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An immune cocktail therapy to realize multiple boosting of the cancer-immunity cycle by combination of drug/gene delivery nanoparticles

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Science Advances  30 Sep 2020:
Vol. 6, no. 40, eabc7828
DOI: 10.1126/sciadv.abc7828
  • Fig. 1 Schematic diagram of the immune cocktail therapy to realize multiple boosting of the cancer-immunity cycle by the combination of drug/gene delivery nanoparticles.

    We constructed a cocktail therapy that consisted of an acid-responsive drug delivery system (DOX NPs) and an acid-responsive dual gene delivery system [(shPD-L1 + Spam1) NPs]. DOX NPs could induce tumor cell ICD and promoted tumor antigens uptake of DCs. (shPD-L1 + Spam1) NPs could generate shPD-L1 and HAase in situ at tumor areas, worked as checkpoint blockade and ECM elimination, respectively. The immune cocktail therapy held the following superiorities: (I) facilitating T cell activation, (II) reprogramming the immunosuppressive TME into an immune active TME, (III) increasing the infiltration of peripheral CD8+ T cells into the tumor, and (IV) inducing strong immune memory effect.

  • Fig. 2 Combination of immunogenic chemotherapy and ICT on the subcutaneous B16F10 model.

    (A) Combinational treatment scheme of DOX NPs and shPD-L1 NPs. Mice received shPD-L1 NPs on days 12 and 16 via intravenous injection. Mice received DOX NPs on days 14 and 18 via intravenous injection. (B) Tumor growth curves in phosphate-buffered saline (PBS), DOX NPs, shPD-L1 NPs, and DOX NPs + shPD-L1 NPs treated groups. (C) CD45+CD3+CD8+ T cell ratios in the tumor of different treatment groups on day 21. (D) Immunofluorescence staining in tumor tissues for CD8 of different treatment groups. Sections were stained with anti-CD8 antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 50 μm. Significant differences in (B) and (C) were assessed using t test (ns represented not significant, **P < 0.01, ***P < 0.001). Results were expressed as means ± SD (n = 4).

  • Fig. 3 Immune cocktail therapy on the subcutaneous B16F10 model.

    (A) Treatment scheme of different combinational formulations. Mice received gene therapies on days 10 and 14 via intravenous injection. Mice received DOX NPs on days 12 and 16 via intravenous injection. (B) Tumor growth curves of different treatment groups. (C) Tumor weight of different treatment groups on day 19. (D) Individual tumor growth curves of each mouse after treated with different groups. PR represented tumors’ longer diameter was reduced by more than 30% compared with their initial longer diameter. (E) Tumor photos and H&E of different treatment groups. Group 1, PBS; group 2, DOX NPs; group 3, shPD-L1 NPs; group 4, DOX NPs + shPD-L1 NPs; group 5, Spam1 NPs; group 6, DOX NPs + Spam1 NPs; group 7, (shPD-L1 + Spam1) NPs; group 8, DOX NPs + (shPD-L1 + Spam1) NPs. Scale bar in tumor photos represented 5 mm. Scale bar in H&E represented 50 μm. Significant differences in (B) and (C) were assessed using t test (***P < 0.001). Results were expressed as means ± SD (n = 6). Photo credit (E): Jiayan Wu, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences.

  • Fig. 4 The immune cocktail therapy induced an immune-active TME.

    Flow cytometry analyses of (A) CD80+MHC II+ matured DCs of the CD11c+ DCs ratios in TDLNs. (B) CD45+CD3+ T cell ratios in the tumor. (C) CD45+CD3+CD8+ T cell ratios in the tumor. (D) CD45+CD3+CD4+ T cell ratios in the tumor. (E) CD8+ T/CD4+ T cell ratios in the tumor. (F) CD206+F4/80+ M2 macrophages (M2φ) of the CD11b+ macrophages ratios in tumor. ELISA results reflected the relative fold change of (G) PD-L1, (H) HAase, (I) IFN-γ, (J) granzyme B, (K) IL-6, and (L) TNF-α in tumor tissues compared with the PBS group. (M) Immunofluorescence staining in tumor tissues for CD8 (green) and granzyme B (purple) of different treatment groups. Scale bars, 50 μm. (N) The heatmap of multiple gene expression levels according to RT-qPCR results measured from tumor tissues in different treatment groups. Each square indicated one individual test. Group 1, PBS; group 2, DOX NPs; group 3, shPD-L1 NPs; group 4, DOX NPs + shPD-L1 NPs; group 5, Spam1 NPs; group 6, DOX NPs + Spam1 NPs; group 7, (shPD-L1 + Spam1) NPs; group 8, DOX NPs + (shPD-L1 + Spam1) NPs. Significant differences in (A) to (L) were assessed using t test (ns represented not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Results were expressed as means ± SD (n = 5).

  • Fig. 5 Cocktail therapy increased peripheral T cell infiltration.

    (A) Treatment scheme for peripheral T cells depletion experiment. (B) Left: Tumor growth curves in different treatment groups. Right: Amplified region of tumor growth curves. (C) Relative fold change of peripheral CD45+CD3+CD8+ T cells in blood compared with the PBS group. (D) CD45+CD3+CD8+ T cells in tumor compared with the PBS group. (E) Immunofluorescence staining in tumor tissues for CD8 (green) and granzyme B (purple) of different treatment groups. Scale bars, 50 μm. (F) Immunofluorescence staining in tumor tissues for α-SMA (green) and CD31 (purple). Scale bars, 100 μm. (G) RT-qPCR results of Cxcl9, Cxcl10, Icam1, and Vcam1 mRNA expression level in tumor tissues. (H) Quantitative measurement of ICAM1 and VCAM1 in tumor tissues. (I) Immunofluorescence staining in tumor tissues for HA (green). Scale bars, 100 μm. (J) The schematic diagram for suggested mechanisms that could affect peripheral T cell infiltration. We concluded four crucial steps: vessel normalization, chemokines level, adhesion molecules level, and ECM elimination. The score table for the comprehensive contributions to peripheral T cell infiltration was also shown in the figure. We supposed that the cocktail therapy group gained 3.5 “points,” whereas the DOX NPs + shPD-L1 NPs group gained 0.5 points in the score table. Significant differences in (B) to (D), (G), and (H) were assessed using t test (ns represented not significant, *P < 0.05, **P < 0.01, ***P < 0.001). Results were expressed as means ± SD (n = 5 or 3).

  • Fig. 6 Cocktail therapy largely increased immune memory cells in the spleen, prolonged medium survival, and could inhibit lung metastasis.

    Flow cytometry results for (A) CD3+CD4+ T cell ratios, (B) CD3+CD8+ T cell ratios, and (C) CD3+CD8+ CD44+CD62 effective memory T cell ratios of different treatment groups in the spleen (n = 5). (D) Treatment scheme of survival experiment. (E) Mouse survival curves of different treatment groups (n = 6). (F) Treatment scheme of lung metastasis experiment. (G) H&E results for lungs of naive mouse group (control) and cocktail therapy group. Scale bars, 500 μm. Significant differences in (A) to (C) were assessed using t test (ns represents not significant, **P < 0.01, ***P < 0.001). Significant differences in (E) were assessed using log-rank (Mantel-Cox) test (*P < 0.05, ***P < 0.001).

Supplementary Materials

  • Supplementary Materials

    An immune cocktail therapy to realize multiple boosting of the cancer-immunity cycle by combination of drug/gene delivery nanoparticles

    Jiayan Wu, Jie Chen, Yuanji Feng, Sijia Zhang, Lin Lin, Zhaopei Guo, Pingjie Sun, Caina Xu, Huayu Tian, Xuesi Chen

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