Fig. 4: Human brain microvascular endothelial cells and microglia modulate the formation of multicellular aggregates in co-culture and influence the growth of DIPG cells.

A Representative bright field and fluorescence images of cultures on day 5 in 96-well flat-bottom (A, C and U-bottom E, G plates forming multi- and/or single cell aggregates. T-6 & T-13 denote neurospheres formed from tumor cells expressing nuclear restricted-GFP only (SU-DIPG-6 and SU-DIPG-13 cell lines, respectively; green). TEM-6 & TEM-13 denote 3D TTA comprised of DIPG cells (green), microglia (nuclear restricted tagRFP; red) and endothelial cells that do not carry any fluorescent label. Cluster area (μm²) calculated after measuring diameter in confocal images with the line tool of LAS X software and displayed as violin plots, for flat-bottom B, D and U-bottom F, H plates (n = 16–25 per sample set; **** p < 0.0001). Scale bar=100 μm. B Growth metrics from live cell imaging in 96-well flat-bottom plates with tumor (GFP-expressing DIPG-6) and stromal cell types incubated in different combinations and cell density are displayed over time, including number of aggregate clusters (cluster threshold=2000 μm²) (Top), cluster area (Middle), and tumor cell count (Bottom). Neurosphere formation by tumor cell only cultures at 30,000 cells/well [T(2)] served as control, in comparison to tumor:endothelial cell (TE) and tumor:microglial cell (TM) combinations. C Overlay of phase contrast and fluorescence images (Incucyte) of multicellular aggregation in 96-well U-bottom plates using two DIPG cell types, with variable plating density and variable cell combinations of Tumor, Endothelial, and Microglial cell cultures, monitored over time (Day 0-2 d). The annotations denote: T(1): 15,000 cells/well; T(2), EM, TE, TM: 30,000 cells/well and T(3), TEM: 45,000 cells/well.