Figure 1: Identification, isolation and characterization of PECAM1⁺ tumour cells from B16F10 melanoma.

(a) PECAM1 antibodies coupled to immunomagnetic beads were used to enrich PECAM1⁺ tumour cells from collagenase-digested B16F10 tumours. (b) The enriched cells (eluate) are ~99% PECAM1⁺ by flow cytometry. (c) Purity of the cells was further confirmed by semi-quantitative RT–PCR. The PECAM1⁺ fraction from B16F10 expresses Pecam1 and Tyr, but not VE-cadherin. (d) Retrieval of PECAM1⁺/GFP⁺ cells from GFP tumours engrafted in wild-type (WT) hosts and PECAM1⁺/GFP⁻ cells engrafted in GFP hosts. Following immunomagnetic separation with PECAM1 antibodies, increasing amounts of cDNA template were analysed by semi-quantitative RT–PCR, indicated by the wedge. (e) Detection of PECAM1⁺/GFP⁺ tumour cells in vivo after injecting B16F10-GFP tumour cells into WT hosts. CD45⁺ haematopoietic cells were excluded by out-gating. The upper right quadrant are PECAM1⁺ tumour cells, which comprise ~0.1% of each tumour. (f) The percentage of PECAM1⁺/GFP⁺ tumour cells are shown for two time points when tumours were different sizes (n=3 mice per group; error bars=s.e.m.).