Sphere formation by CD44+ cells isolated from tongue and neck node tissues. A: CD44+ cells from tumor and neck node tissue (Magnetic activated bead isolation followed by cell culture in conditioned media in non-adhesive 0.1% agarose coated plastic ware. (a) The proliferating CD44+ cells from OTSCC (lymph node metastatic and non-metastatic) formed spheres or spheroids (b) CD44- cells differentiated during 1 week (c) CD44+ cells isolated from neck nodes (Level I) (d) CD44- cells from neck nodes (Level I) B: Clonogenic assay for self-renewal. Purified CD44+ cells were plated at 250cells/mm2 and cultured for 1 week. (a) holoclone comprising of compact cells (b) meroclone containing loosely arranged cells on adhesive plastic ware (c) paraclone comprising of sparsely arranged cells on adhesive plastic ware (d) No colonies were formed in nodes secondary cultures. No cell spheres were formed from non-cancer tissues (not shown). Magnification 40x.