English: (A) Cell suspensions form small, non-adherent clusters 1 wk after plating (top left). After ~10 passages, 1% of spheres persist as larger, symmetric, prototypical spheroids (top right). Typical spheroids contained ~100 cells and could be serially passaged for >6 mo (bottom left). Under differentiating conditions, sphere-forming cells adhere to plates and form symmetric holoclones (bottom right). (B) Injection of ~100 OCICs per mouse from patient tumor 1 (left) or patient tumor 2 (right) dissociated spheroids generated xenograft tumors with 2/2 efficiency. (C) Staining of anti-CD44 monoclonal antibodies (red) in ovarian tumor spheroid (top left); immunofluorescence staining of anti-CD117 monoclonal antibodies (green) in ovarian tumor sphere cells (top right); CD44+ sphere cells colocalize with CD117+ cells (orange overlay, bottom left) and additionally stained with DAPI (blue; bottom right). Magnification, 200x. (Adapted with permission from Zhang, S., Balch, C., Chan, M.W., Lai, H.C., Matei, D., Schilder, J.M., Yan, P.S., Huang, T.H., Nephew, K.P. (2008). Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res 68, 4311–4320. Figures 1A, 3A and C.).