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Nanoparticle-enhanced chemo-immunotherapy to trigger robust antitumor immunity

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Science Advances  28 Aug 2020:
Vol. 6, no. 35, eabc3646
DOI: 10.1126/sciadv.abc3646
  • Fig. 1 Design, optimization, and characterization of PS3D1@DMXAA nanoparticles.

    (A) Schematic illustration of triblock copolymer PEG-b-PSN38-b-PDEA structure and preparation of DMXAA-loaded nanoparticles driven by electrostatic interactions, and the proposed mechanism of redox-triggered SN38 release from nanoparticles. (B) Characterization of self-assembly behaviors of copolymers and DMXAA EE. (C) Representative snapshots of PSN38-b-PDEA with DMXAA after MD simulation. PEG content was not included in the molecular dynamic simulation. Color scheme: blue, PSN38 block; green, PDEA block; red, DMXAA molecules. Solvent molecules have been removed for clarity. (D) Number of polymer clusters formed during the MD simulation. (E) DMXAA release profiles in nanoparticles immersed in NaCl solution (n = 3, mean ± SD). (F) Transmission electron microscopy image of PS3D1@DMXAA nanoparticles. (G) Variation of hydrodynamic particle diameter change recorded for self-assembly of PS3D1 with various amounts of DMXAA. (H) SN38 and DMXAA release profiles in PBS with/without DTT (D = DMXAA, S = SN38, n = 3, mean ± SD).

  • Fig. 2 Nanoparticle encapsulation enhances the uptake and immunostimulatory potency of DMXAA.

    (A and B) High-performance liquid chromatography (HPLC) analysis of intracellular DMXAA concentrations in DC2.4 and B16.F10 cells after treatment with different formulations for 2 and 6 hours. (C to E) Confocal laser scanning microscopy images (E) of DC2.4 and B16.F10 cells upon incubation with free TBF and PS3D1@TBF for varying time intervals (scale bars, 50 μm), and mean fluorescence intensity analysis of TBF uptake in DC2.4 (C) and B16.F10 cells (D). (F and G) qPCR analysis of Ifnb (F) and Cxcl10 (G) gene expression in DC2.4 after treatment with different formulations for varying time intervals. n = 3 biologically independent samples. (H and I) Dose-response curves of the Ifnb response elicited by indicated PS3D1@DMXAA nanoparticles and DMXAA in DC2.4 (H) and BMDCs (I). (J and K) Dose-response curves of the Cxcl10 response elicited by indicated PS3D1@DMXAA nanoparticles and DMXAA in DC2.4 (J) and BMDCs (K). n = 3 biologically independent samples. PS3D1@D, PS3D1@DMXAA; D, DMXAA. Data are means ± SD, and statistical significance was calculated by two-tailed Student’s t test: ***P < 0.001, **P < 0.01, and *P < 0.05; ns, not significant.

  • Fig. 3 PS3D1@DMXAA has therapeutic effects in different murine tumor models.

    (A) Intravenous treatment scheme for mice with established B16.F10 tumor. (B) Tumor growths are shown [n = 5, data are means ± SD, two-way analysis of variance (ANOVA)]. (C) Ex vivo tumor images represented from each treatment group on day 14. (D and E) Intravenous (i.v.) treatment scheme for mice with established B16.F10 tumors. Mice with 50-mm3 subcutaneous (s.c.) tumors were administered with different treatments intravenously for four times, 2 days apart, and sacrificed on day 7 for the splenocytes. Five million of splenocytes from each group were transferred to naïve mice (n = 5) followed by challenging with B16.F10 cells, and tumor growths were monitored (E) (data are means ± SD). (F) Treatment scheme for IFNARwt and IFNARko mice with established B16.F10 tumors. Mice were intravenously treated with PS3D1@DMXAA or PS3D1 for five times, 2 days apart. (G) Tumor growths of different groups are shown (n = 4 mice per group). (H) Schematic representation of AOM/DSS-induced colitis-associated colon cancer mouse model. Mice were intravenously treated with PBS (1), free DMXAA (2), PS3D1 (3), or PS3D1@DMXAA (4) on day 74 for five times, 2 days apart. The mice were sacrificed on day 84 for tumor growth analysis. (I to L) Representative images of tumors (I, red arrows), the number (J) and size (K) of colon tumors, and hematoxylin and eosin (H&E) staining (L) of representative tumors from each group (n = 5). Data are means ± SEM unless otherwise indicated. Statistical significance was calculated by two-tailed Student’s t test. ***P < 0.001, **P < 0.01, and *P < 0.05. Scale bars, 200 μm. Photo credits for (C) and (K): Jingjing Liang, Department of Radiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine.

  • Fig. 4 PS3D1@DMXAA amplifies the immunostimulation activity of DMXAA and promotes the formation of a T cell–inflamed TME.

    Mice bearing B16.F10 tumors were intravenously treated with PBS (1), free DMXAA (2), PS3D1 (3), a physical mixture of empty PS3D1 and DMXAA (4), or PS3D1@DMXAA (5) for five times, 2 days apart. (A) Selected gene expression levels in the tumors were analyzed by qPCR on day 14 (n = 5, two-way ANOVA with Tukey’s test). (B and C) IFN-β, IFN-γ, tumor necrosis factor–α (TNF-α) (B, n = 4), and selected chemokines (C, n = 5) within the B16.F10 TME were measured by bead-based multiplex LEGENDplex (two-tailed Student’s t test). (D and E) Secretion of IFN-γ (D) and TNF-α (E) in sera of mice on day 7 (n = 5). (F and G) Representative flow cytometric analysis of DC maturation (CD80+CD86+ DCs of CD45+CD11c+ DCs) (F) and quantification (G) in the tdLNs on day 14 (n = 5). (H to J) Representative flow cytometric plots (H) and the corresponding quantification of tumor-infiltrating CD8+ (I) and CD4+ (J) T cells on day 14 (n = 4). (K) Ratio of CD8+ to CD4+ T cells in the TME. (L) Quantification of tumor-infiltrating IFN-γ+CD8+ in CD8+ T cells (n = 4). (M) Flow cytometric quantification of the counts of macrophages (MΦ; CD11b+F4/80+), monocytic (m-MDSC; CD11b+Ly6c+Ly6g) and granulocytic MDSC (g-MDSC; CD11b+Ly6c+Ly6g+SSChi), neutrophils (CD11b+Ly6c+Ly6g+SSClo), natural killer cells (NK1.1+), and DCs (CD11c+MHC-II+) in the TME on day 14 (n = 4, two-way ANOVA with Tukey’s test). (N and O) Representative flow cytometric plots (N) and the corresponding quantification (O) of M2 macrophages (CD206+) in F4/80+CD11b+CD45+ cells (n = 4). Data are means ± SD, and statistical significance was calculated by one-way ANOVA with Tukey’s test unless otherwise indicated. ***P < 0.001, **P < 0.01, and *P < 0.05.

  • Fig. 5 PS3D1@DMXAA elicits TAA-specific CD8+ T cell priming.

    (A) Schematic illustrating the process of DC activation in the TME and DC migration to the tdLN for antigen presentation and activation of T cells. Mice bearing B16.F10 tumors were treated with PBS (1), free DMXAA (2), PS3D1 (3), a physical mixture of empty PS3D1 and DMXAA (4), or PS3D1@DMXAA (5) intravenously for five times, 2 days apart. (B) Percentages of CD103+CD11c+ DCs of CD45+ cells in tumors were measured by flow cytometry on day 14 (n = 4). (C and D) Representative flow cytometric analysis (C) and the corresponding quantification (D) of CD103+CD11c+ DCs of CD45+ cells in tdLN on day 14 (n = 4). (E) Intravenous treatment scheme for mice with established B16-OVA tumor. Mice bearing B16-OVA tumors were treated as in (A). Then, the mice were sacrificed on day 7 for different analysis. (F and G) Percentages of OVA (SIINFEKL)–specific CD8+ T cells in TME (F) and spleen (G) were measured by flow cytometry (n = 3). (H) ELISpot analysis of IFN-γ spot-forming cells among splenocytes after ex vivo restimulation with OVA (SIINFEKL) peptide on day 7. (I) Flow cytometric quantification of granzyme B+CD8+ T cells among splenocytes of mice after ex vivo restimulation with SIINFEKL (OVA) peptide. (J) Granzyme B protein levels in the supernatant of splenocytes after ex vivo restimulation with OVA (SIINFEKL) peptide for 24 hours on day 7 (n = 4). (K) Flow cytometric quantification of CD107a+CD8+ T cells among splenocytes after ex vivo restimulation with SIINFEKL (OVA) peptide on day 7. Data are means ± SD, and statistical significance was calculated by one-way ANOVA with Tukey’s test. ***P < 0.001 and **P < 0.01.

  • Fig. 6 PS3D1@DMXAA inhibits breast tumor metastasis and synergizes with ICB to inhibit B16-melanoma.

    (A) Schematic diagram of the orthotopic breast tumor model and administration method. Mice bearing 4T1-Luci breast tumors were treated as in Fig. 5A. (B) Tumor growths are shown (n = 8). (C) Survival curves were compared using log-rank test (n = 8). (D) In vivo bioluminescence images of 4T1-Luci lung metastatic tumors. (E) Number of the lung metastatic nodules on day 25. One-way ANOVA with Tukey’s test. (F) Combined treatment scheme for mice with established B16.F10 tumors. (G) Tumor growths of B16.F10 tumor-bearing mice are shown (n = 8, means ± SEM). (H) Survival curves were compared using log-rank test (n = 8). Data are means ± SD, and statistical significance was calculated by two-tailed Student’s t test unless otherwise indicated. ***P < 0.001 and **P < 0.01. (I) Schematic illustration of the self-assembly of PS3D1@DMXAA nanoparticles with redox-responsive drug release in tumor cells. The electrostatic interaction between the tertiary amine group and DMXAA provides efficient drug loading. (1 and 2) Redox stimuli trigger SN38 release in tumor cells. SN38 induces tumor cell death and release of chemokine CCL4 that drives the infiltration of CD103+ DCs in the TME. (3) Meanwhile, PS3D1@DMXAA elicits efficient cytosolic delivery of DMXAA for STING activation in CD103+ DCs. Together, these enhance the maturation and TAA uptake of CD103+ DCs. (4 and 5) STING activation enhances the migration of mature CD103+ DCs into the tdLN and stimulates TAA cross-presentation by CD103+ DCs for cross-priming of TAA-specific effector CD8+ cytotoxic T cells. (6) PS3D1@DMXAA modulates the immunosuppressive TME and facilitates TAA-specific effector CD8+ cytotoxic T cell recruitment through CXCL9/CXCL10. All these eventually amplify the antitumor therapeutic effects.

Supplementary Materials

  • Supplementary Materials

    Nanoparticle-enhanced chemo-immunotherapy to trigger robust antitumor immunity

    Jingjing Liang, Huifang Wang, Wenxiu Ding, Jianxiang Huang, Xuefei Zhou, Huiyang Wang, Xue Dong, Guangyao Li, Enguo Chen, Fei Zhou, Hongjie Fan, Jingya Xia, Bo Shen, Da Cai, Pengxun Lan, Hanliang Jiang, Jun Ling, Zhen Cheng, Xiangrui Liu, Jihong Sun

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