Fig. 5
Effects of HMGB1 Release by Tumor Cells Treated with ADVNE or ADVPPE on Macrophage Polarization and TEM/TE Infiltration. (A) Schematic of the in vitro experimental model shown in 5B-5E: MC38 cells were treated with PBS, ADVCtrl, ADVNE, or ADVPPE, and after 24 h, the tumor cell supernatant was collected to stimulate BMDMs. Flow cytometry, qPCR, and ELISA were performed 24 h after stimulation. (B) Polarization state of BMDMs after 24 h of stimulation with supernatants from different treatment groups, as assessed by flow cytometry (n = 3 biological replicates). (C) qPCR analysis of gene expression in BMDMs for CD86, iNOS, TNF-α, IL-6, IL-1β, Arg-1, CD206, TGF-β, IDO1, and IL-10, with expression levels presented as a heatmap (n = 3 biological replicates). (D) qPCR analysis of M1 macrophage-related chemokines CXCL10 and CXCL11 in BMDMs (n = 3 biological replicates). (E) ELISA of IL-6 and IL-10 levels in the supernatants of BMDMs after 24 h of stimulation with different treatments (n = 3 biological replicates). (F) Knockdown of HMGB1 in MC38 cells via lentivirus carrying knockout constructs; qPCR and Western blotting were used to assess the knockdown efficiency. The shRNA construct with the highest knockdown efficiency, sh-629, was used to establish the shHMGB1-MC38 cell line. (G) Subcutaneous tumor models were established in mice via the use of shHMGB1-MC38 and NC-MC38 cells, followed by treatment with PBS, ADVNE, or ADVPPE. Subcutaneous tumor volume and survival were monitored every two days (n = 8 mice per group). (H-I) Subcutaneous tumor models were reestablished using shHMGB1-MC38 and NC-MC38 cells, followed by various treatments. On day 7 after the first virus treatment, flow cytometry was used to assess the infiltration of M1 macrophages, M2 macrophages (H), and TEM/TE cells (I) in tumor tissues (n = 6 mice per group). (J) MC38 cells (shHMGB1-MC38 and NC-MC38) were treated with PBS, ADVNE, or ADVPPE, and after 24 h, the tumor cell supernatant was collected to stimulate BMDMs. After an additional 24 h, the polarization of the BMDMs was assessed via flow cytometry (n = 3 biological replicates). (K-M) BMDMs were stimulated with supernatants from the various treatment groups for 24 h, followed by experiments as described in C-E (n = 3 biological replicates). The data are presented as the means ± SDs. NS, no significant difference; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001