Fig 1: The supernatants derived from breast cancer cells four days after drug withdrawal enhance CSC phenotypes and chemoresistance. (a) Experimental design for the study of autocrine factors in supernatants derived from chemotherapy-treated breast cancer cells. SUM190, SUM149 and MDA-MB-231 cells were treated with 15 nM paclitaxel for 4 days. After medium change and wash with PBS, the cells were cultured in fresh medium in the absence of any drug for additional 4 days. At day 8, the cell-free and drug-free culture media (Supernatants) were collected and applied to the same line of fresh cells respectively for 4 days. (b) Flow cytometric analysis of the percentage of CD44high/CD24-/low cells in SUM190, SUM149 and MDA-MB-231 cells after exposure to the supernatants for 4 days. The 7-AAD-negative live cells were pre-gated. Data represent means±S.D., n=3; * P<0.05. Veh-SNs: supernatants (SNs) derived from vehicle (Veh)-pretreated cells; Pa-SNs: supernatants derived from 15 nM paclitaxel (Pa)-pretreated cells. (c) Western blot analysis of stem cell-like markers (ALDH1, CD44 and OCT4) in SUM149 and SUM190 cells after exposure to the supernatants for 4 days. Supernatants (SNs) were collected as described in a and b. Pa 5 nM-SNs or Pa 15 nM-SNs: pretreated with 5 or 15 nM paclitaxel. ß-actin was used as an internal loading control. (d) Representative images from mammosphere assays. SUM190, SUM149 and MDA-MB-231 breast cancer cells were treated for 4 days with vehicle (Veh)-derived or paclitaxel (Pa)-derived supernatants (SNs), and then reseeded in ultra-low attachment plate (2 × 103/well, 6-well plate) and cultured for 8 days. Scale bar, 100 µm. Data represent means±S.D., n=3; *P<0.05. (e)qPCR analysis of stemness-associated (ALDH1, SOX2, OCT4, NANOG, CD44 and c-MYC), epithelial-to-mesenchymal transition (EMT)- (SLUG, MMP9, ZEB1, SNAIL and E-CADHERIN) signature genes in SUM190, SUM149 and MDA-MB-231 cells after exposure to the supernatants (SNs) for 4 days. Data represent means±S.D., n=3; *P<0.05 compared with vehicle-supernatants group. See also Supplementary Figure 1
Fig 2: ALDH1high cells displayed increased tumorogenic properties.(A) Flow cytometry cell sorting of ALDEFLUOR high (ALDH1high) and low (ALDH1low) populations in T-5H-O cells. (B) Protein levels of the indicated factors in sorted populations. ß-actin levels were used as loading control. (C) Relative quantification of the soft agar colony formation (anchorage independent growth) ability of ALDH1high and ALDH1low populations. Representative images of colonies are shown (top panels). (D–F) Sorted cells were growth as tumorspheres for two successive passages (D), disaggregated and assayed for soft agar colony formation (E) and finally, recovered from soft agar cultures and assayed for ALDEFLUOR activity (F). (G) H&E (left panel) and ALDH1A1 immunostaining (right panel) of tumors arising in mice inoculated with 400 ALDH1high cells. Scale bars = 500 µm in panels (C–E) and = 200 µm (100 µm for right panel inset) in panel G. Error bars represent the standard deviation and asterisks indicate a statistically significant difference between the indicated conditions (*p < 0.05, **p < 0.001; two-sided Student’s t-test).
Fig 3: Depletion of ALDH1 isoforms result in reduced ALDEFLUOR activity.(A) Protein levels of ALDH1A1 and ALDH1A3 48 h after the transfection of the indicated siRNAs in T-5H-O cells. ß-Actin levels are presented as a loading control. (B,C) ALDEFLUOR assay of T-5H-O cells depleted for ALDH1A1 or ALDH1A3. The percentage of reduction achieved by each ALDH1 isoform depletion respect to the control siRNA values (n = 3 independent experiments) (B) and a representative assay (C) is presented.
Fig 4: Combination therapy with YAP and Wnt small molecule inhibitors effectively retards tumor growth and reduces CSC enrichment and tumorigenesis in vivo; low expression of CTNNB1 and YAP1 genes correlates with low expression of CD44+ and ALDH1A1+ genes in patients’ tumor samples while inversely correlates with improved survival in breast cancer patients. (A,B) Mesenchymal-like (Mes) and epithelial-like (Epi) MDA-MB-231 TNBC cells were injected into the mammary fat pads of athymic nude mice. When the tumors reached a mean diameter of 3 mm, mice were separated into four groups and intraperitoneally injected daily with the vehicle (DMSO), ICG-001 (100 mg·g-1 per day), simvastatin (5 mg·g-1 per day), and ICG-001 + simvastatin for 15 days. After conclusion of treatments, tumors were harvested from the mice, photographed, and weighed. Data represent means ± SD; n = 4 mice for each group; *P < 0.05, **P < 0.01. Scale bar = 1 cm. (C, D) Flow cytometric analysis of the living CD44high/CD24low and ALDH + CSC subpopulations of dissociated mesenchymal-like (Mes) and epithelial-like (Epi) tumors after 15 days of treatment with vehicle (DMSO), ICG-001, and/or simvastatin. Insets within flow cytometric plots depict DEAB control for ALDH baseline determination. Data represent means ± SD; n = 4 mice for each group; *P < 0.05, **P < 0.01, in comparison with the indicated groups or vehicle control. (E) Mesenchymal-like (Mes) and epithelial-like (Epi) MDA-MB-231 xenografts were dissociated into single cell suspension and retransplanted into the mammary fat pads of new nude mice in serial limiting dilutions (105, 104, 103, or 102 cells per injection). Tumor formation was observed for 6 weeks. (F) Low levels of CTNNB1 (a pivotal effector of the canonical Wnt signaling pathway) and YAP1 (YAP signaling) gene expression in breast cancer patients’ samples (Alt) correlates with low levels of CD44 and ALDH1A1 gene expression in comparison with those patients’ samples without showing the reduced expression of CTNNB1 and YAP1 genes (Ctrl). N = 2509 patients with invasive breast cancer, *P < 0.05, ***P < 0.001. (G) Kaplan–Meier curves for overall survival of the patients with low levels of CTNNB1 (Wnt) and YAP1 (YAP) protein expression in cancer samples (red curve) in comparison with those patients with unaltered expression (blue curve). n = 887, ***P < 0.001, log-rank test.
Fig 5: Increased expression of SOX2 and ALDH1 in CSC subpopulations during tumor progression.(A) Protein levels of ALDH1A1 and SOX2 in adherent (A) and tumorsphere (T) cultures of MSC-5H-FC and T5H-FC#1. ß-actin levels were used as loading control. (B,D) Immunofluorescence staining of SOX2 (B) and ALDH1A1 (D) in adherent cultures or tumorsphere disaggregates and allowed to attach to glass slides of MSC-5H-FC and T5H-FC#1 cells. Scale bars = 50 µm (B) or = 40 µm (D). (C,E) Quantification of nuclear SOX2 (C) or cytosolic and nuclear ALDH1A1 (E) staining in each sample. (F) Simultaneous immunofluorescence staining of SOX2 and ALDH1A1 in GFP-expressing MSC-5H-FC- and T5H-FC#1-derived tumorspheres. Scale bars = 200 µm. (G) Protein levels of ALDH1A1, ALDH1A3 and ß-actin in adherent (A) and tumorsphere (T) cultures of the indicated cell types. (H,I) ALDEFLUOR assay showing the activity of ALDH1 in adherents and tumorspheres cultures of MSC-5H and T5H-O cells. ALDH1 activity was blocked with the specific inhibitor DEAB to establish the basal level. The summary of 3 independent experiments (I) and a representative assay (H) is presented. Error bars represent the standard deviation and asterisks indicate a statistically significant difference between the indicated conditions (*p < 0.05, **p < 0.001; two-sided Student’s t-test).
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