Fig. 1

EV miR-20b-5p derived from ESCC cells induced lymphangiogenesis of lymphatic endothelial cells facilitate lymphatic metastasis by remodeling the tumor microenvironment. a Heat-map of small RNA sequencing in ESCC serum EVs (4 patients with LNM and 4 patients without LNM) and ESCC tissue (4 patients with LNM and 4 patients without LNM). b Schematic model of VEGFC/VEGFR3-mediated lymphangiogenesis and LNM. c Representative IHC staining of D2-40(lymphatic vessel) in ESCC patients with low and high miR-20b-5p level. Scale bar, 100 μm. d Electron microscopy images of EVs secreted by KYSE30 and KYSE450 cells. Scale bar, 100 nm. e RIP assay to assess the effect of hnRNPA2B1 on miR-20b-5p relative to IgG in KYSE30 and KYSE450 cells. Gel electrophoresis of the PCR products from the RIP assay. f Fluorescence observation of HDLECs at 24 h after incubation with PKH67-labeled (green) EVs carried biotin-miR-20b-5p (red) from KYSE30. Representative fluorescence images and phase-contrast images are shown. Scale bar, 30 μm. g Western blot analysis of VEGFR3 in HDLECs and VEGFC in culture medium (CM). h Representative images of HDLEC-Lenti-miR-20b-5p-KD or HDLEC-Lenti-KD-NC and cultured with EVs from KYSE30 cells and VEGFC. Matrigel tube formation assay (scale bar, 2 mm) and migration assay (Original magnification, ×100). i Volumes of popliteal lymph nodes and rates of lymphatic metastasis as determined by HE analysis. KYSE150 cells were injected into the foot pads of nude mice to establish the LNM model. Seven days after cell injection, the mice were randomly assigned to one of two groups receiving tail injections of EVs from either KYSE150-Lenti-OV-NC or KYSE150-Lenti-miR-20b-5p-OV cells. Lymphatic metastasis of representative tumors was determined by HE analysis. Scale bar, 200 μm. j Representative images of HDLECs cultured with CM (devoid of EVs) from KYSE150 miR-20b-5p-overexpressing cells and corresponding control cells. Matrigel tube formation assay (scale bar, 2 mm) and migration assay (original magnification, ×100). k Immunofluorescence analysis of NF-κB sublocalization (green) as determined by confocal microscopy. Scale bar, 30 μm. l Model of EV miR-20b-5p remodeling the tumor microenvironment by inducing lymphangiogenesis of LECs in ESCC LNM. The data are representative of three independent experiments. The error bars represent the SEM. ***P < 0.001, two-tailed unpaired Student’s t-test