Combination Therapy for Tumor Suppression

Female BALB/c and female C57BL/6 mice (6–8 weeks old) were from The Jackson Laboratory. CT26 (~5 × 10⁵ cells) in RPMI or B16-F10 (2.5 × 10⁵ cells) in DMEM was subcutaneously injected into the right hind flank of 8- to 10-week-old mice. Treatment was initiated when tumors reached about 60–120 mm³ (CT26) or about 55–135 mm³ (B16-F10). For CT26 tumor irradiation, immobilized mice received 2 fractions of 2 Gy (6 mV photon energy, 2 cm field) on days 1–2. For B16 tumor irradiation, isoflurane-anesthetized mice received 2 fractions of 4 Gy using an image-guided Precision SmART+ (225 kV) with 10 mm collimator and Cu treatment filter. Mice received 75 mg/kg AZD6738 45–60 minutes before irradiation on days 1–2 and a third dose, approximately 18 hours later (CT26) or 24 hours later (B16), on day 3. Subsequent doses of AZD6738 for prolonged daily treatment experiments were every 24 hours thereafter. For FTY720 Q2Dx4 experiments, FTY720 was administered alongside AZ6738 before radiation on day 1, and again on days 3, 5, and 7. For anti–PD-L1 tumor response experiments, anti–PD-L1 was administered on days 7, 9, and 11. Tumors were measured at least twice weekly with calipers, and volumes calculated as volume = (length × width²)/2. The survival endpoint was designated as tumor volume >1,000 mm³.