Effect of Wnt3a overexpression on in vitro clone-initiation and invasion abilities and in vivo tumor growth and metastasis. (A) Wnt3a overexpression promoted HCT116 anchorage-independent growth in soft agar. Colonies in soft agar culture were stained (left), 200×. Histogram showing colony formation efficiency (right). (B) Cells invading through matrigel-coated transwell inserts were stained (left), 200×. Invading cells were counted in five pre-determined fields (right), 400×. (C) Photograph of representative tumors from mice injected with control or Wnt3a transfected HCT116 cells (clone7) (left). Wnt3a-overexpressing cells produced larger tumor masses than control cells (right). (D) Immunohistochemical staining of β-catenin expression in harvested mouse tumor samples. Tumors overexpressing Wnt3a (clone7) exhibited increased nuclear β-catenin expression compared with control tumors, 400×. (E) Mice injected with Wnt3a-overexpressing cells (clone7) showed lymph node metastasis and lung metastasis (red arrows); hematoxylin and eosin staining, 200 × .