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Association of human breast cancer CD44-/CD24- cells with delayed distant metastasis

  1. Xinbo Qiao
  2. Yixiao Zhang
  3. Lisha Sun
  4. Qingtian Ma
  5. Jie Yang
  6. Liping Ai
  7. Jinqi Xue
  8. Guanglei Chen
  9. Hao Zhang
  10. Ce Ji
  11. Xi Gu
  12. Haixin Lei
  13. Yongliang Yang
  14. Caigang Liu  Is a corresponding author
  1. Department of Oncology, Shengjing Hospital, China Medical University, China
  2. Dapartment of Urology, Shengjing Hospital, China Medical University, China
  3. Department of Breast Surgery, Liaoning Cancer Hospital and Institute, Cancer Hospital of China Medical University, China
  4. Department of General Surgery, Shengjing Hospital, China Medical University, China
  5. Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, China
  6. Center for Molecular Medicine, School of Life Science and Biotechnology, Dalian University of Technology, China
Research Article
Cite this article as: eLife 2021;10:e65418 doi: 10.7554/eLife.65418
7 figures and 6 additional files

Figures

Figure 1 with 3 supplements
Association of a high frequency of CD44-/CD24- cells in breast cancer tissues with delayed tumor distant metastasis.

(A) Immunofluorescent analysis of the percentages of CD44-/CD24- cells in tissue samples from breast cancer patients with or without tumor metastasis. The P-value was determined by Student’s t-test. N = 105 and 250 for with and without metastasis, respectively. (B) ROC analysis of the sensitivity and specificity of 19.5% of CD44-/CD24- cells as a cutoff value for evaluating delayed distant metastasis. (C) Metastasis rates in patients with different molecular subtypes of breast cancer after they were stratified into high (≥19.5%) or low frequency of CD44-/CD24- cells. (D) Metastasis rates in patients with ≥2% CD44+/CD24- cells (n = 105) vs. C1 patients with <2% CD44+/CD24- cells and ≥19.5% CD44-/CD24- cells (n = 69). (E) Kaplan–Meier analysis of the DFS of breast cancer patients after they were stratified, based on the indicated measure. (F) Kaplan–Meier analysis of the DFS of TNBC patients with standard chemotherapy after they were stratified into ≥19.5% CD44-/CD24- cells (n = 52); <19.5% CD44-/CD24- cells (n = 80). (G) Kaplan–Meier analysis of the DFS of luminal breast cancer patients with standard endocrine therapy after they were stratified into ≥19.5% CD44-/CD24- cells (n = 24) vs. <19.5% CD44-/CD24- cells (n = 23).

Figure 1—figure supplement 1
Immunofluorescent analysis of CD44+/CD24- and CD44-/CD24- cancer cells in human breast cancer specimens.

(A, B) Representative H and E staining and immunofluorescent images of consecutive breast cancer tissue sections that had been stained with anti-CD24 and anti-CD44 antibodies. Scale bar, 50 μm; high indicate (>2% of CD44+/CD24- cells or ≥19.5% of CD44-/CD24- cells while low represents <2% of CD44+/CD24- cells or <19.5 of CD44+/CD24- cells).

Figure 1—figure supplement 2
A higher frequency of CD44-/CD24- cancer cells, like CSCs, is associated with delayed distant metastasis and a high frequency of CSCs is related to worse.

DFS in patients with different subtypes of breast cancer. (A) Immunofluorescent staining analysis of the percentages of CD44+/CD24+, CD44-/CD24-, CD44-/CD24+, and CD44+/CD24- cells in breast cancer tissue specimens (n = 355). ****p<0.0001 vs. CD44+/CD24- cells and CD44-/CD24- cells, determined by Student’s t-test. (B) The distant metastasis rates of patients with different subtypes of breast cancers 4 years post tumor resection after they were stratified into high or low frequency of CD44-/CD24- cells. N = 10/44 and 4/45 for high and low luminal A; N = 15/30 and 7/38 for high and low HER-2+; N = 10/24 and 6/40 for high and low TNBC, respectively. (C) The dynamic metastatic rates of patients with high or low frequency of CSCs in their tumor specimens. (≥ 2%; n = 105 vs. < 2%; n = 250). (D) Kaplan–Meier analysis of the DFS of patients with luminal breast cancer (≥2% CD44+/CD24- cells; n = 47 vs. <2%; n = 112), HER-2+ breast cancer (≥2% CD44+/CD24- cells; n = 14 vs. < 2% one; n = 25), and TNBC breast cancer (≥ 2% CD44+/CD24- cells; n = 44 vs. < 2%; n = 113).

Figure 1—figure supplement 3
A high frequency of CD44-/CD24- cells is associated with worse DFS of breast cancer patients.

Individual types of breast cancer patients were stratified into high or low frequency of CD44-/CD24- cells in their tumor tissue specimens and their DFS was estimated by Kaplan-Meier method and analyzed by log-rank. (A) Luminal (≥19.5% CD44-/CD24- cells; n = 71 vs. <19.5%; n = 88), HER-2+ (≥19.5% CD44-/CD24- cells; n = 17 vs. <19.5%; n = 22), and TNBC (≥19.5% CD44-/CD24- cells; n = 58 vs. <19.5%; n = 99). (B) Breast cancer patients with chemotherapy (≥19.5% CD44-/CD24- cells; n = 118 vs. <19.5%; n = 161). (C) Breast cancer patients with low frequency of CSCs in their specimens were treated with chemotherapy and stratified into ≥19.5% CD44-/CD24- cells; n = 54 vs. <19.5%; n = 138. (D) TNBC patients with low frequency of CSCs and chemotherapy were stratified into the C1 group (≥19.5% CD44-/CD24- cells) or the C0 group (n = 21 vs. <19.5%; n = 70).

Figure 2 with 1 supplement
The higher frequency of CD44-/CD24- tumor cells is associated with delayed distant metastasis and worse DFS in the testing group of patients.

(A) Postoperative metastasis rate in the test group of patients with different molecular subtypes of breast cancer after they were stratified by 19.5% of CD44-/CD24- cells. (B and C) Kaplan–Meier analysis of the DFS (B) and OS (C) of all patients in the testing group after they were stratified into ≥19.5% of CD44-/CD24- (n = 100) vs. <19.5% of CD44-/CD24- tumor cells (n = 121). (D) Longitudinal measurements of metastasis rates among all breast cancer patients (n = 211), patients with ≥2% CD44+/CD24- cells (n = 68) and the C1 group of patients with <2% of CD44+/CD24- and ≥19.5% CD44-/CD24- cells (n = 153). (E) Kaplan–Meier analysis of the DFS in the testing group of TNBC patients with chemotherapy after they were stratified into ≥19.5% of CD44-/CD24- (n = 24) vs. <19.5% of CD44-/CD24- tumor cells (n = 35). (F) Kaplan–Meier analysis of the DFS in the testing group of luminal breast cancer patients with endocrine therapy after they were stratified into ≥19.5% of CD44-/CD24- (n = 22) vs. <19.5% of CD44-/CD24- tumor cells (n = 19). p-Values were determined by log-rank test.

Figure 2—figure supplement 1
Kaplan–Meier and the log-rank test analyses of DFS and OS in the testing group of breast cancer patients stratified by percentage of CD44-/CD24- cells.

(A, B) The DFS and OS of stratified luminal cases (≥19.5% CD44-/CD24- cells; n = 44 vs. <19.5%; n = 45), HER-2+ cases (≥19.5% CD44-/CD24- cells; n = 31 vs. <19.5%; n = 37), and TNBC cases (≥19.5% CD44-/CD24- cells; n = 25 vs. <19.5% ones; n = 39). (C) The DFS of breast cancer patients with a low frequency of CSCs in their specimens were treated with chemotherapy and stratified into ≥19.5% CD44-/CD24- cells; n = 47 vs. <19.5%; n = 94. (D) The DFS and OS of breast cancer patients with a low frequency of CSCs after they were stratified into >19.5% CD44-/CD24- cells; n = 51 vs. <19.5% ones; n = 181.

Figure 3 with 1 supplement
Spontaneous conversion of CD44-/CD24- TNBC cells into CD44+/CD24- CSCs.

(A) A diagram Illustrated the experimental protocol for testing the spontaneous conversion of primary human breast cancer CD44-/CD24- cells into CD44+/CD24- CSCs in vitro. (B, C) Flow cytometry analysis of the spontaneous conversion of primary human or xenograft breast cancer CD44-/CD24- cells into CD44+/CD24- CSCs in vitro. The primary human and xenograft breast cancer CD44-/CD24- cells were purified from human fresh TNBC tissue cells or MDA-MB-231 xenograft tissue cells by flow cytometry sorting and cultured in L15 medium for 7 and 21 (specifically for cells from xenograft tissue cells) days, respectively. The percentages of CD44+/CD24- CSCs were analyzed by flow cytometry. (D) A diagram illustrated the experimental protocol for testing the spontaneous conversion of CD44-/CD24- cells from TNBC cells into CD44+/CD24- CSCs. (E) Flow cytometry analysis of the percentages of CD44+/CD24- CSCs. CD44-/CD24- MDA-MB-231 cells were purified by flow cytometry sorting and cultured in the indicated medium for the indicated duration, followed by flow cytometry analysis.

Figure 3—figure supplement 1
The spontaneous conversion of CD44-/CD24- TNBC cells into CD44+/CD24- CSCs in vitro.

(A) Primary human breast cancer cells were cultured for 5 and 10 days. Breast cancer cells were isolated from fresh human TNBC tumors (N = 3). (B) Flow cytometry analysis of CD44-/CD24- cells and their spontaneous conversion into CSCs. CD44-/CD24- cells were purified from MDA-MB-468 cells by flow cytometry sorting and cultured in SC or L15 medium for 7 days. The percentages of CD44+/CD24- CSCs in CD44-/CD24- MDA-MB-468 cells were analyzed by flow cytometry.

Figure 3—figure supplement 1—source data 1

The picture of primary human breast cancer cells.

https://cdn.elifesciences.org/articles/65418/elife-65418-fig3-figsupp1-data1-v2.zip
Both WT CSCs and CD44-/CD24- CSCs from TNBC cells have similar biological behaviors in vitro.

(A) CD44-/CD24- CSCs and WT CSCs displayed similar ability to form mammosphere in vitro following culturing them for 7 days. (B) CCK-8 assay analysis of WT and CD44-/CD24- CSC proliferation. (C) Flow cytometry analysis of the frequency of CSCs after culture WT and CD44-/CD24- CSCs in SC medium for 7 days. (D) Flow cytometry analysis of WT and CD44-/CD24- CSC differentiation after culturing them in SC medium for 7 days. Data are representative images or expressed as the mean or mean ± SD of each group from three independent experiments. (E) Western blot analysis of stemness marker expression in WT and CD44-/CD24- CSCs.

Both WT and CD44-/CD24- CSCs from MDA-MB-231 cells exhibit similar tumorigenicity and comparable abilities to differentiate and induce lung metastatic in vivo.

(A) Both WT and CD44-/CD24- CSCs had similar tumorigenicity to induce comparable sizes of tumors in BALB/c nude mice following subcutaneous implantation for 21 days (n = 5–8 per group). (B) Flow cytometry analysis of different subtypes of TNBC cells in xenograft tumors. (C) Lung metastasis. NOD/SCID mice were intravenously injected with the same number of WT or CD44-/CD24- CSCs and 21 days later, the mice were euthanized and their lungs were dissected for weighing and histological H and E staining to examine lung metastatic morphology and nodule sizes. Data are representative images or expressed as the mean ± SD of each group (n = 5–8 per group), and samples were analyzed from three independent experiments.

Figure 6 with 1 supplement
RHBDL2 expression is up-regulated during the process of CD44-/CD24- MDA-MB-231 cell conversion into CD44+/CD24- CSCs.

(A) Representative images of micrograft plates for culture of CD44-/CD24- MDA-MB-231 cells at a single cell level. CD44-/CD24- MDA-MB-231 cells were purified by flow cytometry sorting and transfected with pGL3-OCT4-EGFP, followed by culturing them in micrograft plates at a single cell level for 24, 72, and 120 hr, and the average of the gene expression profile of three cells are photographed. (B) RNA-seq analysis and heatmap displayed the top DEGs during the process of CD44-/CD24- MDA-MB-231 cell conversion into CD44+/CD24- CSCs. (C) Kaplan-Meier estimation of the association of the expression of DEGs with DFS in breast cancer patients in TCGA database. (D) RT-qPCR analysis of the relative levels of gene mRNA transcripts in WT CD44+/CD24-, CD44-/CD24- CSCs, and unconverted CD44-/CD24- cells after culture for 7 days. Data are representative images or expressed as the mean ± SD of each group from at least three separate experiments. *p<0.05, ***p<0.001; ##p<0.01, ###Pp<0.001 vs. the CD44-/CD24- cells, determined by Student’s t-test.

Figure 6—figure supplement 1
Bioinformatic analysis of DEGs between 24 hr converted CSCs and CD44-/CD24- MDA-MB-231 cells.

(A, B) GO analysis of KEGG enrichments in the different.

RHBDL2 silencing inhibits the spontaneous conversion of CD44-/CD24- cells into CSCs by attenuating the YAP1/NF-kB signaling through enhancing USP31 expression in TNBC cells.

(A) Western blot analysis exhibited that RHBDL2 silencing decreased RHBDL2 and YAP1 expression, but increased YAP1 phosphorylation in TNBC cells. (B) Western blot analysis displayed that RHBDL2 silencing decreased RHBDL2 and YAP1 expression, NF-kB activation, but increased USP31 expression and YAP1 phosphorylation in the indicated TNBC CD44-/CD24- cells. (C) Western blot analysis of cytoplasmic and nuclear YAP1 protein levels in MDA-MB-231/MDA-MB-468 cells following RHBDL2 silencing indicated that RHBDL2 silencing mitigated the nuclear translocation of YAP1 in TNBC cells. (D and E) RHBDL2 silencing inhibited the spontaneous conversion of CD44-/CD24- cells into CSCs in vitro. CD44-/CD24- cells were purified from MDA-MB-231 (D and E) RHBDL2 silencing inhibited the spontaneous conversion of CD44-/CD24- cells into CSCs in vitro. CD44-/CD24- cells were purified from MDA-MB-231 (D) and MDA-MB-468 (E ) cells and transfected with the control or RHBDL2-specific siRNA, followed by culturing them in L15 medium for 7 days. Subsequently, the percentages of CD44+/CD24- CSCs in each group of cells were analyzed by flow cytometry. (F) RHBDL2 silencing attenuated mammosphere formation of CD44-/CD24- CSCs. Following transfected with the control or RHBDL2-specific siRNA, CD44-/CD24- CSCs were cultured in L15 medium for 7 days and the formed mammospheres were photoimaged (magnification x 400) and their numbers and sizes were measured in a blinded manner. Data are representative images or expressed as the mean ± SD of each group from at least three separate experiments. *p <0.05, **p <0.01, determined by Student t-test. (G) A diagram illustrates the possible mechanisms underlying the action of RHBDL2 in enhancing the spontaneous conversion of CD44-/CD24- cells into CD44+/CD24- CSCs, contributing to delayed distant metastasis of breast cancer.

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