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.