Co-culture CRISPR screens reveal ATG9A as a regulator to macrophage-mediated cytotoxicity in cancer

This study investigates the role of cancer cell surface proteins in modulating the response of cancer cells to macrophage-induced cytotoxicity. Utilizing two targeted CRISPR screens using OVCAR-8 cell line and a targeted CRISPR library for cancer cell surface proteins, ATG9A was identified as a key regulator. ATG9A KO increased the sensitivity of cancer cells to macrophage-induced cytotoxicity. Subsequent RNA sequencing and other analysis elucidated the underlying mechanisms; ATG9A KO in cancer cells not only enhances inflammatory cytokines production but also plays a crucial role in the plasma membrane repair following macrophage co-culture. These findings reveal novel targets for enhancing macrophage-mediated cytotoxicity in cancer, offering additional avenues for therapeutic intervention. mRNA-seq was performed in control sgGAL4 and sgATG9A OVCAR-8 ovarian cancer cells. In this study, two million sgGAL4 or sgATG9A SKOV3 cells were subcutaneously injected into the flanks of NSG mice. Tumor growth was monitored bi-weekly and calculated using the formula: (length ×h²Once tumors reached approximately 50 mm³e were randomized into treatment groups and received intraperitoneal injections of Trastuzumab (10 mg/kg) or human IgG isotype control every 2-3 weeks. Additionally, mice were fed a diet containing the CSF1R inhibitor PLX5622 at 1200 ppm. After 6-7 weeks of treatment, mice were euthanized, and tumors were harvested for downstream analyses.