Fig. 6

Relationship between HMGB1 released by tumor cells treated with ADV^NE or ADV^PPE and the induction of M1 macrophage polarization via the TLR4‒MyD88‒NFκB‒NLRP3 (ASC) pathway. (A) MC38 cells were treated with PBS, ADV^Ctrl, ADV^NE, or ADV^PPE. After 24 h, the supernatant was collected to stimulate BMDMs and RAW264.7 cells. After another 24 h, qPCR was used to analyze the expression of the HMGB1-related receptors TLR2, TLR4, TLR9, and RAGE on the surfaces of BMDMs and RAW264.7 cells (n = 3 biological replicates). (B) Co-IP experiments were performed to examine the interaction between HMGB1 and the TLR4 receptor. The cell lysates were immunoprecipitated with anti-HMGB1 and anti-IgG antibodies, followed by immunoblotting with anti-HMGB1 and anti-TLR4 antibodies. Additionally, the lysates were immunoprecipitated with anti-TLR4 and anti-IgG antibodies, followed by immunoblotting with anti-HMGB1 and anti-TLR4 monoclonal antibodies. (C) Subcutaneous MC38 tumor models were established in TLR4 knockout mice and wild-type mice, followed by treatment with PBS, ADV^NE, or ADV^PPE. Subcutaneous tumor volume and survival were monitored every two days (n = 8 mice per group). (D) Following the protocol shown in C, flow cytometry was used on the 7th day after oncolytic virus treatment to assess M1 macrophage, M2 macrophage, and T_EM/T_E infiltration in tumor tissues (n = 6 mice per group). (E) MC38 cells were treated with different viruses, and 24 h later, the supernatant was collected to stimulate BMDMs and RAW264.7 cells. After another 24 h, qPCR was used to assess MyD88, NLRP3, and ASC expression in BMDMs and RAW264.7 cells (n = 3 biological replicates). (F) BMDMs and RAW264.7 cells were stimulated with supernatants from different treatment groups for 48 h, followed by Western blot analysis to detect the protein expression levels of TLR4, MyD88, NFκB, p-NFκB, NLRP3, and ASC. (G) shHMGB1-MC38 and NC-MC38 cells were treated with different viruses. After 24 h, the supernatant was collected to stimulate BMDMs and RAW264.7 cells, and Western blotting was performed 48 h later to detect the expression levels of TLR4, MyD88, NFκB, p-NFκB, NLRP3, and ASC. (H) BMDMs were stimulated with supernatants from different treatment groups in the presence of vehicle or TAK-242. After 24 h, flow cytometry was used to assess the macrophage polarization status (n = 3 biological replicates). (I) BMDMs and RAW264.7 cells were stimulated with supernatants from different treatment groups in the presence of vehicle or TAK-242 for 48 h, followed by Western blot analysis of the proteins mentioned above. (J) BMDMs were stimulated with supernatants from different treatment groups in the presence of vehicle or TAK-242 for 24 h. qPCR was then used to assess M1 and M2 gene expression, and the expression levels are presented in a heatmap (n = 3 biological replicates). (K) Following the protocol shown in J, qPCR was used to analyze the M1 macrophage-related chemokine expression levels of CXCL10 and CXCL11 in BMDMs (n = 3 biological replicates). (L) Supernatants from different treatment groups were used to stimulate BMDMs in the presence of either the vehicle control or TAK-242. After 24 h, ELISA was performed to measure IL-6 and IL-10 levels in the BMDM culture supernatants. (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