2.3. Tumor Establishment and Treatments

344SQ-P tumors were subcutaneously established in the right and left hind legs of 129Sv/Ev mice, 4 d apart, to establish primary (0.5 × 10⁶) and secondary (0.1 × 10⁶) tumors, respectively. Both primary and secondary tumors were measured twice per week using digital calipers. When primary tumors reached around 7 mm in diameter, they were locally irradiated using a cesium source (36Gy total, divided into 3 fractions of 12Gy each). For the RadScopal experiments, secondary tumors were also irradiated with L-XRT, 3 d after the last fraction of H-XRT to a dose of 2Gy total, divided over 2 fractions of 1Gy each. Anti-TIGIT and anti-PD1 were given on Days 5, 9, 12, 16, and 20 at 200 µg/i.p. injection. According to our protocol, mice were euthanized when the average tumor diameter reached 14 mm. Wherever specified, lungs were collected, stained with Bouin’s fixative solution, then enumerated for lung metastases.