A-B. Tumor growth curves were plotted for different numbers of cells injected (for MDA-MB-453: 50,000 cells, 5,000 cells, and 500 cells and for SUM159: 100,000 cells, 10,000 cells, and 1,000 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. C-D. H&E staining of ALDEFLUOR-positive cells' injection site, revealing presence of tumor cells (Ca: MDA-MB-453 ALDEFLUOR-positive cells' injection site, and Da: SUM59 ALDEFLUOR-positive cells' injection site). Cb, Db. The ALDEFLUOR-negative cells' injection site contained only residual Matrigel, apoptotic cells, and mouse tissue (Cb: MDA-MB-453 ALDEFLUOR-negative cells' injection site, and Db: SUM59 ALDEFLUOR-negative cells' injection site). Data represent mean ± SD.

A. Effect of IL8 treatment on tumorosphere formation of three different cell lines (HCC1954, SUM159, MDA-MB-453). IL8 treatment increased the formation of primary and secondary tumorospheres in a dose-dependent manner. B. Effect of IL8 treatment on the ALDEFLUOR-positive population of four different cell lines cultured in adherent conditions. IL8 increased the ALDEFLUOR-positive population in a dose-dependent manner in each of the four cell lines analyzed (* p<0.05/ ** p<0.01, statistically significant differences from the control group).

Hierarchical clustering of 33 breast cell lines and 13,550 genes/ESTs based on mRNA expression levels. A. The dendrogram of samples represents overall similarities in gene expression profiles. Two large groups of samples are evidenced by clustering. Name of cell lines is colored as follows: blue for luminal (n=12), red for basal (n=10), brown for mesenchymal (n=6) cell lines according to the correlation of expression profile of each cell line with each Ross and Perou centroid (i.e. molecular subtype). Five cell lines were not attributed any subtype (name in grey). The ER, ERBB2, and ALDEFLUOR status of breast cell lines are represented according to a color ladder (for ER and ERBB2 status: negative, white; positive, black; unavailable, oblique feature; for ALDEFLUOR status: a color scale shown at the bottom of the dendrogram relates the percentage of ALDELFUOR-positive cells found in each breast cell line). B. Comparison of the ALDEFLUOR status with the molecular subtypes of breast cell lines revealed a strong correlation between the basal/mesenchymal subtypes and the presence of ALDEFLUOR-positive cells (p-value=0.0006).

A-B. To confirm our gene expression results, we measured in a set of five breast cancer cell lines sorted for the ALDEFLUOR phenotype the expression of five discriminator genes overexpressed in ALDEFLUOR-positive populations (CXCR1/IL8RA, FBXO21, NFYA, NOTCH2 and RAD51L1) by quantitative RT-PCR. The quantitative RT-PCR expression levels of CXCR1 and FBXO21 are presented in this figure and the ones of NFYA, NOTCH2, and RAD51L1 are presented in Supplementary Fig. 8. Gene expression levels measured by quantitative RT-PCR confirm the results obtained using DNA microarrays with an increase of CXCR1 and FBXO21 mRNA level in the ALDEFLUOR-positive population compared to the ALDEFLUOR-negative population (p<0.05).

A. The IL8/CXCR1 axis is involved in cancer stem cell invasion. The role of the IL8/CXCR1 axis in invasion was assessed by a Matrigel invasion assay using serum or IL8 as attractant for three different cell lines (HCC1954, MDA-MB-453, SUM159). ALDEFLUOR-positive cells were 6- to 20-fold more invasive than ALDEFLUOR-negative cells (p<0.01). When using IL8 (100 ng/ml) as attractant, we observed a significant increase of ALDEFLUOR-positive cells invading through Matrigel compared to serum as attractant (p<0.05). In contrast IL8 had no effect on the invasive capacity of the ALDEFLUOR-negative population. B-C. The ALDEFLUOR-positive population displayed increased metastatic potential. Ba-c. Quantification of the normalized photon flux measured at weekly intervals following inoculation of 100,000 luciferase infected cells from each group (ALDEFLUOR-positive, ALDEFLUOR-negative, unseparated). Mice inoculated with ALDEFLUOR-positive cells developed several metastasis localized at different sites (bone, muscle, lung, soft tissue) and displayed a higher photon flux emission than mice inoculated with unseparated cells, which developed no more than one metastasis per mouse. In contrast, mice inoculated with ALDEFLUOR-negative cells developed only an occasional small metastasis, which was limited to lymph nodes. C. Histologic confirmation, by H&E staining, of metastasis in bone (Ca), soft tissue (Cb) and muscle (Cc) resulting from injection of ALDEFLUOR-positive cells.