| Background:Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death worldwide.Clinically,the therapeutic strategies for advanced HCC are fairly limited.More recently,checkpoint blockade immunotherapy targeting programmed cell death protein 1(PD-1)has produced promising efficacy in HCC patients.Unfortunately,the clinical efficacy of the anti-PD-1 as monotherapy is limited to partial patients,with an objective response rate(ORR)of 20%or less.Considering the unprecedented rates of long-lasting anti-tumor responses following anti-PD-1treatment,methods to enhance its therapeutic efficacy are urgently required.The immune checkpoint molecule PD-1 is well-known to bind to its ligand programmed cell death ligand 1(PD-L1),leading to T cell exhaustion.Thus,antibodies targeting PD-1 can prevent the PD-1–PD-L1 interaction,alleviating the inhibitory effect of PD-1 on T cells,including CD8~+T cells.The number of CD8~+T cells is used to predict the response to anti-PD-1 therapy,but this remains suboptimal in a clinical setting.Emerging evidence indicates that the failure of anti-PD-1 in cancer treatment partly results from the imbalance between CD8~+T cells and tumor burden,and the therapeutic efficacy of anti-PD-1 is positively ssociated with the ratio of CD8~+T cell invigoration to the tumor burden.When the number of CD8~+T cells is comparable between small and large tumors,CD8~+T cells in the larger tumors may not be sufficient to effectively inhibit tumor growth.Methods to reduce the tumor burden and simultaneously increase the number of intratumoral CD8~+T cells can therefore enhance the therapeutic efficacy of anti-PD-1 for the treatment of HCC.Tumors are supplied with oxygen and nutrients through blood vessels.Blocking the tumor blood vessels will make the tumor smaller and reduce the tumor burden.Studies have shown that anti-vascular drugs combined with PD-1 immune checkpoint inhibitors have been successfully used in patients with unresectable liver cancer.As HCC has a hyper-vascular structure,vascular disrupting agents(VDAs)can reduce HCC tumor burden by selectively disrupting established tumor blood vessels.CA4 is a representative VDA,and its phosphate prodrug CA4P has entered phase III clinical trials.Compared with CA4P,poly(L-glutamic acid)-graft-methoxy poly(ethylene glycol)/combretastatin A4(CA4-NPs)display enhanced tumor blood vessel targeting and tumor inhibition because of the low permeability of nanoparticles in tumor blood vessels.Thus,CA4-NPs reduce the tumor burden of HCC tumor-bearing mice more effectively.The abnormal tumor vascular system of formed under the action of pro-angiogenesis over anti-angiogenesis factors which inhibits the rolling and adhesion of T cells to endothelial cells,and finally reduces the infiltration of T cells in tumor.The structure and function of normalized tumor blood vessels are closer to normal blood vessels,and more T cells were infiltrated in the tumor.We want to use vascular normalization to increase the number of T cells in the HCC.By increasing anti-angiogenic factors,we can break the imbalance between anti-angiogenic factors and pro-angiogenic factors in the tumor.Recent studies have suggested that blocking the VEGF/VEGF receptor(VEGFR)2 signaling can transiently normalize the tumor vasculature and increase the number of intratumoral CD8~+T cells.VEGF/VEGFR2inhibitors potentially block the function of VEGF in response to CA4-NPs,temporarily normalizing the tumor vasculature,and increasing the number of intratumoral CD8~+T cells.Accordingly,the combination of CA4-NPs and VEGF/VEGFR2 inhibitors has the potential to reduce tumor burden of H22 tumor-bearing mice,simultaneously increasing the number of intratumoral CD8~+T cells.In this study,CA4-NPs plus a VEGF/VEGFR2 inhibitor DC101 reduce tumor burden of H22 tumor-bearing mice and increase the number of intratumoral CD8~+T,which synergized with anti-PD-1 antibody enhancing the therapeutic effect in HCC.Objective:In this study,the efficacy of CA4-NPs plus DC101 synergizing with anti-PD-1antibody in enhancing HCC treatment and its mechanism were verified in the H22subcutaneous tumor model.The effect on tumor burden of H22 tumor-bearing mice of CA4-NPs was revealed and effects of DC101 on H22 tumor vascular normalization and chang of the number of intratumoral T cells were evaluated.Furthermore,the effect on tumor burden of H22 tumor-bearing mice and chang of the number of intratumoral T of CA4-NPs plus DC101 was studied.Finally,the tumor suppressing and chang of the number of intratumoral CD8~+T cells of anti-PD-1 synerging with CA4-NPs plus DC101 were verified.Methods:1.The effect of CA4-NPs on the tumor burden of H22 tumor-bearing mice:The murine H22 cell was inoculated to the syngenic Balb/c mice to generate HCC tumor model.CD31 immunohistochemical staining was used to evaluate the effect of CA4-NPs on tumor blood vessels.Ki67 immunohistochemical staining was used to evaluate the effect of CA4-NPs on tumor cell proliferation.H&E staining was used to observe the morphological changes of tumor tissues after CA4-NPs administration.Tumor burden was assessed by H22 tumor length diameter.2.The effect of DC101 on normalizing H22 tumor-bearing mice of blood vessels and changing of the number of intratumoral CD8~+T cells:The effects of DC101 on normalizing H22 tumor blood vessels were evaluated by pericyte coverage,tumor blood vessel perfusion and tumor hypoxia.Flow cytometry was used to detect the changes of the number of intratumoral T cells after DC101 administration.3.The effect of CA4-NPs combined with DC101 on tumor burden of H22tumor-bearing mice and the number of intratumoral CD8~+T cells:Immunohistochemical staining of Ki67 was used to evaluate the effect of CA4-NPs combined with DC101 on H22 tumor cell proliferation;H&E staining was used to observe intratumoral necrosis after CA4-NPs combined with DC101administration;immunofluorescence assay was used to evaluate the effect of CA4-NPs combined with DC101 on normalizing H22 tumor blood vessels;flow cytometry was used to detect the changes of the number of intratumoral CD4~+T cells and CD8~+T cells after CA4-NPs combined with DC101 administration.4.Evaluation of the efficacy of CA4-NPs plus DC101 synergizing with anti-PD-1 antibody in the treatment of HCC:In an H22 tumor model,the tumor inhibition,weight change and survival time of H22 tumor-bearing mice were observed;flow cytometry was used to detect the effect of CA4-NPs plus DC101 synergizing with anti-PD-1 antibody on changing of the number of intratumoral CD8~+T cells;ELISA was used to detect the intratumoral cytokine.Results:1.The effect of CA4-NPs on the tumor burden of H22 tumor-bearing mice:Compared with the PBS group,the CA4-NP group had significantly fewer CD31 staining blood vessels in the H22 tumor tissue sections(P<0.0001),while the H22 tumor cell proliferation was significantly inhibited(P<0.01),and the H22tumor cell necrosis significantly increased(P<0.001).CA4-NPs effectively reduced tumor burden of H22 tumor-bearing mice(P<0.01).2.The effect of DC101 on the number intratumoral CD8~+T cells of HCC:Compared with the PBS group,the DC101 group increased pericyte coverage(P<0.05),increased FITC-lectin perfusion(P<0.001)and increased Hoechst33342 perfusion(P<0.01),and decreased intratumoral hypoxia(P<0.05);compared with the PBS group,DC101 increased the number of intratumoral CD4~+T cells(P<0.01)and CD8~+T(P<0.001)cells.3.The effect of CA4-NPs combined with DC101 on the tumor burden of H22tumor-bearing mice and the number of intratumoral CD8~+T cells:Compared with the PBS group,CA4-NPs+DC101 inhibited tumor cell proliferation(P<0.05)and caused massive H22 tumor cell necrosis(P<0.01),while CA4-NPs+DC101 increased H22 tumor pericyte coverage(P<0.01),increased FITC-lectin perfusion(P<0.01),increased Hoechst 33342 perfusion(P<0.01),and reduced tumor hypoxia(P<0.05).Compared with the PBS group,CA4-NPs+DC101 increased the number of intratumoral CD4~+T cells(P<0.05)and CD8~+T cells(P<0.001).4.Evaluation of the efficacy of CA4-NPs plus DC101 synergizing with anti-PD-1 antibody in the treatment of HCC:The H22 tumor inhibition rate of CA4-NPs+DC101+anti-PD-1 group reached 86.4%on day 10,which was significantly higher than CA4-NP+DC101group(63.5%)and anti-PD-1 group(16.8%),the Q value calculated was 1.24,indicating that CA4-NPs+DC101 have a strong synergistic effect with anti-PD-1.CA4-NP+DC101+anti-PD-1 treatment significantly prolonged the median survival time(23 days)of H22 tumor-bearing mice,which was about 1.9 times of that in anti-PD-1 treatment(12 days).The Ki67 immunohistochemical staining of H22 tumor tissues on day 11 showed that the number of positive cells in the triple drug combo group was the least,and the quantitative analysis of H&E staining of H22 tumor tissue revealed that the triple drug combo group had the largest necrosis area.The number of intratumoral CD8~+T cells in the triple drug combo group was significantly higher than that in PBS group,CA4-NP+DC101 group and anti-PD-1group on day 11.ELISA results showed intratumoral IFN-?,TNF-?,and IL-2significantly increased in triple drug combo group.Conclusions:1.CA4-NPs disrupted H22 tumor blood vessels and inhibited tumor cell proliferation,leading to tumor cell necrosis,efficiently reducing tumor burden of H22 tumor-bearing mice.2.DC101 normalized the H22 tumor vasculature and increased the number of intratumoral CD8~+T cells.3.CA4-NPs combined with DC101 reduced tumor burden of H22 tumor-bearing mice while increasing the number of intratumoral CD8~+T cells.4.Anti-PD-1 synergizing with CA4-NPs plus DC101 increased the number of intratumoral CD8~+T cells of HCC.The combination of CA4-NPs+DC101 and anti-PD-1 had a strong synergistic effect,significantly inhibiting tumor growth of H22tumor-bearing mice,at the same time significantly extended the survival of H22tumor-bearing mice. |
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