Figure 3.

Combinations of dietary agents are superior in reducing spheroid formation compared to single agents. (A) MIA-PaCa2 cells were seeded at clonal density in low-adhesion plates for spheroid formation. Three days later the cells were treated as described in Fig. 2B. Seven days after treatment spheroids were dissociated to single cells and the number of living cells was determined by counting (1st generation). The number of spheroids in the control was set to 100%. (B) Thereafter, equal numbers of live cells were seeded at clonal density in low-adhesion plates resulting in 2nd-generation spheroids, which were treated again 3 days after seeding. The 2nd-generation spheroids were grown until day 7 after treatment and photographed at ×100 magnification. (C) MIA-PaCa 2 and BxPc-3 cells were treated with GTC alone or combined with quercetin or sulforaphane as indicated. Seventy-two hours later ALDH1 activity was measured with the non-toxic ALDEFLUOR assay kit and FACS analysis. The data were quantified and statistically analyzed as described in Fig. 2.