Fig. 1: Breast cancer stem-like cells are highly enriched within the arteriolar niche.

a CD44⁺ cancer cells within a vascular niche (white arrows), as shown by staining with α-SMA (red) in human ER⁺ breast cancer. DAPI (blue) is used for nuclei staining. Normal tissue control (upper panel) refers to the tissues adjacent to breast cancer. Tissue structures are shown by H&E staining. Scale bar = 400 µm (upper panel) or 50 µm (lower panel). b BCSCs in a human BC specimen within TME. Human ER + BC specimen was co-stained with anti-CD44 and anti-ALDH1A1 antibodies, followed with appropriate second antibodies, and mounted with DAPI. Images were acquired using an Eclipse Ti2^®Nikon microscope and overlaid using NIS Elements software. BCSCs are indicated by white arrows and the blood vessel-like structure is shown by a white star. No CD24 was detected. Shown are representative images. Bar = 25 µm. c Ephrin B2 and ALDH1A1 co-staining in human ER⁺BC specimens. Ephrin B2 was stained green and ALDH1A1 red. White arrows show ephrin B2 positive arteriole, yellow arrows indicate ALDH1A1⁺ cancer cells, and tumor nest is indicated by the blue dashed circle. Cells in the cancer nests as indicated did not show significant ALDH1A1 staining (blue arrow). Scale Bar = 10 µm. d Quantification of ALDH1A1⁺ BC cells within the arteriolar niche. The arteriole is indicated by the white arrow, ALDH1A1⁺ BCSCs were shown by yellow arrows, and tumor nest was indicated by the blue dashed circle and BCs with low ALDH1A1 expression occurred within the tumor nest (blue arrow). H&E staining as a tissue structure control. Scale bar = 10 µm. BC cells were randomly counted under an Olympus BX60 fluorescence microscope linked with a CCD camera. The standard for examining cell locations was: (1) closed to the artery is defined as ALDH1A1⁺ BC cells within 50 µm from the SMA⁺ small arteries, and (2) close to the cancer nest refers to ALDH1A1⁺ BC cells locating cancer cell nest more than 50 µm from the SMA⁺ small artery. Up to 30 cancer cells were randomly counted within 50 µm × 50 µm area with five repetitions. The yellow arrows indicate ALDH1A1⁺ BCSCs. The dotted blue circle indicates a tumor nest containing BC cells to determine BCSCs that were localized more than 50 µm from the α-SMA⁺ arterioles. The bar graph shows relative numbers of ALDH1A1⁺ BCSCs that were related to either arteriolar niche or tumor nests in the BC tissues as detected by immunofluorescence microscopy. ***P < 0.001 compared with cells in the tumor nest.