FIGURE 3.

Cell viability and endothelial cell content in triple heterotypic spheroids: effect of agitation. (A) Fluorescence microscopy of triple heterotypic spheroids at day 30 of culture in agitation (2 days of static culture followed by 28 days of agitation-based culture, rotational speed of 100 rpm) or in static (30 days of static culture) conditions. Spheroids generated by seeding 6,000 cells per well, at distinct ratios of HCC1954 breast tumor cells (TC); human dermal fibroblasts, labeled with PKH26 (hF, red); and HUVEC, labeled with CellTracker Deep Red (EC, cyan). Live cells were stained with FDA (green). Images are representative of the 5–10 spheroids assessed per condition, in each of the three to four independent experiments performed. Scale bar, 250 μm. Quantification of (B) fold change in hF area, (C) fold change in EC area, and (D) total spheroid area at the equatorial axis, for spheroids from A. The EC and hF areas were normalized for the total spheroid area at the equatorial axis; the fold change was determined relative to the condition with a TC:hF:EC of 1:1:1, agitation. Data are presented as mean ± SD from N = 3–4 (independent experiments); in each experiment, at least 20 spheroids were quantified per condition. Statistical analysis was performed using unpaired t-test two-tailed comparing agitation to static for each cell ratio; in B, *p = 0.015 for 1:1:1 and *p = 0.047 for 1:3:10; in C, **p = 0.0068 for 1:3:10; and in D, **p = 0.0017 for 1:1:1, **p = 0.0014 for 1:3:10.