Research ArticleHEALTH AND MEDICINE

Elimination of established tumors with nanodisc-based combination chemoimmunotherapy

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Science Advances  18 Apr 2018:
Vol. 4, no. 4, eaao1736
DOI: 10.1126/sciadv.aao1736
  • Fig. 1 Schematic of doxorubicin-loaded sHDL (sHDL-DOX) for chemo-immunotherapy.

    (A) sHDL-DOX is formulated by incubation of lipid-DOX with preformed sHDL. (B) The ultrasmall size and prolonged circulation of sHDL enable intratumoral delivery of DOX, followed by internalization by tumor cells and pH-responsive release of DOX in the endosomes/lysosomes. Released DOX kills tumor cells and triggers ICD, promoting up-regulation of CRT (the “eat me” signal) and release of danger signals such as HMGB1. DCs recruited to the immunogenically dying tumor cells phagocytose them, process tumor antigens, and cross-prime tumor antigen–specific T cells. Antitumor immunity primed with sHDL-DOX synergizes with immune checkpoint blockade, leading to efficient elimination of established tumors and prevention of tumor relapse. R.T., room temperature.

  • Fig. 2 Preparation and characterization of sHDL-DOX.

    (A) GPC of blank sHDL, the physical mixture of sHDL + DOX, and sHDL covalently attached with DOX (sHDL-DOX) at 220 and 485 nm. (B) TEM of blank sHDL and sHDL-DOX. Scale bars, 50 nm. (C) Sizes of sHDL-DOX before and after lyophilization/reconstitution measured by DLS. (D) Release of DOX from sHDL at pH 5 and pH 7.5. Data represent mean ± SD (n = 3). (E) CT26 cells were incubated with 40 μM free DOX or sHDL-DOX for indicated lengths of time, and the intracellular distribution of DOX was imaged by confocal microscopy. Scale bars, 20 μm. (F to H) CT26 tumor cells (F) or MC38 tumor cells (G) were incubated with serial dilutions of free DOX or sHDL-DOX for 72 hours, and cellular viability was measured by the cell counting kit. (H) Release of HMGB1 was quantified by enzyme-linked immunosorbent assay (ELISA) after CT26 tumor cells were treated with indicated formulations (equivalent to 50 μM DOX). (I and J) BALB/c mice or (K and L) C57BL/6 mice were subcutaneously inoculated with 2 × 105 CT26 (I and J) or 2 × 105 MC38 cells (K and L) on day 0 and treated with DOX (4 mg/kg) in the indicated formulations on days 8 and 11. On day 15, the animals were euthanized and tumor tissues were harvested for analyses of ICD markers. Shown are (I and K) the levels of CRT on tumor cells (DAPICD45) and (J and L) the amount of released HMGB1 per tumor volume. *P < 0.05, **P < 0.01, and ***P < 0.001 analyzed by one-way analysis of variance (ANOVA) (H to L) with Tukey’s multiple comparisons post test. Data in (D) and (F) to (H) represent mean ± SD (n = 3), and data in (I) to (L) are represented as box plots (whiskers, 5th to 95th percentile; n = 4) from a representative experiment from two to three independent experiments. MFI, mean fluorescence intensity.

  • Fig. 3 Antitumor efficacy and T cell immunity exerted by sHDL-DOX monotherapy.

    (A) CT26 tumor–bearing mice were intravenously (iv) injected with sHDL-DiR, and the biodistribution of sHDL-DiR at different time points was imaged by the IVIS optical imaging system. (B) At 72 hours after injection, major organs were harvested and imaged ex vivo, and (C) fluorescence signal was quantified. (D) BALB/c mice were intravenously injected with free DOX or sHDL-DOX at DOX (4 mg/kg). Shown are the serum concentrations of DOX fitted to the two-compartment model. Data represent mean ± SD (n = 3) from a representative experiment from two to three independent experiments. (E) BALB/c mice were subcutaneously inoculated with 2 × 105 CT26 cells on day 0. On days 8, 11, and 14, tumor-bearing mice were treated with indicated formulations at DOX (4 mg/kg). (F and G) The average and individual CT26 tumor growth curves for mice treated with indicated formulations. CR, complete tumor regression. (H) Body weights of CT26 tumor–bearing mice treated with indicated formulations. (I) Hematoxylin and eosin (H&E) staining of the hearts and livers harvested on day 20 from tumor-bearing mice treated with indicated formulations. (J and K) The percentage of tumor cell-reactive T cells (IFN-γ+CD8+) among PBMCs on day 20 was measured by ICS. Shown are (J) the percentage of IFN-γ+CD8+ among PBMCs on day 20, and (K) the representative scatterplots. (L) The percentage of CT26 tumor antigen peptide AH1-specific CD8+ T cells among PBMC on day 20, and (M) the representative scatterplots. Data in (J) and (L) are represented as box plots (whiskers, 5th to 95th percentile; n = 5) from a representative experiment from two independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001 analyzed by one-way ANOVA (J and L) or two-way ANOVA (G and H) with Tukey’s multiple comparisons post test.

  • Fig. 4 Potentiation of αPD-1 immunotherapy with sHDL-DOX for treatment of CT26 tumors.

    (A) BALB/c mice were subcutaneously (sc) inoculated with 2 × 105 CT26 cells on day 0. On days 8, 11, and 14, tumor-bearing mice were treated with indicated formulations at DOX (4 mg/kg). αPD-1 was injected intraperitoneally (ip) at 100 μg per dose on days 9, 12, and 15. (B) The percentage of CT26 tumor antigen AH1-specific CD8+ T cells among PBMCs on day 20, and (C) the representative scatterplots. Data are represented as box plots (whiskers, 5th to 95th percentile). n = 5 from a representative experiment from two independent experiments. (D) Individual growth curves for mice treated with indicated formulations. (E) The average tumor growth curves for mice treated with indicated formulations. Data represent mean ± SD (n = 8) from a representative experiment from two independent experiments. (F and G) On day 60, sHDL-DOX + αPD-1–treated animals in (E) were re-challenged by subcutaneous or intravenous injection of 2 × 105 CT26 cells. For the control groups, naïve BALB/c mice were re-challenged with the same number of CT26 cells. Shown are the animal survival (F) and lung metastasis (G) of CT26 cells on day 22 after re-challenge. Naïve mice were used as control and inoculated with the same number of tumor cells. *P < 0.05, **P < 0.01, and ****P < 0.0001 analyzed by one-way ANOVA (B) or two-way ANOVA (E) with Tukey’s multiple comparisons post test or log rank (Mantel-Cox) test (F).

  • Fig. 5 Chemo-immunotherapy for induction of neoantigen-specific T cell responses and elimination of MC38 tumors.

    (A) C57BL/6 mice were inoculated subcutaneously with 2 × 105 MC38 cells on day 0. On days 8 and 11, tumor-bearing mice were treated with indicated DOX-containing formulations at DOX (4 mg/kg). For the combination immunotherapy, αPD-1 was injected intraperitoneally at 100 μg per dose on days 9 and 12. On day 18, the percentage of Adpgk-specific CD8+ T cells among PBMCs was measured. Data are represented as box plots (whiskers, 5th to 95th percentile). n = 5 for no treatment and n = 8 for other groups, from a representative experiment from two independent experiments. (B and C) The percentage of Adpgk-specific CD8+ T cells among PBMCs (B) and the representative scatterplots (C). (D) Individual tumor growth curves of mice treated with indicated formulations. (E) The average tumor growth curves of mice treated with indicated formulations. Data represent mean ± SD. n = 8 to 10, from a representative experiment from two independent experiments. (F and G) On day 60, sHDL-DOX + αPD-1–treated animals in (E) were re-challenged by subcutaneous or intravenous injection of 2 × 105 MC38 cells. For the control groups, naïve C57BL/6 mice were re-challenged with the same number of MC38 cells. Shown are the survival (F) and lung metastasis of MC38 cells (G) on day 26 after re-challenge. Naïve mice were used as control and inoculated with the same number of tumor cells. *P < 0.05, **P < 0.01, and ****P < 0.0001 analyzed by one-way ANOVA (B) or two-way ANOVA (D) with Tukey’s multiple comparisons post test or log rank (Mantel-Cox) test (F).

  • Fig. 6 Chemo-immunotherapy for orthotopic colon cancer.

    (A to E) BABL/c mice were inoculated with 2 × 106 CT26-FL3-Luc cells in the cecum wall on day 0. On days 8, 11, and 14, mice were injected intravenously with DOX (4 mg/kg) in the indicated formulations. On days 9, 12, and 15, mice were injected intraperitoneally with 100 μg per dose of αPD-1. Shown are (A) the whole-animal in vivo imaging over time and (B) quantification of the bioluminescence signal. (C and D) On day 22, major organs were harvested and imaged ex vivo. Shown are (C) representative bioluminescence images and (D) quantification of the signal in each organ. Data in (D) are represented as box plots (whiskers, 5th to 95th percentile; n = 4) from a representative experiment from two independent experiments. (E) Shown are the animal survival curves with n = 8 combined from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 analyzed by two-way ANOVA (B and D) with Tukey’s multiple comparisons post test or log rank (Mantel-Cox) test (E). avg, average.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/4/eaao1736/DC1

    fig. S1. Schematic for the synthesis of the lipid-DOX conjugate.

    fig. S2. Mass spectroscopy confirmed the conjugation of DOX to PTD.

    fig. S3. sHDL-DOX was lyophilized and stored for 2 months before reconstitution by adding water to the lyophilized powder.

    fig. S4. Cellular uptake of DOX or sHDL-DOX by CT26 cells in vitro.

    fig. S5. Confocal images of CT26 cells and their CRT staining after sHDL-DOX treatment.

    fig. S6. Biodistribution of Lipo-DiR, PEG-Lipo-DiR, and sHDL-DiR in CT26 tumors in vivo.

    fig. S7. Intratumoral uptake of DOX or sHDL-DOX in CT26 tumors in vivo.

    fig. S8. Analysis of DCs in TDLNs after treatment of CT26 tumor-bearing mice with DOX or sHDL-DOX.

    fig. S9. Antitumor immune responses in the tumor microenvironment.

    fig. S10. Efficacy of sHDL-DOX + αPD-1 therapy against MCA205 fibrosarcoma.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Schematic for the synthesis of the lipid-DOX conjugate.
    • fig. S2. Mass spectroscopy confirmed the conjugation of DOX to PTD.
    • fig. S3. sHDL-DOX was lyophilized and stored for 2 months before reconstitution by adding water to the lyophilized powder.
    • fig. S4. Cellular uptake of DOX or sHDL-DOX by CT26 cells in vitro.
    • fig. S5. Confocal images of CT26 cells and their CRT staining after sHDL-DOX treatment.
    • fig. S6. Biodistribution of Lipo-DiR, PEG-Lipo-DiR, and sHDL-DiR in CT26 tumors in vivo.
    • fig. S7. Intratumoral uptake of DOX or sHDL-DOX in CT26 tumors in vivo.
    • fig. S8. Analysis of DCs in TDLNs after treatment of CT26 tumor-bearing mice with DOX or sHDL-DOX.
    • fig. S9. Antitumor immune responses in the tumor microenvironment.
    • fig. S10. Efficacy of sHDL-DOX + αPD-1 therapy against MCA205 fibrosarcoma.

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