Figure 5.

Live proteolysis by 3D prostate tumor cell cultures in the absence or presence of infiltrating macrophages. (A) Schematic representation of experimental design. 3D cultures were seeded on rBM containing DQ-Collagen IV substrate and grown alone or in transwell coculture with adipocytes for 3 days prior to addition of bone marrow macrophages and culture for an additional 2 days. (B–E) 3D reconstruction of DQIV proteolysis by tumor spheroids cultured alone (B) or infiltrated 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 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.