Fig. 2: ATG9A protects ovarian cancer cells from CAR-M-induced plasma membrane damage.

A Individual guides KO co-culture experiment to validate top hits of the screen. CD19⁺ OVCAR-8 cells were cultured alone or with UTD, α-EphA2-CAR or α-CD19-CAR macrophages for 3 days. Live cancer cell % was calculated by normalizing cell counts in the co-cultured group by the cancer-cell-only group at day three (n = 6 biological replicates; mean ± SEM). Box plots showed the median (center line), 25th–75th percentiles (box), and 2.5th–97.5th percentiles (whiskers), with all data plotted. B, C Nuclight Green (NL Green) labeled-OVCAR-8 cells were infected with control guides (sgGAL4); Nuclight Red (NL Red) labeled-cells were infected with test guides. Green cells and red cells were mixed at a one-to-one ratio and were cultured alone or with macrophages for 6 days. n = 3 biological replicates; mean ± SEM). One-way ANOVA followed by Dunnett’s multiple comparisons test was used to determine statistical significance. D sgGAL4 and sgATG9A OVCAR-8 cells were co-cultured with α-EphA2-CAR-Ms in regular 12-well plate or in a 12-well plate with 0.4 μM Transwell inserts for 3 days. (n = 3 biological replicates; mean ± SEM). Statistical significance was determined using two-tailed unpaired Student’s t-tests. E NL Red-labeled sgGAL4 or sgATG9A OVCAR-8 cells and GFP-labeled α-EphA2-CAR-Ms were co-cultured for 2 h. Flow cytometry was used to quantify the percentage of double-positive cells out of total GFP+ macrophages (n = 3 biological replicates; mean ± SEM). Statistical significance was determined using two-tailed unpaired Student’s t-tests. F PI (propidium iodide) PM integrity assay after 2 h co-culture. (n = 3 biological replicates; mean ± SEM). Statistical significance was determined using two-tailed unpaired Student’s t-tests. *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/hv2dnxb.