 h light/12 h dark cycle before initiating the experiment. Then, H460 cells were harvested and injected subcutaneously (5.0 × <sup>10</sup><sup>6</sup> cells in 100 <sup>μ</sup>L 1:1 PBS:Matrigel solution) into the right flank of the mice. When the xenografts reached a volume of 80–100 mm3, the animals were randomized into 2 groups (*n* = 6): the control group (saline) and the JI017 treatment group (500 mg/kg). The animals in the JI017 treatment group were treated with JI017 by oral administration every 2 days. Tumor sizes were measured every 2 days for changes in tumor growth, and tumor volumes were calculated using a standard formula: 1/2 (length × width2). Mice were sacrificed using carbon dioxide, followed by cervical dislocation, and the tumor tissue was isolated for further study, such as immunohistochemical (IHC) analyses.
