Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers

Abstract

Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44(+)/CD24(-/low) and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR(+)/ESA(+) cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44(+)/CD24(-/low), ESA(+), CD133(+), CXCR4(+) and PROCR(+) in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

Figure 1. Soft agar colony forming efficiency of bulk cells or marker expressing or nonexpressing subpopulations from breast cancer cell lines.

Soft agar colony formation assay of cells isolated from ZR-75 (A), HCC1937 (B), SKBR-3 (C), MDA-MB-231 (D) and MDA-MB361 (E) cells with the indicated cell surface markers expressed. The unsorted cells (Bulk) were used as control. Shown is the percentage of colony formation. Results are means ± SD of triplicate samples from one representative experiment.

Figure 2. PROCR/ESA marker profiling of PROCR⁺/ESA⁺ and PROCR⁻/ESA⁻ MDA-MB-231 cells in vitro to evaluate their asymmetric division.

PROCR⁺/ESA⁺ (B) and PROCR⁻/ESA⁻ (C) cells were sorted and cultured for four passages in an attachable culture system. The PROCR/ESA marker profile was evaluated at passage 2 (E, F) and 4 (H, I). Isotype control cells were assayed at each time point as indicated (A, D, G). The PROCR⁺/ESA⁺ subpopulation was able to asymmetrically divide into other subpopulations in vitro; however, the PROCR⁻/ESA⁻ subpopulation was not capable of asymmetric division.

Figure 3. Cell cycle profiling and NUMB expression of PROCR⁺/ESA⁺ and PROCR⁻/ESA⁻ MDA-MB-231 cells.

Bulk cells (A) were synchronized by serum starvation using DMEM supplemented with 0.5% FBS for 48 h. At 20, 24 and 48 h after release from serum starvation, PROCR⁺/ESA⁺ (B) and PROCR⁻/ESA⁻ (D) cells stained with propidium iodide and the cell cycle profile analyzed. C. The expression level of NUMB in PROCR⁺/ESA⁺ relative to PROCR⁻/ESA⁻ cells were assayed using real-time PCR. GAPDH was used as an internal control. Results are means ± SD of triplicate experiments.

Figure 4. PROCR⁺/ESA⁺ cells express markers associated with epithelial-mesenchymal transition (EMT).

Gene expression levels of vimentin, E-cadherin, Slug and FOXC2 in PROCR⁺/ESA⁺ relative to PROCR⁻/ESA⁻ cells were assayed using real-time PCR. GAPDH mRNA was used as an internal control. Results are means ± SD of triplicate experiments.

Figure 5. Stem cell marker profiling of primary human breast cancer specimens.

Known breast cancer stem cell markers CD44⁺/CD24^−/low (A), ALDH (B) and ESA (C), and other solid cancer stem cell markers CD133 (D), CXCR4 (E), PROCR (F) and ABCG2 (G) were detected in primary human breast cancer specimens using flow cytometry after cell labeling. Each panel shows the percentage of cells from each primary human specimen expressing the indicated stem cell marker. Results are from one experiment since the limited material allowed only one analysis.