Fig. 2

Construction and characterization of ADV^NE and ADV^PPE and investigation of their in vitro antitumor mechanisms. (A) Differences in ELANE gene expression between tumor and adjacent normal tissues and overall survival of colorectal cancer patients with high versus low ELANE expression were analyzed using the TCGA database. (B) Schematic structure of the recombinant oncolytic ADVs ADV^NE and ADV^PPE. (C-D) To evaluate the oncolytic activity of the recombinant ADVs, MC38 and CT26 cells were infected with ADV^Ctrl, ADV^NE, or ADV^PPE at different multiplicities of infection (MOIs). Cytotoxicity was assessed 48 h post infection via a CCK-8 assay (C) and crystal violet staining (D) (n = 3 biological replicates). (E) To assess the replication capacity of oncolytic ADVs, MC38 and CT26 cells were infected at an MOI of 1, and viral titers were measured at 12 h, 24 h, 48 h, 72 h, and 96 h post infection via a TCID₅₀ assay (n = 3 biological replicates). (F) Expression of NE and PPE in MC38 and CT26 cells infected with different ADVs, as detected by Western blotting with an anti-His antibody. (G) MC38 and CT26 cells were infected with different viruses, and apoptosis was assessed 48 h later via Annexin-V and 7AAD staining, followed by flow cytometry (n = 3 biological replicates). (H) Levels of cleaved Caspase-3, N-GSDME, and HMGB1 in MC38 and CT26 cells after infection with different ADVs, as detected by Western blotting. (I-J) Following infection of MC38 and CT26 cells with different viruses for 48 h, LDH release was determined using an LDH Release Assay Kit (I), and HMGB1 levels in the supernatant were measured via ELISA (J) (n = 3 biological replicates). The data are presented as the means ± SDs. NS, no significant difference; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001