Spalax fibroblasts suppress colony formation of human breast cancer cells MDA-MB-231 and MCF7 in soft agar. (A) MDA-MB-231 cells (5 × 103 cells) cells were suspended in 0.35% agar and added as the cancer cell top layer to base layer either empty (blank) or containing the Spalax or rat fibroblast monolayer. At Day 21, colonies larger than 50 μm were counted under an inverted microscope and photographed (×40). Representative microscopic images out of 15 fields are shown. (B) Average number of colonies counted in soft agar (n = 15). The experiment was performed in duplicate plates at least three times; mean ± S.D. (C) A representative colony in soft agar was formed by MDA-MB-231 only, or by co-culturing with a Spalax fibroblast monolayer. The size bar shows equivalent magnification in both images (× 200). (D) MCF7 cells (5 × 103 cells) were grown in soft agar on top of a monolayer of mouse newborn (MNbF), or Spalax newborn fibroblasts (SpNbF) in 35-mm culture dishes. After 5 and 11 days of incubation colonies containing >20 cells were counted by using an inverted microscope (× 200), mean ± S.D.