Fig. 2: NPs triggered enhanced ferroptosis of CAFs through the synergistic effect of iron and copper ions.

a Images displaying the isolation of NFs from adjacent normal tissues and CAFs from CRC tumor tissues. b Western blot confirming successful isolation of NFs from three normal colonic tissues and CAFs from three CRC specimens, validated by antibodies against fibroblast activation protein-α (FAP), Vimentin, and α-smooth muscle actin (SMA); β-actin as a loading control. c Cytotoxicity assessment of NPs in NFs and CAFs for 24 h at varying concentrations. d Bio-TEM images of NFs and CAFs incubated with NPs for 24 h; Red dashed boxes marking NPs location. e Bright-field and fluorescent images of CAFs stained with Calcein-AM (live cells, green fluorescence) and PI (dead cells, red fluorescence) after treatment with phosphate buffer saline (PBS), Laser, NPs, and NPs+Laser. PBS as control (NC) (left); Statistical analysis of cell viability (right). f, g Fluorescent images of (f) intracellular Fe²⁺ levels and (g) LPO in CAFs treated without or with NPs for 24 h; Nuclei stained with Hoechst 33342 (blue). h Western blot of GPX4 expression in CAFs treated with increasing concentrations of NPs for 24 h; β-actin as a loading control. i Relative cell viability of CAFs treated with NPs alone, NPs with DFO, NPs with TM, and NPs with both DFO and TM for 24 h by CCK8 assay. j, k Fluorescent images of (j) intracellular Fe²⁺ levels and (k) LPO in CAFs treated as in (i); Nuclei stained with Hoechst 33342 (blue). l Western blot of GPX4 expression in CAFs treated as in (i); β-actin as a loading control. n = 3 independent experiments per group in (c–g) and (i–k). Western blot experiments in (b, h, l), FerroOrange and Liperfluo staining experiments in (f, g, j, k) are repeated independently three times with similar results. Data are presented as means ± standard deviation (SD). Statistical analyses are performed using one-way analysis of variance (ANOVA) with multiple comparisons (e). Source data are provided as a Source Data file.