Figure 3.
Tumor sphere formation and tumor initiation by ex vivo NKG2D+ HGS EOC cells. (A) Phase contrast micrographs of tumor spheres formed by NKG2D+ or NKG2D− cancer cells FACSAria-sorted from EOC16, EO17, and EO18 specimens. Cell numbers plated are indicated below each micrograph pair. (B) Display of sphere (>100 μm) numbers formed by NKG2D+ or NKG2D− cancer cells from specimens EOC16 through EOC23 (serial passages for EOC22 and EOC23) and plated at the indicated cell numbers. Specimen size limitations precluded plating of all cell concentrations per EOC sample. (C) Flow cytometry dot plots showing NKG2D and NKG2DL expression on single cells dispersed from spheres formed by NKG2D+ cancer cells. Vertical lines separate positive from negative NKG2D staining and are drawn based on isotype Ig fluorescence. (D and E) Bar graphs displaying average numbers (from triplicate wells seeded with 5 × 103 cells) of spheres (>100 μm) formed by NKG2D+ cancer cells (D) untreated (gray bars) or transduced with NKG2D RNAi (black bars) or control scrRNAi (hatched bars), and (E) in the presence of control isotype Ig (gray bars), anti-NKG2DL Abs (black bars), or anti-NKG2D Ab (hatched bars). Open bars represent average numbers of spheres formed by NKG2D− cancer cells. (F) Xenograft tumor incidence expressed as numbers of tumors developed per number of inoculations of 5 or 10 × 103ex vivo NKG2D+ or NKG2D− cancer cells into NSG mice. Frequencies of tumor initiating cells (numbers above brackets) and P values were computed using ELDA. EOC34 and EOC35 are HGS EOC cases; EOC36 represents a type I EOC. (G) Flow cytometry dot plots of NKG2D and NKG2DL expression on single cells isolated from xenograft tumors derived from NKG2D+ cancer cells. Vertical lines separate positive from negative NKG2D staining and are drawn based on isotype Ig background fluorescence.