Fig. 2: Anti-cancer mechanism of aECM.

a Cytotoxicity of different concentrations of aECM or aECM-Trail (0.1–1000 ng mL⁻¹) towards CT26 cells. Six biological replicates are shown. b Cytotoxicity of different concentrations of aECM or aECM-Trail (0.1–1000 ng mL⁻¹) towards CT26 MTS. CT26 MTS were treated with aECM or aECM-Trail for 24 h. Six biological replicates are shown. c Cytotoxicity of aECM towards 2D cell culture system and 3D MTS model in 4T1, MCF-7, and HT-29 cells for 24 h. Five biological replicates are shown. d ATP level in CT26 MTS after being treated with aECM or PBS. The ATP levels were measured at the different time points during the treatment. Five biological replicates are shown. e Real-time glucose and lactate concentrations in the culture medium of aECM or PBS treated CT26 MTS. Six biological replicates are shown. The upper curves represent glucose, and the lower curves represent lactic acid. f Oxygen content in CT26 MTS after being treated with PBS or aECM. ROS-ID was used to visualize the hypoxia levels in CT26 MTS. Five biological replicates are shown. g Schematic diagram of the diffusion assay based on Transwell insert. A high concentration solution was added in the lower chamber and DI water was placed in the upper chamber. aECM or collagen was coated on the porous membrane. h The effect of aECM coating on suppressing mass transports (spermidine, lactate, glucose, glutathione, and O₂). Collagen-coated Transwell inserts were set as a control (black). Three biological replicates are shown. Significance between two groups (a–f, h) was calculated by using two-tailed Student’s t-test. The mean values and SD are presented.