Unraveling the roles of CD44/CD24 and ALDH1 as cancer stem cell markers in tumorigenesis and metastasis

Abstract

CD44/CD24 and ALDH1 are widely used cancer stem cell (CSC) markers in breast cancer. However, their expression is not always consistent even in the same subtype of breast cancer. Systematic comparison of their functions is still lacking. We investigated the expression of CD44, CD24 and ALDH1 in different subtypes of breast cancer cells, and explored their relationship with cancer progression. We defined a parameter CD44/CD24 ratio to present the expression level of CD44 and CD24 and found that high CD44/CD24 ratio and ALDH1⁺ are both indicators for cancer malignancy, but play different functions during tumor progression. High CD44/CD24 ratio is more related to cell proliferation and tumorigenesis, which is confirmed by mammosphere formation and tumorigenesis in xenotransplanted mice. ALDH1⁺ is a stronger indicator for cell migration and tumor metastasis. Suppression of CD44 and ALDH1 by siRNA led to decreased tumorigenicity and cell migration capacity. The combination of high CD44/CD24 ratio and ALDH1⁺ would be a more reliable way to characterize CSCs. Moreover, both high CD44/CD24 ratio and ALDH1⁺ were conserved during metastasis, from the primary tumors to the circulating tumor cells (CTCs) and the distant metastases, suggesting the significant value of these CSC markers in assisting cancer detection, prognostic evaluation, and even cancer therapeutics.

Figure 1

The expression of CD44, CD24 and ALDH1 in different molecular subtypes of breast cancer. Four breast cancer cell lines were compared: MCF-7 (Luminal A subtype), SK-BR-3 (HER2 over expression subtype), MDA-MB-468 (basal-like epithelial subtype), and MDA-MB-231 (basal-like mesenchymal subtype). (A) Flow cytometry analysis of the expression of CD44 and CD24 in different breast cancer cell lines. Cells were double stained with anti-CD44-PE and anti-CD24-FITC. The threshold lines were set according to the isotype control. The accuracy of the double immunostaining was confirmed by comparison with single immunostaining for CD44 and CD24 respectively. (B) Immunofluorescence images showing the expression of CD44 and CD24 in different subtypes of breast cancer cell lines. Cells were labeled with anti-CD44-PE, anti-CD24–FITC and the nuclei stain DAPI. Scale bar: 20 μm. (C) The ratio of CD44/CD24 in different subtypes of breast cancer cell lines. Ratios were calculated from the percentage of CD44 and CD24 positive subpopulations in the flow cytometry analysis. Data represent means ± SD (n = 3), **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) Immunofluorescence images showing the expression of ALDH1 in different breast cancer cell lines. Cells were labeled with anti-ALDH1 (magenta) and DAPI (blue), scale bar: 20 μm. (E) The average fluorescent intensities of ALDH1 in different breast cancer cell lines, data were calculated from three parallel immunofluorescence images. Data represent means ± SD (n = 3), ***P < 0.001, ****P < 0.0001.

Figure 2

CD44/CD24 ratio positively correlates with cell proliferation and tumorigenesis. (A,B) The cell proliferation potential of different breast cancer cell lines as indicated by the expression of Ki67. Cells with high CD44/CD24 ratio exhibit high proliferation potential. (A) Immunofluorescence images showing the expression of Ki67 in different breast cancer cell lines. Cells were labeled with anti-Ki67 (red) and DAPI (blue), scale bar: 20 μm. (B) The expression level of Ki67 in in different breast cancer cell lines. The average fluorescence intensities were calculated from three parallel immunofluorescence images. Data represent means ± SD (n = 3), **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) Breast cancer cells with high CD44/CD24 ratio exhibit strong tumorigenic ability. Tumor growth curved of the four subtypes of breast cancer cells injected into female BALB/c nude mice at the amount of 4 × 10⁶ cells. Data represent means ± SD (n = 3).

Figure 3

The expression of CD44, CD24, and ALDH1 in mammosphere formed by MDA-MB-231 cells. (A) Optical microscope image showing MDA-MB-231 cells growing as non-adherent mammospheres in medium after 10 d of cultivation. Scale bar, 100 μm. (B) Representative immunofluorescence images showing the expression of CD44, CD24 and ALDH1 in mammosphere formed by MDA-MB-231, in accordance with the expression of cells. Cells were labeled with anti-CD44 (green), anti-CD24 (red) and anti-ALDH1 (magenta), the nuclei stain DAPI (blue). Scale bar: 20 μm.

Figure 4

ALDH1 expression positively correlates with cell migration and tumor metastasis. (A) High ALDH1 expression promotes liver metastasis of breast cancer. Representative hematoxylin and eosin (H&E) staining images of livers isolated from mice six weeks after being injected with different subtypes of breast cancer cells. Mice injected without cells were used as control. Scale bar, 1mm. (B) The calculated liver metastasis area of mice injected with breast cancer cell lines. Data represent means ± SD (n = 3), **P < 0.01, ***P < 0.001, ****P < 0.0001. (C–E) Transwell migration assay. (C) Schematic illustration of the transwell migration assay. Transwell chambers with 8-μm-pore polycarbonate membranes were placed in a 24 well plate, 2 × 10⁵ of cells were seeded in the upper chamber of the assay model. Cells migrated to the bottom chamber were counted after 24h by staining with crystal violet. (D) The representative microscope images of the cells migrated to the bottom chamber, scale bar, 200 μm. (E) The average number of migrated cells in different breasts cancer cell lines (n = 3), ****P < 0.0001. (F,G) Wound healing assay. (F) Representative images showing the changes of wounds of the four breast cancer cells in the six-well culture plate. Images were obtained at 0 and 48 hours after the creation of the wounds. Scale bar 200 μm. (G) The quantification of cell migration distance in the four breast cancer cells. Data represent means ± SD (n = 3), ****P < 0.0001.

Figure 5

ALDH1 expression positively correlates with the expression of the tumor metastatic marker CXCR4. (A) Immunofluorescence images showing the expression of CXCR4 in different breast cancer cell lines. Cells were labeled with anti-CXCR4 (green) and DAPI (blue), scale bar: 20 μm. (B) The expression level of CXCR4 in in different breast cancer cell lines. The average fluorescence intensities were calculated from three parallel immunofluorescence images. Data represent means ± SD (n = 3), **P < 0.01, ***P < 0.001. (C) Western blot analysis of the expression of ALDH1, CXCR4 in different breast cancer cell lines. β-actin was used as control. The bolts were cropped from their original images and the full-length blots were presented in Supplementary Figure S5.

Figure 6

Suppression of CD44 decreases the expression of Ki67, and the capacities for mammosphere formation and tumorgenesis. (A) Analysis of CD44 mRNA levels following siRNA treatment. The mRNA expression decreased to 0.41 after CD44 siRNA treatment compared with control, Data represent means ± SD (n = 3), ****P < 0.0001. (B) Immunofluorescence images showing the expression of CD44, Ki67 in cells treated with control siRNA or CD44 siRNA, scale bars: 20 μm. (C) MDA-MB-231 cells transfected by control siRNA, CD44 siRNA or ALDH1 siRNA growing as non-adherent mammospheres after 10 d of mammosphere cultivation. Scale bar, 100 μm. (D) MDA-MB-231cells are tumorigenic while cells transfected by CD44 siRNA or ALDH1 siRNA could not form tumor with amount of 4 × 10⁶ in BALB/c nude mice after two weeks. All the experiments were performed in triplicate.

Figure 7

Suppression of ALDH1 decreases the expression of CXCR4 and the tumor cell migration capacity. (A) Analysis of ALDH1 mRNA levels following siRNA treatment. The mRNA expression decreased to 0.29 after ALDH1 siRNA treatment compared with control, Data represent means ± SD (n = 3), ***P < 0.001. (B) Immunofluorescence images showing the expression of ALDH1, CXCR4 in cells treated with control siRNA or CD44 siRNA, scale bars: 20 μm. (C) Transwell migration assay. Representative microscope images of the MDA-MB-231 cells transfected by CD44 or ALDH1 siRNA migrated to the bottom chamber, scale bar, 200 μm. (D) Wound healing assay. Representative images showing the changes of wounds of the MDA-MB-231 cells transfected by CD44 or ALDH1 siRNA in the six-well culture plates. Images were obtained at 0 and 24 hours after the creation of the wounds. Scale bar 200 μm. All the experiments were performed in triplicate.

Figure 8

CD44/CD24 ratio and ALDH1 remain stable from the primary tumor to the liver metastases. (A) Representative immunofluorescent images showing the expression of CD44, CD24 in the primary tumor and liver metastases of immunodefidient mice injected with MDA-MB-231 cells. Tissues were labeled with anti-CD44 (red), anti-CD24 (green) and the nuclei stain DAPI (blue). Scale bar: 20 μm. (B) CD44/CD24 ratios in the primary tumor and the liver metastases. The average ratios of CD44/CD24 were calculated from three parallel immunofluorescent images. All the experiments were performed in triplicate. Data represent means ± SD (n = 3), **P < 0.01. (C) Representative immunofluorescent images showing the expression of ADLH1 in the primary tumor and liver metastases of immunodefidient mice injected with MDA-MB-231 cells. Tissues were labeled with anti-ALDH1 (magenta) and the nuclei stain DAPI (blue). Scale bar: 20 μm. (D) The average fluorescent intensities of ALDH1 from the primary tumor and the liver metastases. The average fluorescent intensities were calculated from three parallel immunofluorescent images. All the experiments were performed in triplicate. Data represent means ± SD (n = 3).

Figure 9

CTCs exhibit high CD44/CD24 ratio and ALDH1 expression. (A) Schematic illustration of the isolation and detection of CTCs from the blood using membrane filter. The isolation device contains an 8 μm pore filtering membrane and a 25 μm diameter filter holder. (B) Immunofluorescent images showing the expression of CK19, CD45, CD44, CD24 and ALDH1 in CTCs isolated from the immunodeficient mice injected with MDA-MB-231 cells. CTCs captured on the 8 μm polycarbonate membrane were stained with anti-CK19 (green), anti-CD45, anti-CD44 (red), anti-CD24 (green), anti-ALDH1 (magenta) and DAPI (blue), respectively. Scale bar: 20 μm. (C) Immunofluorescent images showing the expression of CD44, ALDH1 and CD45 in CTCs isolated from breast cancer patient. CTCs captured on the 8 μm polycarbonate membrane were stained with anti-CD44 (green), anti-ALDH1 (magenta), anti-CD45 (red), and DAPI (blue), respectively. Scale bar: 20 μm.