CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis

Abstract

Introduction: A subpopulation (CD44+/CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. The aim of this study was to investigate whether this subpopulation of cancer cells has the unique ability to invade, home, and proliferate at sites of metastasis.

Methods: CD44 and CD24 expression was determined by flow cytometry. Northern blotting was used to determine the expression of proinvasive and 'bone and lung metastasis signature' genes. A matrigel invasion assay and intracardiac inoculation into nude mice were used to evaluate invasion, and homing and proliferation at sites of metastasis, respectively.

Results: Five among 13 breast cancer cell lines examined (MDA-MB-231, MDA-MB-436, Hs578T, SUM1315, and HBL-100) contained a higher percentage (>30%) of CD44+/CD24- cells. Cell lines with high CD44+/CD24- cell numbers express basal/mesenchymal or myoepithelial but not luminal markers. Expression levels of proinvasive genes (IL-1alpha, IL-6, IL-8, and urokinase plasminogen activator [UPA]) were higher in cell lines with a significant CD44+/CD24- population than in other cell lines. Among the CD44+/CD24(-)-positive cell lines, MDA-MB-231 has the unique property of expressing a broad range of genes that favor bone and lung metastasis. Consistent with previous studies in nude mice, cell lines with CD44+/CD24- subpopulation were more invasive than other cell lines. However, only a subset of CD44+/CD24(-)-positive cell lines was able to home and proliferate in lungs.

Conclusion: Breast cancer cells with CD44+/CD24- subpopulation express higher levels of proinvasive genes and have highly invasive properties. However, this phenotype is not sufficient to predict capacity for pulmonary metastasis.

Figure 1

Identification of a CD44⁺/CD24^- subpopulation in breast cancer cell lines by flow cytometry. Cells in R1 correspond to CD44⁺/CD24^- cells. An isotype control corresponding to TMD-436 cells is shown.

Figure 2

Proinvasive and 'bone metastasis signature' gene expression patterns in cell lines with variable CD44⁺/CD24^- subpopulation. (a) A representative Northern analysis of bone metastasis signature genes and proinvasive genes is shown. CD44⁺/CD24^- status and ER-α expression pattern in cell lines are indicated. RT-PCR results of IL-8 and the control 36B4 are shown on the right. (b) CD44 and CD24 expression patterns in CD44⁺/CD24^- and CD44⁺/CD24⁺ subpopulations of TMD-436 sorted by flow cytometry. Expression in pre-sorted and post-sorted cells is shown. (c) Expression of IL-8, IL-6, MMP-1, and UPA in CD44⁺/CD24^- and CD44⁺/CD24⁺ subpopulations of TMD-436 cells, as determined by Northern blot analysis. ER, estrogen receptor; MMP, matrix metalloproteinase; UPA, urokinase plasminogen activator.

Figure 3

Cell lines with CD44⁺/CD24^- subpopulation are highly invasive. (a) Matrigel invasion properties of breast cancer cell lines with or without CD44⁺/CD24^- subpopulation. Number of cells invaded through matrigel to serum-free media for each cell line was set as one unit and relative invasion to media containing 10% fetal calf serum is shown. Mean and standard error of the mean is shown. Although invasion by TMD-231 and TMD-436 cells were higher than that of MDA-MB-231 and MDA-MB-436 cells, respectively, differences were not statistically significant. (b) Matrigel invasive properties of CD44⁺/CD24^- and CD44⁺/CD24⁺ subpopulations of TMD-436 cells. P = 0.006.

Figure 4

Lung metastatic properties of cell lines with or without CD44⁺/CD24^- subpopulation. (a) Hematoxylin and eosin staining of lungs in animals injected with TMD-231, TMD-436, MDA-MB-468, and DU4475 cells. Metastasis was not observed with DU4475. (b) Faxitron images of calcified tumor growth in animals injected with TMD-231 but not with TMD-436 cells (left and center). Dual energy X-ray absorptiometry whole-body scan of an animal injected with TMD-231 cells is shown on right.

Figure 5

CD44 and CD24 status and lung metastasis signature gene expression in MDA-MB-468 and LMD-468 cells. (a) Flow cytometry showing CD44 and CD24 expression in MDA-MB-468 and LMD-468 cells. (b) Expression of select lung metastasis signature genes was determined by Northern blot analysis. COX, cyclo-oxygenase; CXCR, CXC chemokine receptor; MMP, matrix metalloproteinase; UPA, urokinase plasminogen activator.