Figure 3: In vivo tumour-initiation by purified human prostate TICs.

(a) Limiting dilution experiments for determining percent tumour-initiation by equal numbers of sphere cells, TRA-1-60-positive cells or triple positives transplanted at OT or SC sites. Blue, 10,000 cells; green, 1,000 cells and red, 200 cells. Data represents the 4-week end-point results following transplantation. (b–h) Unless otherwise stated, 5,000 marker-positive or total tumour (unsorted control) cells were used per transplantation. (b) Representative SC tumour growth at 5 weeks following transplantation. Unsorted represents a control tumour-derived from transplantation of 2×10⁶ total tumour cells. (c) Tumour-initiation efficiency (represented as tumour volume) of the marker-positive cells at 5 weeks following transplantation. (d) Upper panel: Dark field images of primary spheres formed by marker-positive cells. Lower panel: hematoxylin and eosin staining of tumours derived from different marker-positive tumour cells. Scale bar, 100 μm. (e) Relative levels of G1 (yellow), G2 (blue) and S (red) phases of cell cycle in the different marker-positive tumour cells. Unsorted represents total tumour cells. (f) Percent triple-marker expression in sequentially passaged OT-tumours that were initially derived from the triple-marker-positive tumour cells. Percentage triple-marker-positive cells in the parent tumour form the control. (g) Maintenance of triple-marker-positive cells in the secondary passaged tumours derived from indicated marker-specific primary tumour cells. Unsorted is as described in (b). Orange-triple-marker-positive cells and purple-triple-marker-negative cells. (h) Immunoblot analysis of whole cell extracts of the tumours derived from marker-positive tumour cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) represents the loading control. Mean±s.d., n≥3.