Fig. 7

MemTNF-signaling-mediated apoptosis serves as the mechanism of action (MOA) for MTA/S-induced complete regression of human breast cancer. a Schematic experimental design for combinatory use of PTX and LCL161 (LCL) in breast cancer PDX, as described in the “Methods” section. b Clinical information of breast cancer patients used for PDX study. c Effect of PTX and LCL161 combinatory treatment on TNBC PDX. Athymic nude mice bearing 200–300 mm³ TNBC PDX were treated with vehicle or with 20 mg/kg PTX and/or 25 mg/kg LCL161. Upper left panel depicts tumor growth, upper right shows representative image of tumor-bearing mice, lower left documented final tumor weight, and lower right represents the image of the final tumors. Vehicle, PTX, and LCL, n = 5; PTX/LCL, n = 6. Scale bars, 2 cm. d Effect of in vivo TNF neutralization on PTX/LCL co-treatment of TNBC PDX. PDX was developed and treated as described in c. Enbrel (20 mg/kg) was treated every 3 days. n = 8 for each group. Scale bars, 2 cm. e 10 days after treatment as in c, tumors were isolated for histology (H&E staining) and apoptosis (cleaved caspase-3 IHC) analysis. n = 5 for each group. Scale bar, 200 µm. f Quantification of apoptotic cells in tumors (right panel), assessed by flow cytometry (left panel). Numbers in quadrants (top left areas; Annexin V⁺ 7-AAD⁻) indicate apoptotic cell percentage in each group. n = 4 for each group. g Membrane-bound TNF was assayed by flow cytometry after 3-day or 7-day drug treatment. n = 5–6 for each group. h In vivo toxicity test for PTX/LCL161 combinatory treatment on wild-type C57BL/6 mice. Mice were treated with vehicle or with 20 mg/kg PTX and 25 mg/kg LCL161. Body weight, body temperature, AST, and ALT were all measured at the end of the study. n = 6 for each group. PTX, paclitaxel; LCL, LCL161. All graphs show mean ± SEM. p values for c, d, f, and g were determined by the one-way ANOVA test, followed by Tukey’s multiple comparison post-test; p values for body weight and ALT in h were determined by Mann–Whitney U-test; p values for body temperature and AST in h were determined by two-tailed unpaired Student’s t-test with Welch’s correction. NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Results are reported from one representative experiment. Experiments were repeated independently for three (c) or two (d–h) times