Figure 4: Metabolic autofluorescence imaging of mouse macrophages within 3D Stacks tumor co-cultures.

A) Representative autofluorescence images (z-plane distance: 6 µm) demonstrate qualitative differences in the redox ratio, NAD(P)H and FAD mean lifetimes (τ_m) of RAW264.7 macrophages in co-culture with PyVMT mouse mammary tumor cells. Scale bar = 50 μm. B) Gene expression levels of M1-like and M2-like markers were evaluated for monocultured and co-cultured macrophages at 24, 48, and 72 hours using qPCR. Upregulation of both M1-like and M2-like genes were observed in co-cultures by 72 hours, demonstrating heterogeneity in macrophage polarization in response to tumor stimulation. Quantitative analysis of C) redox ratio, D) NAD(P)H τ_m, and E) FAD τ_m highlight dynamic metabolic changes in macrophages during prolonged tumor-macrophage co-culture. Monocultured macrophages were not significantly different (p>0.05) across time points, so only the 24 hour monoculture is shown above. Significant differences in NAD(P)H and FAD τ_m are observed between monocultures and co-cultures, as early as 24 hours post-seeding. Additionally, macrophage co-cultures exhibit a gradual decline in redox ratio over time, consistent with transition to a more oxidized state; **,***,**** p<0.01,0.001,0.001 vs. monoculture, ††† p<0.001. Population density curves of single-cell redox ratios in monocultures and co-cultures at F) 24 hours, G) 48 hours, and H) 72 hours demonstrate variable cellular-level heterogeneity in co-cultured macrophages over time in monocultures.