Fig. 7: Neutrophil-specific silencing of SMAD3 enhances an N1 phenotype in NSCLC.
From: Smad3 is essential for polarization of tumor-associated neutrophils in non-small cell lung carcinoma
A RT-PCR analysis showing that transfection of human PBDN with SMAD3 siRNA (S3KD-PBDN), but not with nonsense control siRNA (NC-PBDN), suppresses basal (control) and A549 conditioned medium (A549 CM) stimulated to increase in SMAD3 expression (**P < 0.01 vs. Control, ###P < 0.001 vs. NC-PBDN, n = 3 independent samples, one-way ANOVA). B Co-culture studies show enhanced cytotoxicity of S3KD-PBDN towards the human NSCLC cell line A549 (**P < 0.01, ***P < 0.001, n = 3 independent samples, two-tailed t-test). C, D NSG immunocompromised mice were inoculated with A549 cells and then received injections of human NC-PBDN or S3KD-PBDN on days 15 and 20 and then killed on day 25. C Adoptive transfer of S3KD-PBDN caused a significant reduction in tumor size and weight, which was not apparent with a transfer of NC-PBDN (***P < 0.001 vs. untreated A549 tumors (Control), ###P < 0.001 vs. NC-PBDN, one-way ANOVA). D Immunostaining for human neutrophil markers showed that adoptive transfer of NC-PBDN resulted in a high proportion of TANs exhibiting an N2 phenotype (CD206+ CD16b+ cells), whereas transfer of S3KD-PBDN resulted in a high proportion of TANs exhibiting an N1 phenotype (iNOS+ CD16b+ cells). Furthermore, p-SMAD3 staining was seen in a high proportion of N2, but not in N1 TANs. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated A549 tumors (Control), ###P < 0.001 vs. NC-PBDN, one-way ANOVA) Scale bars, 50 μm. A, B Data represents mean ± SEM from three independent experiments. C, D Data represents mean ± SEM of 4 mice/group. The exact P values of NC-PBDN vs. S3KD-PBDN are 7A. P = 0.001 (Control), P = 0.0001(A549 CM-treated), P = 0.007 (NC-PBDN, Control vs. A549 CM-treated). 7B. P = 0.017 (1:2), P = 0.0157 (1:5), P = 0.0077 (1:10). Exact P values of PBDN treatment are 7C. P = 0.0002 (S3KD-PBDN vs. NC-PBDN, D20), P = 0.0002 (S3KD-PBDN vs Control, D20), P = 0.0001 (S3KD-PBDN vs. NC-PBDN, D25), P = 0.0001 (S3KD-PBDN vs. Control, D25), P = 0.0001 (S3KD-PBDN vs. NC-PBDN, tumor weight), P = 0.0001 (S3KD-PBDN vs. Control, tumor weight). 7D. P = 0.0001 (S3KD-PBDN vs. NC-PBDN, N1 TAN), P = 0.0001 (S3KD-PBDN vs. Control, N1 TAN), P = 0.0068 (NC-PBDN vs. Control, N1 TAN), P = 0.0001 (S3KD-PBDN vs. NC-PBDN, N2 TAN), P = 0.0404 (S3KD-PBDN vs. Control, N2 TAN), P = 0.0001 (NC-PBDN vs. Control, N2 TAN), P = 0.0001 (S3KD-PBDN vs. NC-PBDN, SMAD3 + N2 TAN), P = 0.0398 (S3KD-PBDN vs. Control, SMAD3 + N2 TAN), P = 0.0001 (NC-PBDN vs. Control, SMAD3 + N2 TAN). Source data are provided as a Source Data file.
