Preclinical Mouse Models for Immunotherapy

Five to seven week old female C57BL/6 and BALB/c mice were obtained from NCI Production (Frederick, MD) and Jackson Laboratory (Bar Harbor, ME) and maintained under pathogen free conditions. All animal experiments were performed according to protocols approved by the Institute of Animal Care and Use Committee of the University of Pennsylvania. For B16-F10 melanoma, 5×10⁴ B16-F10 cells were mixed with an equal volume of Matrigel (BD Biosciences) and subcutaneously injected on the right flank of C57BL/6 mice on day 0 and the left flank on day 2. The right flank tumor site was irradiated with 20 Gy on day 8. Blocking antibodies were given on days 5, 8 and 11. For the concurrent vs. sequential RT experiment, the right flank was irradiated on either day 8 (sequential) or 12 (concurrent), while blocking antibodies were given on days 9, 12, and 15. For TSA breast cancer, 1×10⁵ TSA cells were mixed with an equal volume of Matrigel (BD Biosciences) and subcutaneously injected on the right flank of BALB/c on day 0 and the left flank on day 2. The right flank of the mice was irradiated with 8 Gy on three consecutive days starting on day 10 or 11 post tumor implantation. Blocking antibodies were started 3 days prior to RT and given every 3 days for a total of 3 doses. For the pancreatic cancer model, 4×10⁵ PDA.4662 cells were subcutaneously injected on the right flank. The right flank was irradiated with 20 Gy on day 8. Blocking antibodies were given on days 5, 8, and 11. For melanoma and breast cancer models, we used the optimal dose and fraction of radiation as previously reported^{23 (link),24 (link)}. All irradiation was performed using the Small Animal Radiation Research Platform (SARRP). Antibodies used for in vivo immune checkpoint blockade experiments were given intraperitoneally at a dose of 200 μg/mouse and include: CTLA4 (9H10), PD-1 (RMP1-14), PDL-1 (10F.9G2), CD8 (2.43), and rat IgG2B isotype (LTF-2) (BioXCell). Anti-CD8 was given 2 days prior to tumor implantations (day −2), day 0, then every 4 days for the duration of the experiment. Perpendicular tumor diameters were measured using calipers. Volume was calculated using the formula L × W² × 0.52, where L is the longest dimension and W is the perpendicular dimension.