Figure 5. The ALDEFLUOR positive cell population from human breast tumors xeongrafted in NOD/scid mice has cancer stem cell properties.

A-B. Representative flow cytometry analysis of ALDH activity in cells derived from a human breast tumor, orthotopically xenotransplanted in NOD/scid mice. The ALDEFLUOR assay was performed as described above. In addition mouse of cell origin were eliminated from the analysis (see Supplementary methods 1 and Supplementary Figure 5). (A, B) All the ALDEFLUOR analyses on human breast tumor cells were first gated on PI negative cells (viable cells) which represented 73.6±1.8% (Mean ± SDEV, n=43) of the total population. C-G Only the ALDEFLUOR-positive population was tumorigenic. C. The ALDEFLUOR-positive population was capable of regenerating the phenotypic heterogeneity of the initial tumor after a passage in NOD/scid mice. D. Tumor growth curves were plotted for the numbers of cells injected (50,000 cells; 5,000 cells; 500 cells) and for each population (ALDEFLUOR-positive, ALDEFLUOR-negative, unseparated). Tumor growth kinetics correlated with the latency and size of tumor formation and the number of ALDEFLUOR-positive cells. E. Representative tumor grown in NOD/scid mouse at the ALDEFLUOR-positive cells’ injection site (5,000 cells injected). No tumor was detected at the ALDEFLUOR-negative cells’ injection site (5,000 cells injected). F-G. H & E staining of ALDEFLUOR-positive cells’ injection site, revealing presence of tumor cells (F). The ALDEFLUOR-negative cells’ injection site contained only residual Matrigel, apoptotic cells and mouse tissue (G). All the data presented in this figure were generated by analysis of the MC1 tumor. Similar results were obtained for three other tumors, generated from different patients (UM1, UM2, and UM3) tested (Supplementary Figure 4).