Fig. 5

ENPP1i / DDRi post-IR boosts anti-tumor immune responses. a Immunoblot analysis of protein expression levels of STING, TBK1, phospho-TBK1 and Vinculin from cells lysates of OE-ANV5 and OE-4T1 cells treated with IR (2 Gy), ENPP1i (5 µM) and ATMi (5 µM) extracted for the indicated time post-IR. Data are representative of three independent experiments. b Expression levels assessed by RT-qPCR of the indicated genes in OE-4T1 and OE-ANV5 cells treated as indicated. c Quantification by flow cytometry of tumor-infiltrating immune subpopulations derived from orthotopic tumors at day 4 post-IR from tumor cell inoculation treated with ENPP1 and ATMi daily, showing the reverted immunosuppression. Mean and SD are represented. Kruskal-Wallis test was used for comparison. d Quantification of the multispectral analysis performed for the indicated subpopulations of myeloid M1 and M2 macrophages (top) and (bottom) lymphoid CD4⁺, CD8⁺ T cells and CD4 regulatory T cells. Mean and SD are represented. Representative images from multispectral fluorescence analysis in the indicated groups, showing the spatial resolution of different immune myeloid subpopulations (right), and lymphoid (bottom) infiltrating the tumor core in the group treated with FD or FD plus ENPP1i/ATMi (Triple). Scale bar = 50 µm. Kruskal-Wallis test was used in all panels, except for CD8⁺T cells where Student´s t-test was used for comparison. e Left panel: Tumor volume kinetics after orthotopic inoculation of OE-ANV5 tumor cells. Mice (n = 8 mice/group) were treated when tumors reached a 70 mm³ volume, with vehicle (Control), FD (6.2 Gy × 4) and triple combination of FD/ ENPP1i (6 mg/Kg BID)/ ATMi (5 mg/Kg). In other groups of mice, in addition to the triple combination, mice were treated with anti-CD8, anti-NK1.1, anti-CD4 depleting antibodies (200 µg of antibodies, three times per week) and STING inhibitor (i) (C-176, 5 mg/Kg i.p. daily). Kruskal-Wallis test was used for comparison of depleting antibodies against triple treatment. f Quantification of the indicated immune subpopulations in the blood for each group of mice at the day of sacrifice. *P < 0.05; **P < 0.01; ***P < 0.0001