Figure 4

The stemness of ERα36 breast cancer cells and co-distribution of ERα36 with ALDH1A1 in human breast cancer tissues. (A) The numbers of mammospheres formed by MCF-7-ERα36⁺ and ERα36⁻ cells at the first and second passage. Columns are mean values (± SEM). n = 6. Statistical significance was determined by two-tailed Student's t test. ^*P < 0.01. (B) Flow cytometry showing higher percentage of ALDH1^high cells in MCF-7- ERα36⁺ cells. n = 3. (C) Limiting dilution showing higher tumorigenicity of FACS-sorted ERα36⁺ cells of MDA-MB 436 in NOD/SCID mice as compared with ERα36⁻ cells (n = 7 each group). Black line refers to ERα36⁺ cells and red line refers to ERα36⁻ cells. (D) Increased growth of orthotopical xenograft tumors formed by FACS-sorted MDA-MB 436-ERα36⁺ cells (n = 5 in each group). Tumor volume was measured at indicated time points. Data are presented as means ± SEM. Statistical significance was determined by two-tailed Student's t test. ^*P < 0.01. (E) Flow cytometry showing higher percentage of ALDH1^high cells in ERα36-expressing breast cancer cell variants (MCF-7/ERα36 and MDA-MB 436/shERα36-ERα36) as compared to control cells (MCF-7/mock or MDA-MB 436/shERα36 cells). n = 3. (F) Positive correlation between the expression of ALDH1A1 and ERα36 in breast cancer specimens analyzed with normal P-P plot of regression stand (dependent variable: ERα36 IHC score). P value was calculated with one-way analysis of variance (ANOVA) test. (G) ALDH1A1⁺ cancer cells (black arrow) co-expressing ERα36⁺detected by double IHC staining. The arrowheads indicate double expression of ALDH1A1 and ERα36. Brown staining denotes ERα36. Scale bar, 50 μm. (H, I) Increased primary and secondary generation of mammospheres formed by MCF-7/ERα36 (H) and MDA-MB 436/shERα36-ERα36 (I) cells as compared to control cells (MCF-7/mock and MDA-MB 436/shERα36). ^*P < 0.05. (J) Limiting dilution showing decreased tumor-initiating capacity of MDA-MB 436/shERα36 cells compared to control cells (MDA-MB 436/shControl and /shERα36-ERα36 cells) in NOD/SCID mice (seven mice in each group).