Profiling of normal and malignant breast tissue show CD44high/CD24low phenotype as a predominant stem/progenitor marker when used in combination with Ep-CAM/CD49f markers

Abstract

Background: Accumulating evidence supports cancer to initiate and develop from a small population of stem-like cells termed as cancer stem cells (CSC). The exact phenotype of CSC and their counterparts in normal mammary gland is not well characterized. In this study our aim was to evaluate the phenotype and function of stem/progenitor cells in normal mammary epithelial cell populations and their malignant counterparts.

Methods: Freshly isolated cells from both normal and malignant human breasts were sorted using 13 widely used stem/progenitor cell markers individually or in combination by multi-parametric (up to 9 colors) cell sorting. The sorted populations were functionally evaluated by their ability to form colonies and mammospheres, in vitro.

Results: We have compared, for the first time, the stem/progenitor markers of normal and malignant breasts side-by-side. Amongst all markers tested, we found CD44high/CD24low cell surface marker combination to be the most efficient at selecting normal epithelial progenitors. Further fractionation of CD44high/CD24low positive cells showed that this phenotype selects for luminal progenitors within Ep-CAMhigh/CD49f + cells, and enriches for basal progenitors within Ep-CAM-/low/CD49f + cells. On the other hand, primary breast cancer samples, which were mainly luminal Ep-CAMhigh, had CD44high/CD24low cells among both CD49fneg and CD49f + cancer cell fractions. However, functionally, CSC were predominantly CD49f + proposing the use of CD44high/CD24low in combination with Ep-CAM/CD49f cell surface markers to further enrich for CSC.

Conclusion: Our study clearly demonstrates that both normal and malignant breast cells with the CD44high/CD24low phenotype have the highest stem/progenitor cell ability when used in combination with Ep-CAM/CD49f reference markers. We believe that this extensive characterization study will help in understanding breast cancer carcinogenesis, heterogeneity and drug resistance.

Figure 1

Breast epithelial stem/progenitor cells are limited to CD49f + cell fractions. A) A representative dot plot showing the three Ep-CAM/CD49f epithelial cell populations (designated as A, B and C) after exclusion of stromal (mesenchymal Ep-CAM^neg/CD49^neg, hematopoietic CD45+ and endothelial CD31+) cells and as analyzed by flow cytometry. B) Number of mammospheres formed from 1000 cells, of each of the three mammary epithelial cell populations, seeded in a 96-well low-attachment plate for 14 days (mean ± SEM, n = 2).

Figure 2

CD44^high/CD24^low phenotype, within population A, enriches for basal progenitors. A) A representative dot plot showing the expression of each stem/progenitor cell marker in the gated population A (Ep-CAM^-/low/CD49f+, gated red dots) as analyzed by flow cytometry *numbers in brackets indicates average percentage (n = 10, mean ± SEM), quadrants show positivity while rectangles show "stem/progenitor" marker positive population. B&C) Number of mammospheres (B) and volume (C) of each stem/progenitor subpopulation within population A compared with the remaining bulk (rest) of population A (means ± S.E.M, n = 3). D) Colony formation assay for each cell subpopulation from population A (means ± S.E.M, n = 2). Data in B, C and D were normalized to unfractionated population A (Ep-CAM^-/low/CD49f+), **indicate statistical significance (p < 0.05). ALL = the subpopulation within population A that express all the three stem/progenitor markers simultaneously.

Figure 3

CD44^high/CD24^low phenotype, within population B, selects luminal progenitors. A) A representative dot plot showing the expression of each stem/progenitor cell marker in population B (Ep-CAM^high/CD49f + population,gated red dots) as analyzed by flow cytometry, *numbers in brackets indicates average percentage (n = 10, mean ± SEM). Quadrants show positivity while rectangles show "stem/progenitor" marker positive population. B&C) Mammosphere formation measured by either number (b) or volume (C) of each stem/progenitor subpopulation, compared with the remaining bulk (rest) of population B (means ± S.E.M, n = 3). D) Number of colonies formed of each cell subpopulation from population B (means ± S.E.M, n = 2). Data in B, C &D are normalized to unfractionated population B, **indicate statistical significance (p < 0.05). E) Top) Representative image (x400,) of immunohistochemistry for estrogen receptor (nuclear, brown) in each cell subpopulation of population B compared with population C (Ep-CAM^high/CD49f^neg) cells as a positive control. Gills hematoxylin (nuclear, blue) was used as a counterstain. Bottom) Quantification of estrogen receptor positive cells in each subpopulation of population B. ALL = the subpopulation within population B that express all the three stem/progenitor markers simultaneously.

Figure 4

CSC are abundant in primary CD44^high/CD24^low/Ep-CAM^high/CD49+ cancer cells. A) Representative dot plots for Ep-CAM/CD49f profile of tumor cells from different breast cancer patients as analyzed by flow cytometry (top) and histogram showing percentage of each Ep-CAM/CD49f population (n = 9 for normal breast and n = 13 for breast cancer samples). Lineage negative (CD45^neg, CD10^neg, CD31^neg) cancer cells were gated followed by exclusion (gating out) of Ep-CAM^neg/CD49f^neg mesenchymal fraction. Numbers in the corners indicate percentage of cells in each quadrant. B) Expression level of each of the studied stem/progenitor cell markers in Ep-CAM^high/CD49f^neg and Ep-CAM^high/CD49f + cell fractions in 16 breast cancer patients gated as in A. C) Percentage of CD44^high/CD24^low/Ep-CAM^high cancer cells (top) and after fractionation into CD49f^neg or CD49f + cells (bottom). Cases were stratified to four subtypes of breast cancers based on their estrogen receptor (ER), progesterone receptor (PR) status, as well as overexpression of Her2 as following: ER = ER+/PR+/Her2^neg, ER/Her2 = ER+/PR+/Her2+, Her2 = ER^neg/PR^neg/Her2+, and basal = ER^neg/PR^neg/Her2^neg, D&E) Mammosphere formation, measured by either number (D) or size (E) of tumor cells sorted for the stem/progenitor markers CD44^high/CD24^low or ALDH^high and expressed within Ep-CAM^high/CD49f^neg or Ep-CAM^high/CD49f + breast cancer cell subpopulations (means ± S.E.M, n = 7). **indicates statistical significance.

Figure 5

Similarities/differences between normal and malignant breast epithelial stem/progenitor subpopulations. The diagram summarizes the similarities and differences between the different Ep-CAM/CD49f populations. Each epithelial population was further fractionated into subpopulations based on the expression of other stem/progenitor cell makers. The three Ep-CAM/CD49f epithelial cell populations of the normal breast (A, B, and C), and their subpopulations, on top are compared with their malignant counterpart below. ^♣Percentage of each epithelial population (average ± SEM, n = 9 normal & n = 12 for breast cancer).**NA = not done due to very low cell yield *Mo/CFC = mammosphere/colony forming cells. For mammosphere and colony forming ability, √√√ = high, √√ = medium, √ = low, X = none.