HSC-3 Spheroid Enhances Macrophage Autophagy Through Upregulation of HGF Activator

HSC-3 Spheroid Enhances Macrophage Autophagy Through Upregulation of HGF Activator

Alvin Sherman Library

Autophagy plays a critical role in regulating macrophage function within the tumor microenvironment. Hepatocyte growth factor activator (HGFA) can modulate autophagy-related pathways, potentially affecting macrophage function. This study aimed to test HGFA expression in oral cancer cell cultures and its effect on macrophage autophagy in a co-culture system. THP-1- derived macrophages were co-cultured with human oral cancer cells (HSC-3) in two-dimensional (2D) monolayers and three-dimensional (3D) spheroids generated using the hanging drop method. HGFA expression in HSC-3 cells was quantified by qPCR. After 72 hours of co-culture, macrophage polarization and autophagy were assessed by flow cytometry. To evaluate the functional role of HGFA, HGFA-specific siRNA or recombinant HGFA (rHGFA) was introduced, followed by the analysis of autophagy markers and polarization status using flow cytometry. Co-culture with HSC-3 cells significantly increases M2 macrophage polarization and autophagy compared to macrophages cultured alone (p < 0.05). HGFA expression was approximately 10-fold higher in HSC-3 spheroids than in 2D cultures and was further elevated in the presence of macrophages. HGFA knockdowns significantly reduced macrophage autophagy in both 2D and 3D co-cultures. Conversely, rHGFA supplementation markedly enhanced macrophage autophagy in the 3D co-culture system only (p < 0.05). These findings demonstrate that HSC-3 spheroids promote macrophage autophagy through HGFA, highlighting the HGFA-autophagy axis as a potential therapeutic target to modulate tumor-associated macrophages and restore antitumor immunity in oral cancer.