DC101 inhibits metronomic CPA-induced 9L tumor regression and anti-tumor immunity. A) 9L tumors were treated with metronomic CPA (160 mg/kg i.p. every 6 days; bottom 2 sets of arrows along x-axis), DC101 (22.5 mg/kg BW, i.p. every 3 days up to day 18; top set of arrows), or DC101 in combination with CPA (bottom set of arrows). Shown are mean tumor volumes, mean ± SE for n = 10-12 individual tumors/treatment group. The DC101 + CPA combination group was terminated after 13 injections (day 39) due to toxicity, as indicated by body weight loss (data not shown). CPA vs. CPA + DC101: p < 0.01 by 1-way ANOVA comparing time points after 30 days. B) Immunostaining of endothelial cell marker CD31 in untreated 9L tumors (UT) or 9L tumors treated with metronomic CPA ± DC101 and isolated on treatment day 24. Representative images are shown at 20X magnification, with background color tone adjusted to light blue to highlight the CD31-immunostained blood vessels and their near total absence in the CPA + DC101 treated tumors. Signal intensities quantified by ImageJ indicate that DC101 decreases CD31 staining to 21% of control (p < 0.001) (data not shown). C) qPCR analysis of mouse marker genes CD68 (macrophages), CD74 (dendritic cells), NKp46 (NK cells), NK cell cytotoxic effectors perforin (Prf1) and granzyme B (GzmB), and NK cell chemoattractant Cxcl14 in 9L tumor xenografts treated (as in panel A) and extracted on treatment day 15 (untreated tumors, UT), and at a time point corresponding to 4 CPA cycles (DC101 day 21, CPA day 24, CPA + DC101 day 24). All qPCR data are mean ± SE values for n = 10-16 tumors/group. **, ***, p < 0.01 or 0.001, treatment versus UT; ^^, ^^^, p < 0.01 or 0.001, for CPA + DC101 versus CPA alone, by 1-way ANOVA. Results shown are representative of two independent studies.