Fig. 3: Cancer stemness contributes to the suppression of metastases.

a CMT cell lines were stained with CD44 and CD24 antibodies and analyzed by flow cytometry. b Growth rates of the CHMp and CHMm cells. Cell proliferation was measured via manual cell counting. c ALDH activity was examined using an ALDEFLUOR assay in CHMp and CHMm cells. Fluorescence images were examined using a 40x objective lens; the scale bar indicates 130 μm. d Representative confocal microscopy images of mammospheres formed by CHMp and CHMm. Scale bar, 400 μm. e Mammospheres were stained with antibodies against the CSC markers CD44 (green) and CD24 (red). DAPI (blue) was used as a nuclear marker. Scale bar, 100 μm. f Whole-body IVIS bioluminescence images on Day 21. Mice were injected with equal numbers of CHMp (top) and CHMm (bottom) cells on the mammary fat pads (n = 5 per mouse group). g Graph showing the total flux of IVIS-treated mice used to measure their growth ability. The primary tumor volume was quantified once a week. h Picture of surgically removed CHMp (top) and CHMm (bottom) primary tumor masses. i Flow cytometry analysis of CMT cell lines with CD44 and CD24 antibodies. j Schematic illustration of the isolation of the CSC population (CD44+/CD24−) from the CIPp cell line. k An MTT assay was conducted to compare the viability of cells treated with CIPp exosomes and CIPp-CSC exosomes. l The proliferation rate of cells treated with PBS or exosomes was determined by manual cell counting. CIPp-CSC exosomes resulted in a greater decrease in the proliferation rate of CIPm cells than did CIPp exosomes. All the data are presented as the mean ± SEM. Experiments were performed in triplicate unless otherwise indicated. The statistical analysis is presented. **P < 0.01 and ***P < 0.001. ns not significant. The results are presented as the mean ± SD (5 mice were used for each group).