Caspase-8 deficiency renders human CRC cells and xenograft human tumors hypersensitive to SMAC mimetic–induced necroptosis. (A–D) In vitro effect of SMAC mimetic on HT-29 cells and caspase-8–knockout C8KO cells generated with CRISPR/Cas9 plasmids. (B and D) Error bars indicate +SD. (A) Validation of caspase-8–knockout efficiency with immunoblots. β-Actin served as a loading control. (B) Cell death assay with propidium iodide staining followed by flow cytometry analysis. (C) Immunoblot of phospho-MLKL (S358) in the indicated cells treated with DMSO or 100 nM LCL161 for 18 h. An HT-29 cell treated with 20 ng/ml TNF (T) + 100 nM SMAC mimetic (S) + 20 µM zVAD (Z) served as a positive control. (D) Cell death assay with propidium iodide staining followed by flow cytometry analysis. Enbrel, Nec1, and NAS were added 30 min before LCL161. (E–I) Mice with established subcutaneous HT-29/HT-29-luc-D6 tumors (left side tumors) and C8KO/C8KO-luc-D6 tumors (right side tumors) were treated with PBS (mock) or 50 mg/kg LCL161 (oral) every second day. Tumor growth was monitored for 12 d by caliper (E) and in vivo imaging (F) measurements. Error bars indicate ±SD; *, P < 0.05; **, P < 0.01. (G–I) Tumors from mice treated with LCL161 for 3 d were stained with H&E (bars, 100 µm), human β-catenin (bars, 100 µm), or TUNEL (bars, 75 µm). Results from at least three biological replicates are presented unless otherwise specified. Data are derived from two independent animal experiments (E–I).