Ulex europaeus agglutinin 1 lectin

Paraffin-embedded skin sections (5-μm thick) including both epidermis and dermis were stained with anti-human LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1) antibody (R&D Systems, Minneapolis, Minnesota; 3 μg/ml, overnight, 4°C; matched isotype goat immunoglobulin G as negative control), after overnight low-temperature antigen retrieval (Unitrieve; Innovex Biosciences, Richmond, California) and nonspecific signal blocking. Alexa fluor 488–conjugated secondary antibody (2 μg/ml; Invitrogen, Carlsbad, California) and Ulex europaeus agglutinin I lectin (10 μg/ml; Vector Laboratories, Burlingame, California) were used to stain LYVE-1⁺ lymphatic vessels and blood endothelial cells, respectively. Tiled images of the entire epidermal line and the subpapillary dermis were acquired at 20× magnification. Blinded automated image analysis for microvessels quantification in the 600-μm-thick dermal area starting from the epidermis–dermis junction and spanning the entire length of the biopsy was performed with ImageJ version 1.52q (National Institutes of Health, Bethesda, Maryland), by pre-defined homogeneous thresholding criteria and the “analyze particles” function (Supplemental Figure 1).

PBMNCs and QQMNCs were cultured for seven days in 5% FBS-EGM^®-2 BulletKit depleted hydrocortisone on human fibronectin-coated Primaria dishes. PBMNCs or QQMNCs were seeded in 96-well plates at 5 × 10⁴ cells in 0.2 mL of EGM-2 medium per well.
Early EPCs were cultured as previously described. Briefly, cells were cultured for seven days. To detect the uptake of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine–labelled acetylated LDL (DiI-ac-LDL Biomedical Technologies Inc.), cells were incubated with DiI-ac-LDL (6 μg/mL, 37 °C, 2 h). Cells were then fixed with 4% paraformaldehyde for 10 minutes and incubated with fluorescein-labelled Ulex Europaeus Agglutinin I lectin (Vector Laboratories, Burlingame, CA, USA) for six hours. After staining, cells were examined with an inverted fluorescence microscope (BZ-9000, Keyence, Osaka, Japan), and DiI-ac-LDL and BS-1 lectin double-stained cells (early EPCs) were counted in four random low-power fields.

The samples were fixed with 4% (w/v) paraformaldehyde (Biosesang) in DPBS (WELGENE) for 15 min, followed by permeabilization with a 20 min immersion in 0.20% Triton X‐100 (Sigma). The samples were then treated with 3% BSA (Sigma) for 24 h. Endothelial cell (EC)‐specific staining was performed using 488 fluorescein‐labeled Ulex Europaeus Agglutinin I (Lectin; Vector), which was prepared at a 1:500 ratio of dye in BSA for 12 h at 4℃. Epithelial tumor tissue‐specific surface staining was performed with Alexa Fluor 594‐tagged variants of anti‐epithelial cell adhesion molecule (EpCAM, CD326; Biolegend) using a 1:300 dilution of dye in BSA and incubation for 2–3 days. Image acquisition was performed using confocal microscopy (Nikon Ti‐2) to produce slice and z‐stackable images of tumor spheroid and vasculature. ImageJ (http://fiji.sc.), an open‐access software, was used to examine the confocal images.