Figure 6.

Contribution of bone marrow macrophages to proteolysis of DQ-Collagen IV by tumor cells exposed to bone marrow adipocytes. (A) Schematic representation of experimental design. Tumor cells were mixed with BMMs, and 3D cultures were seeded on rBM containing DQ-Collagen IV substrate and grown alone or in transwell coculture with adipocytes for 3 days. (B–E) 3D reconstruction of DQIV proteolysis by tumor spheroids seeded alone (B) or with BMMs (C) in the absence or presence of adipocytes (D,E); DQ-Collagen IV cleavage products (green), CellTracker Orange: PC3 cell label (red); Hoechst: nuclear label (blue); green arrow (X), red arrow (Y), and blue arrow (Z) indicate orientation of the spheroid in 3D space. (F–I) Middle slice through z-stack showing DQIV fluorescence, CellTracker Orange, and Hoechst staining of PC3 spheroids with or without BMMs grown in control conditions (F,G) or in transwell with adipocytes (H,I); 40× images; bar, 50 μm. (J) Quantification of DQ-Collagen IV proteolysis, quantified in each 3D reconstructed spheroid using Volocity Software, is shown as fluorescence intensity per cell in the entire volume; nuclei were stained with Hoechst 33342 (blue) at the time of imaging and counted. Data are shown as average (±SD) of three independent experiments with at least three independent spheroids measured/experiment. *p < 0.05 and ***p < 0.001 are considered statistically significant.