Fig. 6
TRIM28 Promotes MDSCs recruitment in autochthonous tumors from KP mice. (A) Migration of MDSCs toward conditioned medium (CM) from CMT-167 cells were transfected with the indicated plasmids, treated with or without immunoglobulin G (IgG) control or CXCL1-neutralizing antibody. Data are representative of results obtained in three independent experiments. (B) Correlation of CXCL1 expression, tumor purity, and MDSCs infiltration in TCGA lung adenocarcinoma and lung squamous cell cancer. The expression of CXCL1 positively correlates with MDSCs infiltration in NSCLC. (C) Schematic representation of lentiviral TRIM28 or GFP overexpression in KP mice. KP mice were anesthetized with sodium pentobarbital followed by intranasal injection of Ad-Cre (1.5 × 106 pfu/mouse). (D) Representative images of HE staining in tumor-burdened lungs of KP mice were analyzed. (E-F) Representative IHC (E) and IHC quantification (F) for immune cell markers (CD8, CD8+T cells; Gr-1, S100A8, S100A9, MDSC cells) in indicated in indicated tumor models. The scale bar represents 50 μm. Statistics calculated using a one-way ANOVA post hoc Tukey test. (G) Kaplan-Meier survival analysis of KP mice infected with Lenti-GFP-Cre or Lenti-TRIM28-Cre. Log-rank test. (H-I) Flow cytometry gating strategy of immune cells. Quantification of flow-cytometry data for CD8+T, CD4+T, MDSCs as a percentage of leukocytes (CD45+) in tumor-burdened lungs from indicated tumor models. (J) Real-time qPCR and western blotting showed expression of representative MDSCs immunosuppressive gene in the indicated cell lines. Data are representative of results obtained in three independent experiments. (K) Quantification of the proliferation of CFSE-labelled CD8+T cells cocultured with MDSCs from tumor-burdened lungs of KP or KP-TRIM28 mice, analyzed by flow cytometry. Data are representative of results obtained in three independent experiments. Statistics calculated using one-way ANOVA post hoc Tukey test for multi-group or two-tailed Student’s t-test for two-group comparisons. **p < 0.01