Research ArticleTUMOR IMMUNOLOGY

A melanin-mediated cancer immunotherapy patch

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Science Immunology  10 Nov 2017:
Vol. 2, Issue 17, eaan5692
DOI: 10.1126/sciimmunol.aan5692
  • Fig. 1 Schematic of melanin-mediated cancer immunotherapy through a transdermal MN-based vaccine patch.

    (A) Schematic illustration of MN-based transdermal vaccination. TH cell, T helper cell; Tc cell, cytotoxic T cell. (B) Photograph of representative MN patches without (W/o) melanin and with (W/) melanin (scale bar, 4 mm). (C) Scanning electron microscopy image of the MN patch (scale bar, 400 μm). (D) Fluorescence cross-sectional images of a representative MN. Actin filaments in cells were visualized by Alexa Fluor 488 phalloidin (green), cell DNA fragments were stained with Hoechst (blue), and hyaluronic acid polymer matrix was labeled with rhodamine B (red) (scale bar, 200 μm). (E) Fluorescence imaging of a representative MN patch that contained the Alexa Fluor 488 phalloidin–labeled tumor lysate and rhodamine B–labeled hyaluronic acid (scale bar, 400 μm).

  • Fig. 2 Characterization of the light-responsive transdermal MNs.

    (A) Surface temperature changes of the MNs with or without tumor lysate in real time with continuous 808-nm NIR irradiation at 1.0 W/cm2, characterized by an infrared thermal camera (scale bars, 1 mm). (B) Quantitative surface temperature changes of representative MNs with continuous NIR irradiation at 1.0 W/cm2 (n = 3). (C) Quantitative temperature changes of representative MN patch with increasing laser power flux (n = 3). (D) In vitro collective release of tumor lysate proteins from the MN patch (n = 3). (E) In vitro activation of DCs in response to MNs loaded with tumor lysate and GM-CSF or LPS and exposed to NIR irradiation for different time (n = 3). (F) LIVE/DEAD assay of DCs after treatments with (i) blank MN, (ii) 10 min of NIR, (iii) MN, and (iv) MN and 10 min of NIR. Live cell (green), dead cell (red). (G) Quantification of DC viability after treatments. Data points represent mean ± SD (n = 3). Error bars indicate SD. Statistical significance was calculated by Student’s t test [not significant (NS), P > 0.05; *P < 0.05].

  • Fig. 3 Vaccine MN confer protective innate and adaptive immunity.

    (A) Schematic illustration of MN cancer immunotherapy. (B) Characterization of the MNs after insertion. (i) Photo of mouse dorsal skin (the area within the red line) that was treated transdermally with one MN patch and (ii) fluorescence signals of Cy5.5-labeled MN patch over time. (C) Surface temperature changes of individual animal after MN insertion into the skin measured by an infrared thermal camera. (D) Schematic representation of the B16F10 vaccine tumor model. (E) Average tumor volumes in treated mice after tumor challenge. (F) Kaplan-Meier survival curves for treated and control mice. Data points represent mean ± SD (n = 8). Error bars indicate SD. Statistical significance was calculated by Student’s test and log-rank test (**P < 0.01; ***P < 0.001). (G) In vivo bioluminescence imaging of the B16F10 melanoma in different experimental groups and at different time points after tumor challenge. Three representative mice per treatment group are shown. The tumor growth in (E) to (G) was measured 10 days after tumor cell inoculation.

  • Fig. 4 Immune cell recruitment after the NIR-boosted and MN-mediated cancer immunotherapy.

    (A) Representative quantitative analysis of DCs (CD11c+ and PIR-A/B+) infiltrated in the skin 3 days after treatments as assessed by flow cytometry. The indicated samples were treated with blank MN (blank), vaccine MN (MN), MN loaded with tumor lysate without GM-CSF and treated with NIR (NIR), and vaccine MN and treated with NIR (MN + NIR). a.u., arbitrary units. (B) Representative quantitative analysis of NK cells (CD49b+) in the skin upon transdermal cancer immunotherapy as assessed by flow cytometry. (C and D) Immunofluorescence staining and quantitative analysis of (C) CD11c+ DCs (scale bar, 100 μm) and (D) CD49b+ NK cells (scale bar, 100 μm). Statistical significance was calculated by Student’s t test (*P < 0.05; **P < 0.01). Asterisks indicate significant differences between the MN + NIR group and all other treatment groups. Data points represent mean ± SD (n = 3). Error bars indicate SD.

  • Fig. 5 Immunologic responses after the MN-mediated cancer immunotherapy.

    (A) Representative quantitative analysis of T cells (gated on CD3+ T cells) in treated tumors analyzed by flow cytometry. (B) Representative quantitative analysis of activated DCs (CD86+ and CD80+) in the draining lymph nodes analyzed by flow cytometry. Data points represent mean ± SD (n = 8). (C) Immunofluorescence staining of the tumors showing CD4+ T cell and CD8+ T cell infiltration (scale bars, 100 μm). (D) Quantification of IgG1 subtypes in serum collected at day 10. Data points represent mean ± SD (n = 8). (E) Cytotoxic responses of splenocytes against B16F10 cells in vitro. Data points represent mean ± SD (n = 6). (F) Immunofluorescence staining of HSP70 (green) in the regional skin with actin filaments visualized by Alexa Fluor 660 phalloidin (red) and cell nuclei stained with Hoechst (blue) (scale bars, 100 μm). (G) In vivo local detection of cytokines from extracted patches at day 3. Statistical significance was calculated by Student’s t test (*P < 0.05; **P < 0.01). Data points represent mean ± SD (n = 8). Error bars indicate SD. CTL, cytotoxic T lypmphocyte.

  • Fig. 6 Antitumor effect of local cancer immunotherapy treatment toward distant B16F10 tumors.

    (A) Schematic representation of the B16F10 tumor model. (B) Photograph of a mouse before and after MN administration. Red arrows indicate established tumors on both sides. L, left; R, right.The red line indicates the MN injection site. (C) Tumor weights in different experimental groups (n = 3). (D) Tumor-infiltrating CD8+ T cells after treatments in different experimental groups (n = 6). Asterisks in (C) and (D) indicate statistically significant differences between blank MN and other groups. (E) Images of tumors extracted from treated mice indicated by the labels in (D). (F) In vivo bioluminescence imaging of treated B16F10 melanoma at different time points after treatment. One representative mouse per treatment group is shown. (G) Average tumor volumes in treated mice. (H) Body weights of treated mice (n = 6). (I) Average B16F10 tumor volumes in mice treated with MNs loaded with BP lysate with melanin and blank MN (n = 8). (J) Average tumor volumes in vaccinated mice rechallenged with either B16F10 cells or BP cells on day 80. (K) Kaplan-Meier survival curves for rechallenged mice. Data points represent mean ± SD (n = 8). Error bars indicate SD. Statistical significance was calculated by Student’s t test and log-rank test (NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001).

  • Fig. 7 Antitumor effect of local cancer immunotherapy treatment in various tumor models.

    (A) Average tumor growth and Kaplan-Meier survival rate of vaccinated C57BL/6J mice after BP tumor cell challenge. Mice were pretreated with blank MN (blank), MN loaded with BP tumor lysate and melanin (MN), or loaded MN combined with NIR irradiation (MN + NIR). (B) Average tumor growth and Kaplan-Meier survival rate of vaccinated BALB/cJ mice after 4T1 tumor cell challenge. Mice were pretreated with blank MN, MN loaded with 4T1 tumor lysate and melanin, or loaded MN combined with NIR irradiation. (C) Average tumor growth and Kaplan-Meier survival rate of C57BL/6J mice bearing established BP tumors. Mice were treated with blank MN, MN loaded with BP tumor lysate and melanin, or loaded MN combined with NIR irradiation. (D) Average tumor growth and Kaplan-Meier survival rate of BALB/cJ mice bearing established 4T1 tumors. Mice were treated with blank MN, MN loaded with 4T1 tumor lysate and melanin, or loaded MN combined with NIR irradiation. Data points represent mean ± SD (n = 8). Error bars indicate SD. Statistical significance was calculated by Student’s t test and log-rank test (*P < 0.05; **P < 0.01; ***P < 0.001).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/17/eaan5692/DC1

    Materials and Methods

    Fig. S1. Mechanical property of the MN.

    Fig. S2. Characteristics of tumor lysate solution and synthetic melanin.

    Fig. S3. Heating behavior of MN patches by repetitive NIR irradiation.

    Fig. S4. Surface temperature of MN patches with various loadings of tumor lysates upon NIR irradiation.

    Fig. S5. In vitro release profiles of GM-CSF and tumor lysate proteins.

    Fig. S6. Scanning electron microscopy images of MN patch after insertion into the mouse skin over time.

    Fig. S7. DC function evaluation after in vitro activation.

    Fig. S8. Cytotoxicity study of the blank MNs.

    Fig. S9. Characterization of the skin after MN insertion.

    Fig. S10. Melanin-loaded MNs confer protective immunity in vivo.

    Fig. S11. Tumor growth in control and treated mice.

    Fig. S12. Quantified B16F10 bioluminescent tumor signals in control and treated mice.

    Fig. S13. Tumor weights in control and treated mice.

    Fig. S14. Histology and apoptosis analysis of the tumor sections.

    Fig. S15. Tumor growth of mice receiving the transdermal cancer immunotherapy.

    Fig. S16. Measurement of local microcirculatory blood perfusion of mice.

    Fig. S17. Immunologic responses after the transdermal cancer immunotherapy.

    Fig. S18. Quantification of IgG1 subtypes in serum after treatment with blank, MN, or MN + NIR.

    Fig. S19. ROS detection by flow cytometry in tumor sections.

    Fig. S20. HSP90 expression after the transdermal cancer immunotherapy.

    Fig. S21. Cytokine kinetics after the transdermal cancer immunotherapy.

    Fig. S22. Histology analysis after the transdermal cancer immunotherapy.

    Fig. S23. Antitumor effect of the transdermal cancer immunotherapy toward different tumor models.

    Fig. S24. Surface temperature changes of the melanin-loaded MNs.

    Fig. S25. Antitumor effect of the transdermal cancer immunotherapy.

    Fig. S26. HSP70 expression after the transdermal cancer immunotherapy.

    Fig. S27. Representative quantitative analysis of the DC activation.

    Fig. S28. Cytokine kinetics after the transdermal cancer immunotherapy.

    Fig. S29. Average weights of mice after the transdermal cancer immunotherapy in control and treated mice.

    Fig. S30. H&E staining of organs collected after the transdermal cancer immunotherapy.

    Table S1. Melanin content of tumors excised from tumor-bearing mice.

    Table S2. Measurement of total local microcirculatory blood perfusion of mice receiving different treatments using the laser Doppler flowmetry.

    Excel file 1.

    Excel file 2.

  • Supplementary Materials

    Supplementary Material for:

    A melanin-mediated cancer immunotherapy patch

    Yanqi Ye, Chao Wang, Xudong Zhang, Quanyin Hu, Yuqi Zhang, Qi Liu, Di Wen, Joshua Milligan, Adriano Bellotti, Leaf Huang, Gianpietro Dotti, Zhen Gu*

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

    Published 10 November 2017, Sci. Immunol. 2, eaan5692 (2017)
    DOI: 10.1126/sciimmunol.aan5692

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Mechanical property of the MN.
    • Fig. S2. Characteristics of tumor lysate solution and synthetic melanin.
    • Fig. S3. Heating behavior of MN patches by repetitive NIR irradiation.
    • Fig. S4. Surface temperature of MN patches with various loadings of tumor lysates upon NIR irradiation.
    • Fig. S5. In vitro release profiles of GM-CSF and tumor lysate proteins.
    • Fig. S6. Scanning electron microscopy images of MN patch after insertion into the mouse skin over time.
    • Fig. S7. DC function evaluation after in vitro activation.
    • Fig. S8. Cytotoxicity study of the blank MNs.
    • Fig. S9. Characterization of the skin after MN insertion.
    • Fig. S10. Melanin-loaded MNs confer protective immunity in vivo.
    • Fig. S11. Tumor growth in control and treated mice.
    • Fig. S12. Quantified B16F10 bioluminescent tumor signals in control and treated mice.
    • Fig. S13. Tumor weights in control and treated mice.
    • Fig. S14. Histology and apoptosis analysis of the tumor sections.
    • Fig. S15. Tumor growth of mice receiving the transdermal cancer immunotherapy.
    • Fig. S16. Measurement of local microcirculatory blood perfusion of mice.
    • Fig. S17. Immunologic responses after the transdermal cancer immunotherapy.
    • Fig. S18. Quantification of IgG1 subtypes in serum after treatment with blank, MN, or MN + NIR.
    • Fig. S19. ROS detection by flow cytometry in tumor sections.
    • Fig. S20. HSP90 expression after the transdermal cancer immunotherapy.
    • Fig. S21. Cytokine kinetics after the transdermal cancer immunotherapy.
    • Fig. S22. Histology analysis after the transdermal cancer immunotherapy.
    • Fig. S23. Antitumor effect of the transdermal cancer immunotherapy toward different tumor models.
    • Fig. S24. Surface temperature changes of the melanin-loaded MNs.
    • Fig. S25. Antitumor effect of the transdermal cancer immunotherapy.
    • Fig. S26. HSP70 expression after the transdermal cancer immunotherapy.
    • Fig. S27. Representative quantitative analysis of the DC activation.
    • Fig. S28. Cytokine kinetics after the transdermal cancer immunotherapy.
    • Fig. S29. Average weights of mice after the transdermal cancer immunotherapy in control and treated mice.
    • Fig. S30. H&E staining of organs collected after the transdermal cancer immunotherapy.
    • Table S1. Melanin content of tumors excised from tumor-bearing mice.
    • Table S2. Measurement of total local microcirculatory blood perfusion of mice receiving different treatments using the laser Doppler flowmetry.

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