Fig 1: Eliminating tumor‐promoting neutrophils enhances the anti‐melanoma efficacy of VNP. (a) The effect of neutrophils on B16F10 cell viability. On day 9 after VNP therapy, subcutaneous B16F10 melanomas were collagenase‐digested to single‐cell suspensions; tumor‐associated neutrophils were magnetically sorted (#480058, BioLegend) and co‐cultured with B16F10 cells (1:1) in triple‐antibiotic (P/S/G) medium to prevent bacterial contamination. (b) Neutrophil‐mediated effects on B16F10 melanoma cell migration assessed by wound‐healing assay. Scale bar = 400 µm. (c) Neutrophil‐mediated effects on B16F10 melanoma cell migration assessed by trans‐well assay. Scale bar = 100 µm. (d) The effect of neutrophils on B16F10 cell invasion. Scale bar = 100 µm. (e) The effect of neutrophils on HUVECs tube formation. Scale bar = 500 µm. (f) Tumor growth curve after VNP or VNP combined with anti‐Ly6G treatment. (g) VNP plating on organ homogenates. The dilution ratio of the tissue homogenate is indicated in the corresponding panel. (h,i) Organ burden (h) and Bacterial Tumor Targeting Index (BTTI) (i) following VNP or VNP combined with anti‐Ly6G treatment. (j) Changes in the proportion of neutrophils among immune cells in the blood of mice following treatment with VNP combined with neutrophil‐neutralizing antibodies. (k) Relative mRNA expression levels of neutrophil marker genes in tumor tissues. (l) Western blot analysis of neutrophil marker protein expression in tumor tissues. (m) Flow cytometric analysis of the proportion of neutrophils in the tumor immune microenvironment. (n) MPO immunohistochemical staining of tumor tissues. Scale bar = 50 µm. (o) Ly6G immunofluorescence staining of tumor tissues. Scale bar = 200 µm. Data represent the mean ± S.D. in a–e, j,k, m (n = 5), f (n = 6), h,i, l (n = 3). Statistical significance was determined using an unpaired Student's t‐test in b–e. One‐way ANOVA with Tukey test was used in j, k, m. Two‐way ANOVA with Tukey's post hoc test was used in a, f, h,i, l.