Fig. 2: Indirect and direct interactions of breast cancer stem-like cells with the arteriolar endothelium within TME.

a Coculture of breast cancer cells and endothelial cells increased the expression of BCSC markers and stemness-associated genes. Total RNA in MCF-7 cells cultured in a six-well plate was collected for detection of stemness-associated genes at mRNA levels by RT-qPCR. The results of triplicate experiments are shown as mean ± SEM. ^***P < 0.001, ^**P < 0.01, or ^*P < 0.05 compared with the control. b An ALDH1A1⁺ BCSC was attached to the arteriolar endothelium within the vascular lumen. The human BC specimen was stained with ALDH1A1 and alpha-smooth muscle actin (α-SMA) antibodies used, followed by appropriate secondary antibodies and mounted with DAPI. Scale bar = 50 µm. c An ALDH1A1⁺ cell that expresses high levels of Notch1 and DLL4 interacts with DLL4⁺ endothelium in the arteriolar vessel. Human breast cancer specimens were processed and stained with Notch1, DLL4, and ALDH1A1 antibodies followed by appropriate secondary antibodies. Scale bar = 50 µm. d MCF-7 cells transduced with DsRed interacted with an HMVEC. MCF-7 cells transduced with Ds-Red that were grown in complete MammoCult^™ Media for 5 days were cocultured with HMVECi-Ds that expressed with DLL4 at the ratio of 2:1 in DMEM for 48 h. The cells were stained with Notch1 and DLL4 antibodies, followed by appropriate secondary fluorescence antibodies. Scale bar = 50 µm. The images were taken with an Olympus BX60 fluorescence microscope, and representative images are shown.