2.7. In Vivo Tumor Treatment with a Combination of IRE and STING Agonist

The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) (IACUC Approval Number 180103). Seven-week-old C57BL/6 mice were purchased from Orient Bio (Gyeonggi-do, Korea). All experiments were conducted after one week of adaptation. Mice were housed in a breeding environment that maintained a 12 h night and day cycle, at a temperature of 20–24 °C and humidity of 44.5–51.8%.

To generate tumor models, LLC (5 × 10⁵ cells) were subcutaneously injected into C57BL/6 mice. During the treatment, tumor size and body weight were measured every two days. Tumor-bearing mice were randomized to either (1) Non-treated group (Control, n = 5), (2) IRE-treated group (IRE, n = 5), (3) STING agonist-treated group (STING, n = 5), or (4) IRE plus STING agonist-treated group (IRE/STING combination, n = 5). Intraperitoneal anesthesia was performed using the IACUC approved ketamine and xylazine cocktails.

The anesthetized mice were electroporated using a two-needle array electrode with a 3 mm spacing made of medical-grade stainless steel. When the tumor grew to a size of 3 mm (diameter), the needle electrode was inserted entirely into the center of the tumor and then electroporated. Electroporation was applied under the conditions determined in the in vitro cell experiments (voltage: 1000 V, pulse duration: 100 μs, pulses: 40). Following IRE according to a pre-planned schedule, the STING agonist (10 μg) was injected twice in the intra-tumoral route (Figure 4b).