Steps of lymph node metastasis. (A–C) SCS of the sentinel lymph node stained with Abs against VEGFR-3 or CD31 as indicated. (A) Dilation of lymphatic sinuses before the entry of tumor cells. VEGFR-3 immunostaining visualizes LECs of the sinuses and the columns connecting the floor and the roof of the sinus. (B) Single tumor cells (GFP, green) present in the lumen of the SCS or attached to the lymphatic endothelium. (C) Early stage of metastatic growth in the SCS along the lymphatic endothelium. (D–F) Scanning electron microscopy micrographs depicting architecture of the SCS at high resolution. (D) Lymphatic endothelia at the roof and the floor of the SCS are connected by numerous column-like structures. (E) Horizontal bridges between the columns in the subcapsular space. (F) Metastatic lesion confined to the SCS. (G) Metastasis in the afferent lymphatic vessel (dashed line) at the junction with the lymph node (boxed area). Image shown is a composite of three images stitched together. (H and I) High magnification of the boxed area in G, showing metastasis in the SCS at the junction of the lymphatic vessel and the LN and in the adjacent LN cortex. Lymphatic endothelium at the border to the lymph node cortex, i.e., the floor of the SCS, is immunostained for podoplanin (arrows). (J–L) Metastasis in the LN parenchyma. Lymphatic vessels are stained with the Abs to LYVE-1 (J and K), podoplanin (L), MDAcl.6 (A), or MDA cl.13 (B-L). t, tumor; c, capsule. Bars: (D and E) 10 µm; (F) 20 µm; (A–C and H–L) 100 µm; (G) 200 µm.