PMC

Microvessel density and blood perfusion in CSC-low and CSC-high tumors. A, top, CD31⁺ microvessels (Cy3; red), ×200; bottom, blood perfusion (Hoechst; blue), ×100. B, image quantification of microvessel density: number of CD31⁺ (red) structures per field (counted manually). C, image quantification of blood perfusion: total Hoechst fluorescence intensity (total colored pixels × mean fluorescence intensity) per field. Five mice per group, five fields analyzed per tumor.

VEGF- and SDF1-blocking experiments in mice with CSC-low and CSC-high tumors. A and B, image quantification of microvessel density in tumors ± DC101 (anti-VEGFR2) or AMD3100: number of CD31⁺ structures per field (counted manually). Five mice per group, eight fields per tumor. C, quantification of vessel-associated, bone marrow–derived (GFP⁺) endothelial cells (CD31⁺/VEGFR2⁺/CD45⁻) ± AMD3100 per 10,000 tumor cells by flow cytometry. Five mice per group, except CSC-low control (n = 3).

Endothelial cell proliferation and tubule formation in vitro in response to CSC-low and CSC-high conditioned medium. A and B, HUVEC proliferation assays ± IMC1121-b (anti-VEGFR2) or AMD3100. CPM, counts/min, 10 wells per group. C, HUVEC tubule formation assays ± IMC1121-b or AMD3100. Data are the sum of lengths (in pixels) of all tubules per field at ×100. Four wells per group, 10 fields per well.

VEGF and SDF1 expression in CSC-low and CSC-high cultures and tumors. A, ELISA for VEGF in conditioned medium. Three replicate wells per group. B, Western blot for SDF1 in whole-cell lysate (WCL; loading control: β-actin) and conditioned medium. A separate gel was used for conditioned medium. C, VEGF and SDF1 expression in C6 tumors. Top, VEGF (Cy3; red), ×200; bottom, SDF1 (Cy3; red), ×400. Histograms: image quantification of VEGF (top) and SDF1 (bottom). Total fluorescence = total colored pixels × mean fluorescence intensity per field. Six mice per group; VEGF: five fields per tumor and SDF1: four fields per tumor.

Relative CSC content in C6 Ser+ and SFS cultures and tumors. A, tumor sphere-forming assay: 100 cells plated per well. B, cytospins of C6 cultures stained for nestin (Texas red; red). Nuclei stained with 4′,6-diamidino-2-phenylindole (blue), ×200. Histogram: image analysis of nestin staining. Eight fields analyzed per group. Cells displaying Texas red fluorescence above background (established using Texas red–conjugated IgG control) were counted as nestin-positive. % Nestin-positive cells per field calculated as nestin-positive cells/DAPI–stained cells (counted manually). C, ex-vivo tumor sphere-forming assay using established tumors (4 wk post-implantation).

EPC mobilization and vessel incorporation in mice with CSC-low and CSC-high tumors. A, EPC in peripheral blood ± DC101 (#/μL blood). Five mice per group. B, vessel-associated BMDC (GFP⁺; green) in CD31-stained microvessels (Cy3; red; ×200). Top, full field; bottom, zoomed subsection. Bar, 50 μm. Histogram: image quantification of microvessels containing BMDC (number of GFP⁺, CD31⁺ microvessels/field divided by total number of microvessels/field; counted manually). Five mice per group, eight fields per tumor. C and D, GFP⁺ RBCC/hemangiocytes, TEM (C), or endothelial cells (D), per 10,000 tumor cells, assessed by flow cytometry. Mice per group: (C) 8 for CSC-low and 10 for CSC-high and (D) 5 per group.