Fig. 4: ATG9A protects ovarian cancer cells from CAR-macrophage-induced membrane damage and lipid peroxidation.

A Confocal images showing OVCAR-8 cells infected with ATG9A-RFP plasmid and Myr-Palm-GFP plasmid co-cultured alone or with BFP-labeled macrophages. Scale bar = 20 μm. B Quantification of ATG9A-RFP + /Myr-Palm-GFP+ area normalized by ATG9A-RFP+ area in OVCAR-8 cells (n = 30 images, mean ± SEM). Statistical significance was determined using two-tailed unpaired Student’s t-tests. C Control or sgATG9A OVCAR-8 cells were cultured with α-EphA2-CAR-macrophages for 2 h with or without Ca²⁺ (n = 3 biological replicates; mean ± SEM). Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. D, E HaloTag-labeled OVCAR-8 cells were co-cultured with α-EphA2-CAR-macrophages for 2 h with permeable or impermeable ligands (n  = 4 biological replicates; mean ± SEM). Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. F Schematic showing membrane samples isolated from flow-sorted cancer cells were submitted for lipidomics. G Normalized lipid peroxidation was calculated by dividing the abundance of peroxidized lipids by the abundance of lipids prior to transformation (n = 26 lipid species; mean ± SEM). Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. H Lipid peroxidation in control or KO OVCAR-8 cells pre- or post-co-culture was quantified by a lipid peroxidation sensor (n = 35 biological replicates; mean ± SEM). Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. I IncuCyte co-culture experiment with different inhibitors blocking NO or ROS (n = 4 biological replicates; mean ± SEM). Statistical significance was determined using two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001. Some elements of this figure were created with BioRender.com and are included under a publication license in accordance with BioRender’s user agreement. Created in BioRender. DeSelm, C. (2025) https://BioRender.com/7iu7qmq.