Figure 1.

Tumor‐associated neutrophils promote lymphangiogenesis and LN metastasis in BCa. Representative IHC images straining for A) CD66b and B) MPO in MIBC LN (‐) and MIBC LN (+) BCa tissues (left). The quantification of A) CD66b⁺ and B) MPO ⁺ cells infiltrated in NMIBC (n = 26), MIBC LN (‐) (n = 84) and MIBC LN (+) (n = 50) BCa tissues (right). Kaplan‐Meier survival analysis of C) OS and D) DFS of BCa patients with high level (n = 76) versus low level (n = 84) infiltration of CD66b⁺cells in cohort 1. Kaplan‐Meier survival analysis of E) OS and F) DFS of BCa patients with high level (n = 71) versus low level (n = 89) infiltration of MPO⁺ cells in cohort 1. G) Kaplan‐Meier survival analysis of OS of BCa patients’ high level (n = 49) versus low level (n = 316) infiltration of TANs in TCGA cohort. H) Flow cytometry analysis of CD45⁺CD11b⁺CD66b⁺ neutrophils from fresh surgically excised BCa tissues (n = 16). I) Representative bioluminescence images (left) and histogram analysis (right) of the popliteal metastatic LNs from nude mice treated with IgG or neutralizing antibody of Ly6G (1A8, 300 µg per mouse). J) Images of the dissected popliteal LNs (left) and analysis (right) of the LN volume (n = 12). K,L) Representative immunofluorescence images (left) staining for MPO and LYVE1 and quantification (right) of MPO⁺ and LYVE1⁺ cells infiltrated in K) IgG‐ and aLy6G‐ treated footpad tumor or L) LN (‐) and LN (+) human BCa tissues. M) Correlation analysis between immunofluorescent detected MPO ⁺ and LYVE1⁺ cells in IgG‐ and aLy6G‐ treated footpad tumor (n = 12). N) Correlation analysis between CD66b⁺ cells and LYVE1⁺ cells in cohort 2 (n = 173). O) Representative images of tube formation assay of HLECs incubated with CM from neutrophils treated with the indicated supernatant for 6 h, histograms show means and SD for tube length. Statistical significance was assessed using two‐tailed t tests. *p < 0.05, ***p < 0.01, ***p < 0.001, ****p < 0.0001.