Research ArticleCancer

In situ formed reactive oxygen species–responsive scaffold with gemcitabine and checkpoint inhibitor for combination therapy

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Science Translational Medicine  21 Feb 2018:
Vol. 10, Issue 429, eaan3682
DOI: 10.1126/scitranslmed.aan3682
  • Fig. 1 Schematic and characterization of in situ formed ROS-responsive gel scaffold.

    (A) Schematic of combination chemoimmunotherapy using a ROS-degradable hydrogel scaffold to deliver GEM and aPDL1 into the TME. (B) Representative Cryo-SEM image of gel scaffold loaded with GEM and aPDL1. Scale bar, 0.5 μm. Inset: Zoomed-in image of the scaffold. Scale bar, 0.1 μm. (C) Representative fluorescent images of a cryosection of hydrogel in which fluorescein isothiocyanate (FITC) (green) was used as a fluorescent surrogate for GEM and aPDL1 was labeled with Cy5.5 (red). Scale bar, 25 μm. (D) Morphology changes of hydrogels in 1× PBS with and without H2O2 (1 mM) over 7 days. (E and F) Cumulative release profiles of GEM (E) and aPDL1 (F) from hydrogels incubated with PBS with or without H2O2 (1 mM). Data are means ± SEM. IgG, immunoglobulin G.

  • Fig. 2 GEM@Gel implantation for eliciting immunogenic tumor phenotypes.

    B16F10 tumors harvested from mice implanted with hydrogels or GEM@Gel were analyzed by flow cytometry 2 days after treatment. (A) Representative flow cytometric analysis of T cell infiltration within the tumor and (B) corresponding quantification results. UnTx, untreated. (C) Representative flow cytometric analysis images (left) and the corresponding quantification (right) of MDSCs (CD11b+Gr-1+), gating on CD45+ cells. (D) Representative flow cytometric analysis images (left) and the corresponding quantification (right) of M2 macrophages (CD206+) in F4/80+ CD11b+ CD45+ cells. (E) Confocal immunofluorescence images of B16F10 tumor with (right) or without (left) GEM@Gel treatment. Red and blue colors represent aPDL1 signals from Cy3-conjugated aPDL1 and 4′,6-diamidino-2-phenylindole (DAPI) staining of nuclei, respectively. Scale bar, 20 μm. (F) PD-L1 expression of tumor cells and PD-1 expression of TILs after empty hydrogel or GEM@Gel treatment and the corresponding quantification of PD-L1 and PD-1 mean fluorescence intensity (MFI). a.u., arbitrary units; DC, dendritic cell. (G) Systemic IL-6 and IFN-γ concentrations before and after GEM@Gel treatment. Data are means ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.005.

  • Fig. 3 Local gel scaffold for inhibition of B16F10 melanoma growth in vivo.

    (A) In vivo bioluminescence imaging of the B16F10 tumor in control and treated groups. Three representative mice of 7 to 10 mice per treatment group are shown. (B and C) Individual (B) and average (C) tumor growth kinetics in control and treated groups (treatment started at day 0). Inset: Representative mouse photographs 2 weeks after treatment. White arrows indicate the tumors. Growth curves represent means ± SEM; growth curves were stopped when the first animal of the corresponding group died. (D) Survival curves for the treated and control mice (n = 7 to 10). ***P < 0.001. (E) Immunofluorescence of tumors showing CD4+ and CD8+ T cell infiltration. Scale bar, 100 μm. (F and G) Absolute numbers of the CD8+ (F) and CD4+ T cells (G) per gram of the tumor upon various treatments. (H and I) Ratios of the tumor-infiltrating CD8+ T cells (H) and CD4+ T cells (I) to Tregs in the tumors upon various treatments. Teff, effector T cell. Data are means ± SEM. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.005.

  • Fig. 4 Local gel scaffold for systemic anticancer immune response.

    (A) Mice were inoculated with tumor cells in the right and left flanks. Control mice were untreated, whereas treated mice were implanted with hydrogels only on the left flank. (B) PD-L1 expression of cancer cells collected from the tumor sites of the control and treated mice and (C) corresponding quantification of PD-L1 MFI (n = 3). (D) In vivo bioluminescence imaging of B16F10 tumors in response to local aPDL1-GEM@Gel treatment. (E) Left and right tumor growth curves and (F) tumor weight on day 10 in untreated and treated mice. (G) Representative photographs of mice on day 10 after treatment. White arrows indicate the tumors. (H) Percentages and representative dot plots of CD4+ and CD8+ T cells in tumors of control and treated mice and (I) absolute numbers of CD8+ cells per gram of tumor. Data are means ± SEM. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. *P < 0.05; ***P < 0.005.

  • Fig. 5 Local gel scaffold for T cell memory response.

    (A) Splenocytes isolated from tumor-bearing control and treated mice were analyzed for the presence of CD8+CD44+CD122+ and CD4+CD44+CD122+ central memory T cells (TCM). (B and C) Corresponding quantification of CD4 (B) and CD8 (C) TCM in splenocytes. (D) In vivo bioluminescence imaging of mice after rechallenging with intravenous injection of B16F10 cancer cells. (E) Representative lung photographs (day 10) and (F) H&E staining of lungs collected from control (naïve) and treated (cured) mice after rechallenging. The blue arrowheads indicate metastatic tumors in the lungs. Scale bar, 100 μm. (G) Survival curves for naïve and treated mice. Six mice for each group are shown. Data are means ± SEM. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. **P < 0.01.

  • Fig. 6 Local gel scaffold for treatment of low-immunogenic 4T1 carcinoma tumor.

    (A) In vivo bioluminescence imaging of the 4T1 tumor growth in control and treated mice. (B) Tumor growth kinetics in control and treated mice. Growth curves represent means ± SEM; growth curves were stopped when the first animal of the corresponding group was euthanized. (C) Survival curves for control and treated mice (n = 5 to 10). Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 7 Gel scaffold for inhibition of postsurgical recurrence of B16F10 tumors.

    (A) In vivo bioluminescence imaging of the B16F10 tumor growth in C57B6 mice after various treatments as indicated. (B to D) Individual (B) and average quantitative bioluminescence signals of tumors (C) and tumor growth kinetics (D) in control and treated groups. Black arrows indicate the day of the surgery (day 9). (E) Survival curves for different treatments (n = 7 to 10). Growth curves were stopped when the first animal of the corresponding group was euthanized. Data are means ± SEM. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 8 Gel scaffold for inhibition of postsurgical recurrence of 4T1 tumors.

    (A) Individual and (B) average tumor growth kinetics in control and treated groups receiving the indicated treatments. Growth curves were stopped when the first animal of the corresponding group was euthanized or dead. (C) Survival curves for different treatments (n = 5 to 9, as indicated in the figure). (D) Measurements of body weight of control and treated mice. Black arrows indicate the day of the surgery (day 14). Data are means ± SEM. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. *P < 0.05.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/429/eaan3682/DC1

    Materials and Methods

    Fig. S1. Schematic of the H2O2 responsiveness mechanism of the PVA-TSPBA gel.

    Fig. S2. Synthesis route and characterization of TSPBA.

    Fig. S3. Dynamic rheological behavior of PVA before and after gelation.

    Fig. S4. In vivo gel maintenance.

    Fig. S5. Oxidation and hydrolysis of TSPBA.

    Fig. S6. In vivo release of payloads from gel scaffold.

    Fig. S7. Low-dose GEM@Gel for enhancement of lymphocyte infiltration.

    Fig. S8. ROS-responsive scaffold as a scavenger of ROS within the TME.

    Fig. S9. Frequency of CD4+FOXP3+ T cells within the tumors of mice receiving the indicated treatments.

    Fig. S10. Effects of GEM on cancer cells in vitro.

    Fig. S11. In vivo PD-L1 expression in tumor cells at different time points.

    Fig. S12. Effects of GEM@Gel on systemic concentrations of cytokines.

    Fig. S13. Tumor inhibition in mice treated with free drugs compared to aPDL1-GEM@Gel.

    Fig. S14. Characterization of T cell–mediated antitumor immune response.

    Fig. S15. PD-L1 expression in distant tumors.

    Fig. S16. Effects of GEM on 4T1 cells in vitro.

    Fig. S17. Biocompatibility of hydrogel in vivo.

    Fig. S18. Body weight of control and treated mice.

  • Supplementary Material for:

    In situ formed reactive oxygen species–responsive scaffold with gemcitabine and checkpoint inhibitor for combination therapy

    Chao Wang, Jinqiang Wang, Xudong Zhang, Shuangjiang Yu, Di Wen, Quanyin Hu, Yanqi Ye, Hunter Bomba, Xiuli Hu, Zhuang Liu, Gianpietro Dotti, Zhen Gu*

    *Corresponding author. Email: zgu{at}email.unc.edu

    Published 21 February 2018, Sci. Transl. Med. 10, eaan3682 (2018)
    DOI: 10.1126/scitranslmed.aan3682

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Schematic of the H2O2 responsiveness mechanism of the PVA-TSPBA gel.
    • Fig. S2. Synthesis route and characterization of TSPBA.
    • Fig. S3. Dynamic rheological behavior of PVA before and after gelation.
    • Fig. S4. In vivo gel maintenance.
    • Fig. S5. Oxidation and hydrolysis of TSPBA.
    • Fig. S6. In vivo release of payloads from gel scaffold.
    • Fig. S7. Low-dose GEM@Gel for enhancement of lymphocyte infiltration.
    • Fig. S8. ROS-responsive scaffold as a scavenger of ROS within the TME.
    • Fig. S9. Frequency of CD4+FOXP3+ T cells within the tumors of mice receiving the indicated treatments.
    • Fig. S10. Effects of GEM on cancer cells in vitro.
    • Fig. S11. In vivo PD-L1 expression in tumor cells at different time points.
    • Fig. S12. Effects of GEM@Gel on systemic concentrations of cytokines.
    • Fig. S13. Tumor inhibition in mice treated with free drugs compared to aPDL1-GEM@Gel.
    • Fig. S14. Characterization of T cell–mediated antitumor immune response.
    • Fig. S15. PD-L1 expression in distant tumors.
    • Fig. S16. Effects of GEM on 4T1 cells in vitro.
    • Fig. S17. Biocompatibility of hydrogel in vivo.
    • Fig. S18. Body weight of control and treated mice.

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