Fig. 4.

Radiation Enhances Cytotoxicity and Homing of Dog NK Cells Against Dog Osteosarcoma. a – b. Dog osteosarcoma cell lines were irradiated with 10–20 Gy. Twelve to 24 h later, irradiated cells and unirradiated controls were co-cultured with activated NK cells at increasing E:T ratios. Twelve to 16 h later, cytotoxicity was determined using dog CD45−/human CD19−/7AAD+ tumor cells to identify lysed tumor cells. NK cytotoxicity was significantly higher after RT. c – d. A single fraction of focal RT (2 Gy) was administered unilaterally to NSG mice harboring bilateral dog PDX tumors (#465049). Twelve hours later, hydrodynamic rhIL-15 was administered via tail vein followed by intravenous 14-day expanded dog NK cells. Two days later, tumors were harvested and analyzed for the presence of dog CD45+/mouse H2^d−/7AAD- NK cells, demonstrating that significantly more NK cells trafficked to tumors after RT than to un-irradiated control tumors. Data represent one experiment with 3 mice per group. The experiment was repeated a second time with similar results. e. Gating on dog CD45−/ mouse H2^d−/forward scatter-side scatter high to identify tumor cells, we observed a significantly greater fraction of the tumor cells to be non-viable after NK plus RT compared to RT alone. f. The higher percentage of non-viable tumor cells reflected a comparably higher number of non-viable tumor cells after NK plus RT. Representative data from two experiments are shown. g. Representative flow cytometry panels from panel D are shown, demonstrating significantly greater recovery of mouse H2^d-7AAD-dogCD45+ cells in PDX tumors after RT compared to un-irradiated controls. One of 3 replicates is shown. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 via one-way ANOVA with Tukey’s post-test (a, b) and via paired Student’s T-test (d – f)