4‐FPAC‐Modulated Tumor‐Associated Macrophage Conditioned Media Suppresses Malignant Phenotypes and Promotes Cell‐Cycle Arrest and Apoptosis in NSCLC

ABSTRACT

Non‐small cell lung cancer (NSCLC) progression is strongly influenced by tumor‐associated macrophages (TAMs), which frequently acquire an M2‐like phenotype that promotes proliferation, epithelial‐mesenchymal transition (EMT), angiogenesis, stemness, and therapy resistance. Previous studies from our group established the direct cytostatic and macrophage‐polarizing effects of the synthetic coumarin derivative 4‐fluorophenylacetamide‐acetyl coumarin (4‐FPAC), including ROS‐mediated apoptosis, G0/G1 cell‐cycle arrest, suppression of EMT‐associated signaling in A549 cells, and promotion of M1‐like macrophage polarization. In the present study, we investigated whether 4‐FPAC‐modulated TAM‐derived conditioned media could suppress tumor‐promoting behavior in A549 NSCLC cells. THP‐1 monocytes were differentiated into macrophages and polarized using A549‐conditioned medium to generate TAM‐like macrophages. These TAMs were subsequently treated with 4‐FPAC, and conditioned media from control or treated TAMs were applied to A549 cells to evaluate macrophage‐associated anti‐tumor effects. MTT analysis further showed that direct exposure of A549 cells to 4‐FPAC under TAM‐CM‐supported conditions reduced cell viability in a dose‐dependent manner. Functional assays demonstrated that conditioned media derived from 4‐FPAC‐treated TAMs significantly reduced A549 proliferation, inhibited migration and invasion, and impaired angiogenic responses in a chick chorioallantoic membrane model. Flow cytometry revealed G0/G1 cell‐cycle arrest and reduced NANOG‐positive stem‐like cells, while DNA fragmentation and TUNEL assays confirmed enhanced apoptosis. Mechanistically, qRT‐PCR and immunoblotting demonstrated EMT reversal, suppression of AKT signaling, VEGFα, IL8, OCT3/4, and NANOG, and upregulation of p21, p53, and caspase‐3/cleaved caspase‐3. Collectively, these findings demonstrate that 4‐FPAC directly suppresses A549 viability under TAM‐CM‐supported conditions and modulates TAM‐derived conditioned‐media activity, highlighting its potential to target both tumor cells and tumor‐macrophage interactions within the NSCLC microenvironment.