B16 cells were used to evaluate cytotoxic effect of VNP-Dox-Lip. As shown in Fig5A, VNP-Dox-Lip showed the same IC50 in B16 tumor cells as Dox-Lip (Fig 5A), suggesting VNP-Dox-Lip did no change cytotoxicity of Dox. Recent studies showed that chemotherapy could not only directly damage tumor cells but also drive “immunogenic cell death” (ICD) by the release or exposure of potential immunogenic signals to the elimination of residual tumor cells (21).Doxorubicin has been reported to trigger ICD effect to elicit anti-tumor immunity(20).We wonder if VNP-Dox-Lip could drive ICD, just like DOX did. ICD is characterized by active secretion of ATP, exposure of CRT on the outer leaflet of the plasma membrane (22).Therefore, we evaluated the release of ATP and surface-exposed CRT after B16 tumor cells were treated with VNP-Dox-Lip. The results showed that both ATP secretion and surface-exposed CRT were significantly increased in cells treated with VNP-Dox-Lip or Dox-Lip, but not in cells treated with VNP.  In addition, compare to Dox-Lip, VNP-Dox-Lip treatment exhibited significantly enhanced in ATP secretion and surface-exposed CRT (Fig.5B-D). The results indicate that VNP-Dox-Lip can enhance Dox potentials in stimulating ICD.
Tumor-associated macrophages (TAM) are very important in regulation of tumor growth and antitumor immune responses (23). TAMs are classified as M1, which secrete pro-inflammatory cytokines TNFα and IFN-γ, and produce high levels of inducible nitric oxide synthase (iNOS), and M2, which secrete anti-inflammatory cytokines and express scavenger receptors, including CD163, CD206. As activation M1 macrophage within TAM can boost inflammatory reaction as a mechanism for immune surveillance of cancer cells, promoting M1-macrophage polarization would contribute a crucial role in tumor treatment [24].To explored the effects of VNP-Dox-Lip on polarization of M1/M2 macrophages in vitro, mouse macrophages (Raw264.7) were cultured with B16 cells to induce TAM, and then exposure to VNP, Dox-Lip, or VNP-Dox-Lip. VNP-Dox-Lip enhanced M1 macrophage polarization as represented by an increased secretion of pro-inflammatory cytokines TNF-α and iNOS and reduced the expression of M2 markers (CD206, Arg1) (Fig 5E).  Taken together, these results have provided evidence that VNP-Dox-Lip elevated ICD-associated danger signals in melanoma cells and promoted M1 macrophage polarization. 
Fig 6 VNP-Dox-Lip reprograms TME and inhibiting tumor growth
To test whether VNP-Dox-Lip could protect mice from tumor challenge, we inoculated mice with B16 tumor cells followed by treatment with VNP-Dox-Lip (Fig. 6A). The VNP-Dox-Lip treatment inhibited tumor growth (Fig. 6B) and lead to better survival (Fig. 6C). VNP-Dox-Lip was associated with robust lymphcytes infiltration in tumor tissue consistent with the effects of VNP-Dox-Lip (Fig. 6D). We further analysis TME with FACS and found that both CD86+MHCII+CD11c and CD86+MHCII+CD11b increased, suggesting that VNP-Dox-Lip promoted antigen process(Fig 6E，F，G). Of note, there was an apparent decrease the immunosuppressive population of MDSCs and Treg cells in the VNP-Dox-Lip group compared with the control group (Fig 6G). Cytokines in the tumor microenvironment are important for recruiting the infiltration of immune cells. We examined intratumoral cytokines at day? in mice and detected increases in IFNγ, TNFa, IL-12, IL-18,and IL-2.(FigS6). Cytotocix molecules (GzmA and Gzm B) were also increased after VNP-DOX-Lip.
 To further validate the safety of the VNP-Dox chemical delivery platform, we measured the body weight of mice during the treatment process and evaluated the in vivo safety of the medication by weighing the main tissues and organs at the end point of the experiment. At the end point of the experiment, the main tissues and organs of each group of mice were weighed, and there was no difference in the weight of the heart, liver, spleen, lungs, and kidneys between the groups of mice. HE staining showed no cardiac injury in VNP-Dox treated group though cell damage and vacuoles could be found in the heart tissue of the group treated with drug Dox. Taken together, our data indicate that VNP Dox Lip has good safety (Fig. s6D).

Figure7. Evidence that PD1 signaling is capable of inducing T cell exhaustion in the tumor model. 
Tumor cells can upregulate PD-L1 expression in response to IFN-γ produced by tumor infiltrating lymphocytes（）. Indeed, we observed increased levels of PD-L1 via qRT-PCR and IHC in subcutaneous B16 tumors following treatment with Dox and VNP-Dox (Fig7 A and B). 
We therefore investigate to enhance anti-tumor effect by combination VNP-Dox with PDL1 blockade in B16 tumor bearing mice. BMS-8, a small molecule inhibitor, has been shown to induce the formation of dimmers of PD-L1 to offer a promising route forward in the functional inhibition of the PD-1/PD-L1 interaction. To deliver BMS-8 to tumor tissue by VNP, we first constructed VNP-BMS-Lip by conjunct streptavidin modified VNP20009 with BMS-Lip containing biotin on the surface (Fig7c) . VNP-BMS-Lip alone treatment had no effect on induce ICD relative to vehicle control. However, combining VNP-Dox-Lip with VNP-BMS-Lip treatment exhibited significantly enhanced in ATP secretion and surface-exposed CRT (Fig.7C-D). 

Figure 8. Combined PDL1 inhibitor and VNP-DOX therapy results in increased anti-tumor efficacy in vivo（Combination PD-1 blockadewith BMS enhances the ability of VNP-DOX to slow the tumor growth.） 

We sought to investigate whether the combined treatment of BMS and DOX may lead to augmented anti-tumor responses in three melanoma model (Figure 8). We demonstrated that the combination of these agents resulted in decreased tumor volumes and increased survival of mice in  melanoma tumor models. In the first tumor model, C57BL/6J mice bearing  B16 tumors were treated with BMS and/or DOX delivered intraperitoneally at day 8 when big tumor were established. We observed a significant decrease in tumor volume when mice were treated with either single agent. However, treatment with the combination regimen resulted in a further significant reduction of tumor volume, and a significant increase in survival (Figure 8b, d, and f and Supplementary figure 8A, C, E and G). In the second tumor model, BMS and/or DOX delivered intraperitoneally start at day 3 when tumors were small(Fig 8E). Under these conditions,we observed very similar results with reduced tumor volumes and increased survival of mice with the combination being significantly better than untreated control or either single-agent therapy. (Figure 8G and F). 
In the third tumor model, B16 tumor cells were directly injected into the lung of mice, modeling metastatic spread to lung, then treated with BMS and/or DOX at day 3 and day6. Under these conditions, tumor growth in the lung was significantly reduced after BMS and DOX and the combination of BMS and DOX. However, the combination of BMS +DOX was more effective at preventing growth of tumors compared to BMS or DOX alone.
Remarkably, we observed similar responses in these tumor models. In summary, we show here that the combination of BMS and DOX therapy has profound anti-tumor activities and combining both agents leads to significant synergistic effects 

Figure 9 Combination PD-1 blockade with BMS modulates the tumor immune microenvironment to  enhances the ability of VNP-DOX to slow the tumor growth. 
To investigate whether an immune system is involved for VNP-DOX-bms to be effective, we assessed the tumor microenvironment (Fig. 9). Immunohistochemical staining with anti-CRT showed areas of increased CRT staining into the tumor after VNP-DOX-bms treatment. VNP-DOX-bms tumors displayed evidence of cell immunogenic death consistent with the ablative effects (Fig. 2A). Although there was no visible increase in the infiltrating CD4+ T cell population by IHC (Fig. 2A), there was an apparent increase in the infiltration of CD8+ T cells (Fig. 2A). By flow cytometry, there was greater variability in the immune profiles of VNP-DOX-BMS -treated than in control tumors (Fig. 2B), likely due to variability in response. Most of the differences between VNP-DOX-BMS and VNP-DOX group, including CD8+ T cells, effector and CD4+T cells. Pro-inflammatory M1-type macrophages (MHCIIhiCD86loCD11b+F4/80+) were higher in VNP-DOX-BMS compared to VNP-DOX group. Cytokines in the tumor microenvironment recruit or prevent the infiltration of immune cells. We examined intratumoral cytokines at ? in mice to and detected increases in IFNγ,  IL-2, and TNFa ,suggesting Th1 response (Fig. 2C). An increase in IL-12, IL-18, and IL-21, was also observed, indicating. Cytotocix molecules (GzmA and Gzm B )were also increased after VNP-DOX-BMS. 
Prompt: You are now acting as an expert in the field of tumor immunology. From a professional point of view,  please polish the paragraph above to make it more logical, and academic