Xenograft tumor formation assay

CL1-5 cells (1×10⁶) were subcutaneously injected into the right flanks of 6-week-old nude mice, followed by intraperitoneal pemetrexed (50 mg/kg) every 4 days for two cycles. Tumors were collected 1 week after the final treatment. The isolated tumors were cut into small pieces, which were lysed with Radioimmunoprecipitation assay buffer (RIPA buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) containing complete protease inhibitor cocktail (Roche). Protein samples were subjected to immunoblotting with antibodies against PD-L1 (#13684, Cell Signaling) and anti-β-actin (Sigma-Aldrich), the latter being the internal control.