4T1 syngeneic mouse model with resection and vaccination

We have previously developed and validated a mouse model of spontaneous metastasis and surgical resection of primary TNBC.36 37 At day 0, 1×10⁵ 4T1 cells in 100 µL (>98% viability) were injected orthotopically into the fourth mammary fat pad of BALB/c mice. Following tumor implantation, mice were monitored daily by palpation of the injection site, the volume of palpable tumors were measured by a Vernier caliper and the tumor volume was calculated via the equation (width²×length)/2. At days 12–14, a complete resection of the primary tumor was performed (tumor volume=75–80 mm³). During surgery, all mice were kept under anesthesia (3% induction, 1.5% maintenance of isoflurane with 2% O₂). For perioperative pain management, all mice were injected with 0.05 mg/kg of buprenorphine 1 hour before and 4 hours following surgery. Mice were randomized into different cohorts for treatment. At 1 day and 3 days following surgery, irradiated 4T1 cells, infected cell vaccine (4T1-ICV), VSVd51 alone or sterile PBS was injected subcutaneously into the cleared mammary fat pad. For the in vivo depletion of immune cell populations, 6 doses of depletion antibodies (1 dose 24 hours before surgery, followed by 5 additional doses 2–3 days apart) were administered by intraperitoneal injection at 20 µg/dose for anti-Asialo (GM1; Life Technologies) and 250 µg/dose for anti-CD8α (53-6.7; BioXCell). For anti-PD-1 treatment, mice were injected intraperitoneally with 1 dose of anti-PD-1 (RMP1-14; BioXCell) at 100 µg/dose, followed by 1 additional dose 3 days later.