Enrichment and detection of CTCs from orthotopic xenograft models of GBM. A, immunohistologic analysis of coronal sections showing mCherry expressing GBM8.

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Enrichment and detection of CTCs from orthotopic xenograft models of GBM. A, immunohistologic analysis of coronal sections showing mCherry expressing GBM8 and GBM24 tumor xenografts. Enrichment and detection of CTCs from orthotopic xenograft models of GBM. A, immunohistologic analysis of coronal sections showing mCherry expressing GBM8 and GBM24 tumor xenografts. B, bioluminescence imaging of GBM xenografts (n = 6). C, genome-wide expression of GBM and WBCs identifies tumor-specific markers. Left, genes plotted by average expression in GBM and WBCs from publicly available microarrays [GSE15824, 15 GBM tumors and five GBM cell lines; GSE33331, 10 CD14+ monocyte, five mature dendrocyte (mDC), four eosinophil (Eo), five CD19+ B cell (BC), five CD4+ and five CD8+ T cell (T Cell), five CD56+ natural killer T cell (NKC), three neutrophil (NE), and five plasma dendrocyte (PC) samples]. Right, an unsupervised hierarchical cluster analysis of the genes expressed 2-fold greater in tumor cells versus WBCs. D, expression heatmap of candidate CTC markers in GBM and WBCs. E, bar graph showing the recovery of mCherry+ GBM cells from GBM8 and GBM24 tumor cell spiked blood samples processed through the CTC-iChip (left y-axis) and stained with the STEAM antibody cocktail (right y-axis; n = 4). F, images of a GBM8 cell and WBC isolated from GBM8 cell line spiked blood processed through the CTC-iChip and stained with 4,6-diamidino-2-phenylindole (DAPI), mCherry, and the STEAM cocktail (single color images, ×20 magnification; merged image, ×40 magnification). Free and WBC-bound CD45 antibody–conjugated immunomagnetic beads are shown (black arrows) in the merged immunofluorescence and bright-field images at the bottom. Scale bar, 20 µm. G, quantification of mCherry+ cells isolated from sham-operated and GBM8 and GBM24 xenografted mice. Dotted line, baseline set for CTC detection based on mCherry, STEAM, and CD45 staining of blood analyzed from control mice. H, representative images of a STEAM+/mCherry+ CTC and a CD45−/low WBC isolated from the blood of a mouse bearing a GBM8 xenograft. Scale bar, 20 µm. James P. Sullivan et al. Cancer Discovery 2014;4:1299-1309 ©2014 by American Association for Cancer Research