Fig. 1

EV-packaged circTLCD4-RWDD3 positively correlates with LN metastasis of NSCLC. a Flowchart of steps to identify circRNAs upregulated in NSCLC relative to NATs and upregulated in LN-positive NSCLC relative to LN-negative cancer. b, c Heatmap of circRNAs differentially expressed in NSCLC tissues and NATs and in NSCLC tissues with or without LN metastasis. d qRT-PCR analysis of circTLCD4-RWDD3 expression in NSCLC cell lines and human bronchial epithelial cell line. e–h qRT-PCR analysis of circTLCD4-RWDD3 expression in LN-positive and LN-negative NSCLC tissues from PUMCH, SYSMH, SYSUCC, and combined cohort, respectively. i Representative FISH images and percentages of circTLCD4-RWDD3 expression in LN-positive or LN-negative NSCLC tissues and NATs. White arrows indicated the extracellular expression of circTLCD4-RWDD3. Scale bars, 50 µm. j Representative images and percentages of circTLCD4-RWDD3 expression and LYVE-1-indicated lymphatic vessels in NSCLC tissues. Scale bars, 50 µm. k, l Kaplan–Meier curves for OS (k) and DFS (l) of NSCLC patients with low versus high circTLCD4-RWDD3 expression. The cutoff value is the median. m, n TEM (m) and NTA (n) identified the characteristics of A549 cell-derived EVs. Scale bar, 200 nm. o Western blotting analysis of EV markers in cell lysates or A549 cell-derived EVs. p qRT-PCR analysis of circTLCD4-RWDD3 expression in EVs derived from NSCLC tissues and paired NATs. q qRT-PCR analysis of circTLCD4-RWDD3 expression in EVs derived from LN-positive and LN-negative NSCLC tissues. r qRT-PCR analysis of circTLCD4-RWDD3 expression in NSCLC cell lines and human bronchial epithelial cell line and in their corresponding EVs. The statistical difference was assessed through nonparametric Mann–Whitney U test in (e–h, p, and q); and Chi-square test in (i, j); and one-way ANOVA followed by Dunnett tests in (d, r); and unpaired Student’s t-test in (r). Error bars show the SD from three independent experiments. *P < 0.05; **P < 0.01