Podophyllotoxin sensitizes triple-negative breast cancer cells to CD47-targeted immunotherapy.

Triple-negative breast cancer (TNBC) remains a highly aggressive malignancy with limited therapeutic options. Immunotherapeutic strategies are emerging as promising avenues to improve clinical outcomes in TNBC. Among these, CD47, a critical self-protective "don't eat me" immune checkpoint against macrophage immunosurveillance, is frequently upregulated in TNBC, contributing to tumor immune evasion. However, CD47 blockade alone has demonstrated limited efficacy in this context. To identify agents that potentiate CD47-targeted therapy, we conducted a high-throughput small molecule screen in TNBC models. This effort led to the identification of podophyllotoxin (PTOX), a plant-derived microtubule-disrupting agent, as a potent enhancer of macrophage-mediated TNBC clearance. PTOX treatment significantly sensitized TNBC cells to CD47 blockade-induced clearance. Gene set enrichment analysis (GSEA) revealed significant negative enrichment of PI3K_AKT signaling and positive enrichment of TNFα signaling via NF-κB in PTOX-treated TNBC cells. Functional perturbation studies further indicated that EGFR, TNFα, and TNFAIP3 contribute to PTOX-induced macrophage clearance. Together, these findings identify PTOX as a novel phagocytosis-sensitizing agent and support its potential as a combinatorial immunotherapeutic strategy to enhance CD47-targeted therapy in TNBC.