Fig. 4

ENPP1i/DDRi post-IR eradicates local control and impacts disseminated disease. a Left panel: Tumor volume kinetics after orthotopic implantation of OE-ANV5 cells treated with FD (6.2 Gy × 4) alone or in combination with ENPP1i (6 mg/kg daily, BID), ATMi (5 mg/kg daily), or the triple combination (n = 8 mice/group). Right panel: Waterfall plot at the day of sacrifice. Kruskal-Wallis test was applied. Mean ± SEM are represented. **P < 0.01; *** P < 0.0001. b Tumor volume kinetics of orthotopic tumors in Control and dual-treated post-IR treated animals (n = 5 per group) which did not develop tumors were rechallenged by orthotopically implanting OE-ANV5 cells 2 months after treatment interruption. One-way ANOVA was performed. ****P < 0.00001. c Left panel: Similar experiment as in a using 4T1 cells. Treatments included ENPP1i (12 mg/Kg BID) and ATMi (5 mg/Kg daily). Right panel: Waterfall plot at the day of sacrifice. Kruskal-Wallis test was applied. Mean ± SEM are represented. ***P < 0.0001. d Top panels: Quantification of the metastatic surface (left) and the number of pulmonary nodules in histological section of mice treated performed by an in-house developed macro based on ImageJ®. Median and inter-quartile range are represented. Mann-Whitney U test was used for comparison. *P < 0.05; **P < 0.01; ***P < 0.0001. Bottom panels: Representative H/E images of lung lobules in Control and treated-mice with the triple combination. Scale bar = 5 mm. e Left panel: Quantification of Caspase-3 immunostaining in tumor sections of treated animals (n = 5/ group) for 4 days. Right panels: Representative images. Scale bar = 50 µm. f Quantification of hematological parameters in blood samples extracted from naïve animals compared to Control and triple-treated animals for 2 weeks of the indicated cell subpopulations. One-way ANOVA was used for comparison. MPV Mean Platelet Volume. g Quantification of plasma levels of the indicated biochemical markers. ALT Alanine aminotransferase, AST aspartate aminotransferase, HDL High density lipoprotein, BILT Bilirubin, LDL-C LDL-cholesterol. h Left panel: Schematic outline of LF assay. Middle panel: LF-free survival after tumor resection from OE-implanted cells. Mice (n = 15/group) were treated with ATMi (5 mg/Kg/day), ENPP1i (6 mg/Kg BID) or the combination from the day of tumor resection or treated with vehicle (control). Right panel: tumor volume at the day of tumor-resection (Day 0). Log-rank test was used in Kaplan-Meier curves. ** P < 0.001. i Left panel: LF-free survival after surgical resection of tumors derived from OE-ANV5 cells orthotopically implanted as in h. Mice (15 mice/group) were treated with FD (4 × 6.2 Gy) IR alone, on two consecutive days after surgery with an implanted catheter,¹² in combination with ATMi (5 mg/kg daily) or with the dual ENPP1i (6 mg/Kg by oral gavage BID), and ATMi. Right panel: tumor volume at the day of surgery in each group. No differences in tumor margins between groups were detected. Log-rank test was used in Kaplan-Meier curves. **P < 0.01; ***P < 0.0001. j Experimental outline shows the orthotopic tumor cell inoculation in the irradiated mammary gland whereas the contralateral mammary gland was not irradiated. Animals were treated with ENPP1i and ATMi. k Left panels: Orthotopic tumor growth after double simultaneous inoculation of OE-ANV5 cells in opposite inguinal mammary glands in 3 groups of mice (8 mice/group). Treatments include IR only with fractionated dose (FD) performed in one flank, systemic ENPP1i/ATMi treatment, and triple treatment. Tumor volumes were monitored over time in both flanks. Right panels: Tumor volumes of each tumor at the IR flank and the non-IR contralateral flank. Kruskal-Wallis test was used for comparison. *** P < 0.0001