https://orcid.org/0000-0003-2615-617X
Figures & data
Table 1. The origins and mechanism of action for targeting BCSCs of all-trans retinoic acid (ATRA), salinomycin (Sal), and bufalin.
Figure 1. Schematic illustration of (A) transwell migration assay and (B) transwell invasion assay. BT-474 and BT-474R cells were seeded in the upper chamber in the serum-free DMEM and moved through the 8.0 µm pore membrane without or with Matrigel toward the lower chamber, containing DMEM with 10% FBS. Blue cell represents non-BCSCs and orange cells represent BCSCs.
Figure 2. Confirmation of trastuzumab-resistance in BT-474R. (A) Morphology observed under a light microscope. BT-474R has a similar morphology as BT-474; (B) Cell viability in the presence of trastuzumab for 5 days (mean ± SD, three independent experiments, four wells per concentration each time); (C) Cell growth curves and (D) Doubling time in the presence or absence of 10 µg/ml trastuzumab. ****p < 0.0001. ns: no significant difference.
Figure 3. Characterization of BCSCs subpopulation in BT-474 and BT-474R by flow cytometry. CD44+/CD24− BCSCs had minimal expression and ALDH+ BCSCs were elevated in BT-474R. MDA-MB-231 and SKBR3 were used as positive controls for CD44+/CD24− and ALDH+ cells, respectively. (A) CD44+/CD24− cells (in Q3). (B) Expression of ALDH+BCSC population in BT-474, BT-474R. Both experiments were performed in triplicates, and a representative graph is shown.
Figure 4. Representative images of mammospheres (A,B) and SEF (C,D) formed in BT-474 (A,C) and BT-474R (B,D) after treated with free ATRA (1 µM), Sal (1 µM), and Buf (0.02 µM) alone or in combination with Dox (0.1 µM) for 5 days. Data are mean ± SD from three independent experiments.
Figure 5. Physicochemical characteristics of liposome formulations. (A) Cryo-TEM images showing the morphology and structure of Buf-pSL and Buf-CDpSL. Scale bar: 100 nm (B) Dox and (C) Buf release in different mediums (pH 7.4 vs. pH 5.0). (D) Buf-CDpSL demonstrated better stability than Buf-pSL during the first 3 months.
Table 2. Characterization parameters of freshly prepared liposomes (n = 3 experiments).
Figure 6. ALDH+ cell populations in BT-474 and Bt-474R measured by an AldeRed™ ALDH detection assay following 24 h exposure to bufalin (Buf; 100 nM) and doxorubicin (Dox; 1 µM) alone or in combination as free drug(s) or loaded in pSL, with untreated cells as controls. Data are mean ± SD (n = 3 independent experiments). ns: not statistically different by ANOVA; ****p < 0.0001.
Figure 7. Buf And Dox inhibit sphere formation of BCSCs in BT-474 and BT-474R. (A) Morphologies of spheres formed in untreated BT-474 and BT-474R cells (control). (B,C) Morphologies of spheres in BT-474 (B) and BT-474R (C) after being treated with Buf (10 nM) and Dox (100 nM) mono-or combination either as free drug or liposomes for 5 days. Cells were seeded at 4000 cells per well in ultra-low adherent 24 well plate. (D) SFE of BT-474 and BT-474R with or without (control) Buf and Dox treatment (mean ± SD). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The experiments were repeated three times, with two wells for each treatment in each experiment, and images from one experiment are presented.
Figure 8. Buf And Dox inhibit self-renewal of BCSCs in BT-474 and BT-474R. (A) Morphology of secondary spheres formed in untreated BT-474 and BT-474R cells (control) were round. After treatments, the secondary mammospheres in (B) BT-474 and (C) BT-474R lost their integrity. (D) Secondary SFE of BT-474 and BT-474R (mean ± SD from three repeated experiments, with two wells for each treatment each time. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 9. In vitro anti-proliferation effect of Buf and Dox alone or in combination (as free drug or in liposomes) to BT-474 and BT-474R cells. (A) Cell viability after treatment of 24 h (mean ± SD, n = 3 experiments, four wells for each concentration in each experiment). Concentrations of the combined therapy were expressed based on Buf. (B) CI showing synergism of Buf and Dox in BT-474 and BT-474R as both free drug and liposomal formulation.
