Inhibition or absence of IDO triggers widespread complement deposition in tumors after chemo-radiation therapy. GL261 tumors were grown in syngeneic host mice treated with or without IDO-blocking drugs (1MT, 4 mg/mL; or NLG919, 6 mg/mL) in drinking water starting on day 14, plus TMZ (100 mg/kg, i.p.) on day 16 and RT (500 cGy) on day 17. Tumors were harvested on day 18 and frozen for immunohistochemical analysis of complement component C3 deposition (red) endothelial cells (CD31, green), and nuclei (Hoechst, blue). A, representative photomicrographs of control tumors from WT host mice, either untreated or treated with TMZ + RT. B, representative photomicrographs of tumors and adjacent normal brain from WT host mice treated with either TMZ + RT + 1MT or TMZ + RT + NLG919; and from syngeneic IDO-deficient host mice treated with TMZ + RT. Data are representative of at least 3 mice per group, from at least 3 independent experiments. Original magnification, ×400; Scale bars, 25 μm. C to E, quantitative analysis of complement deposition. Photomicrographs of tumors stained for complement C3 (red) were obtained in a grid pattern at magnification ×400, and image analysis software was used to abstract fluorescence intensity histograms from each photomicrograph for quantitative analysis. C, Representative photomicrographs and histograms are shown. Scale bars, 25 μm. Histograms are labeled with mean fluorescence intensity (MFI) and the proportion of pixels occurring downfield from an arbitrarily-chosen negative threshold (channel thirty-two). Comparisons of MFI (D) and Percent Downfield Pixels (E) are shown with means represented by a solid bar. For each experimental group, at least 35 photomicrographs were analyzed from 3 separate mice pooled from 3 independent experiments. *, P < 0.02; **, P < 0.002; ***, P < 10-6 (vs. WT mice treated with TMZ + RT), by ANOVA with Kruskal-Wallis test.