Figure 9.

Treatment of B16 melanoma-bearing mice with SAS reduces tumor volume, decreases tumor cell PD-L1 expression and increases CD8+ cell infiltration into tumors; this effect increases when combined with αPD-1. (A) Male C57BL/6 mice, 7-8 w of age, were inoculated subcutaneously with 8.10⁴ B16 cells/mouse. When the tumors were palpable, the mice were treated intraperitoneally with various concentrations of SAS or PBS every other day in a 0.2 ml volume. Some mice were treated with either αPD-1 antibodies (250 μg/mouse) one day after SAS injection or with an isotype-matched control or SAS+αPD-1 antibodies. The tumor volume was recorded 3-4 times/week (Fig. 9A). In accordance with ethical criteria, the mice were sacrificed when the tumor volume reached 2000 mm³. N=10/group. *p<0.05 vs. PBS; ##p<0.001 vs. Cntr Ab; @ p<0.01 vs. αPD-1 or vs. SAS 1 or SAS 1.5 mg/kg, respectively. For tumor volume analysis, two-way ANOVA with multiple comparisons and repeated measures with Bonferroni corrections were applied. (B) Mice were sacrificed, and their tumors were excised and homogenized to form single cell suspensions. The cells were then stained with an anti-PD-L1 antibody (Fig. 9B). PD-L1 expression was determined by FACS analysis of gated tumor cells, as presented in Figure S4D. The results represent data from one of 3 mice/group (Fig. 9B). (C) Cells were stained with an anti-CD8 antibody. The percentage of infiltrating CD8⁺ cells was determined as the proportion of total cells (malignant + lymphocytes). The data represent the mean+SE of 3 groups of mice. Significance was calculated via one-way ANOVA.