Radiotherapy

All single-fraction RT treatments in this study were applied directly to established primary tumors when their volume reached the designated range (“small”: <100 mm³; “medium”: 100–400 mm³; “large”: 400–600 mm³) or exceeded 200 mm³. To perform RT, tumor-bearing mice were first anesthetized by ketamine (17.5 mg/ml, Henry Schein) and xylazine (2.5 mg/ml, Henry Schein), and were then placed in a customized jig with a lead holder such that only the primary tumor was exposed, followed by irradiation in an RS-2000 biological X-ray irradiator (Rad source technology, Suwanee, GA) with a dose rate of 1.2 Gy/min (160 kV, 25 mA) to reach 4 Gy, 8 Gy or 15 Gy. Mice were monitored post-RT for discomfort and change in tumor volume. Mice that had eradicated their tumors following RT were maintained for an additional 6 months to 1.5 years to monitor tumor recurrence and determine long-term survival rates. For RT combination modalities, primary tumors in WT mice prior to (<3 h) or immediately after IR were intratumorally (i.t.) administered 50 µg rat-anti-mouse CD47 (αCD47, clone miap301), or soluble murine SIRPα extracellular domain fusion protein (mSirpα.ex, also termed mSirpα.ex-Fc). Rat IgG isotype, and soluble human SIRPα extracellular domain fusion protein (hSirpα.ex)⁴⁷. To test RT combined with Sirpα^−/− macrophage infusion, macrophages derived from Sirpα^−/− mouse bone marrow (BMDM) were generated in vitro. Briefly, bone marrow cells were obtained by flushing both femur bones with 1-2 ml of ice-cold PBS. After lysis of RBC using RBC lysis buffer (0.42 g NH₄Cl, 0.05 g NaHCO₃, 0.05 g EDTA in 50 ml cell culture grade water), cells were then placed in culture with RPMI-1640 containing 10% FBS, 1% penicillin/streptomycin, and M-CSF (10 ng/ml, Peprotech) for 5–6 d to generate BMDMs. Prior to or after RT, Sirpα^−/− BMDM were adoptively transferred via i.t. (0.2–1 × 10⁴ cells/mm³ tumor volume) or i.v. (0.5–2 × 10⁷ cells/mouse) into tumor-bearing WT mice. In some cases, the recipient mice received the same Sirpα^−/− BMDM transfusion three days later. Biofluorescence imaging (IVIS Spectrum, Perkin Elmer) was used to trace the biodistribution of i.v. administered GFP-Sirpα^−/− BMDM in tumor-bearing mice. To determine critical immune populations underlying RT efficacy, depletion of intratumoral macrophages or T cells was performed in tumor-bearing Sirpα^−/− mice. To deplete intratumoral macrophages, clodronate-containing liposomes (Cl2MDA-liposomes) or rat-anti-mouse CSF1R antibodies (clone AFS98) were i.t. injected (100 μg/tumor) in 50 μl PBS using a 30 G Ultra-Fine Insulin Syringe (BD). To deplete CD8 or CD4 T cells, rat-anti-mouse CD8α (clone 2.43) or CD4 (clone GK1.5) antibodies were used (100 μg/tumor, i.t.). These liposome or antibody treatments were conducted twice (2×, 2 days and 3 h) prior to IR. Liposomes without clodronate and Rat IgG isotype were used as controls.