Research ArticleHEALTH AND MEDICINE

GSH depletion liposome adjuvant for augmenting the photothermal immunotherapy of breast cancer

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Science Advances  02 Sep 2020:
Vol. 6, no. 36, eabc4373
DOI: 10.1126/sciadv.abc4373
  • Fig. 1 Scheme illustration of GSH depletion adjuvant for augmenting the PA imaging–guided photothermal immunotherapy of breast cancer.

    (A) Preparation of the AHL photothermal liposomes and ML adjuvant. (B) Tumor PTT-induced ICD to stimulate DC maturation and further promote the CD8+ T cell infiltration in tumor, inducing abscopal effect and antimetastasis effect of breast cancer. In tumor cells, ML promotes ROS generation and ABTS activation, thus augmenting the PA imaging (PAI)–guided PTT and ICD-mediated immunotherapy. In DCs, ML relieves the immunosuppression by inducing the ROS generation to promote DC maturation and antigen presentation.

  • Fig. 2 Physicochemical characterization of AHL and ML.

    (A) Scheme of the component in AHL and ML. (B) Particle size of AHL determined by DLS. Inset: TEM images of AHL. Scale bar, 200 nm. (C) Particle size of ML determined by DLS. Inset: TEM images of ML. Scale bar, 200 nm. (D) Colloidal stability of AHL and ML within 7 days by monitoring the DLS particle size (n = 3). (E) Scheme illustration of MA-mediated GSH depletion and its role on enhancing the ABTS activation. (F) Ultraviolet-visible (UV-Vis) spectrum of AHL, AHL + H2O2, and AH + H2O2. (G) Absorbance of AHL at 730 nm in the presence of different concentrations of H2O2 (0 to 200 μM; n = 3). (H) The relative GSH level after treatment with increasing molar ratios of free MA/GSH or ML/GSH (n = 3). (I) Absorbance changing of AHL + H2O2 in the presence of increasing GSH. (J) Absorbance changing of AHL + H2O2 + ML in the presence of increasing GSH. (K) Statistics of the absorbance at 800 nm of AHL + H2O2, AHL + H2O2 + GSH, and AHL + H2O2 + ML + GSH (n = 3). ***P < 0.001. (L) Temperature-changing curve and (M) IR images of DW, AHL + H2O2, AHL + H2O2 + GSH, and AHL + H2O2 + ML + GSH after 808-nm laser irradiation for 120 s. (N) PA signal of AHL + H2O2 or AHL + H2O2 + ML in the presence of different concentrations of GSH (n = 3). d, diameter.

  • Fig. 3 Cellular study of AHL and ML.

    (A) Cell uptake of ABTS&HRP-FITC liposomes and MA&RhB liposomes by 4T1 cells. Scale bars, 10 μm. (B) GSH and ROS levels in 4T1 cells after treatment with phosphate-buffered saline (PBS) or ML. Scale bars, 50 μm. (C) Representative cell viability of 4T1 cells after treatment with AHL + H2O2, AHL + H2O2 + L, AHL + ML + H2O2, and AHL + ML + H2O2 + L (n = 3). ***P < 0.001. (D) Cell apoptosis of 4T1 cells after different treatments determined by flow cytometry (FCM). (E) Statistical analysis of (D). (F) Calreticulin (CRT)–positive cell analysis by FCM (n = 3). ***P < 0.001. (G) High-mobility group protein B1 (HMGB1) secreted from 4T1 cells and detected by an enzyme-linked immunosorbent assay (ELISA) kit (n = 3). **P < 0.01. (H) Adenosine triphosphate (ATP) secretion detection by the Enhanced ATP Assay Kit (n = 3). ***P < 0.001. (I) ROS level in DC2.4 cells after treatment with PBS or ML. Scale bars, 200 μm. (J) DC maturation after incubation with 4T1 cells with different treatments (gated on CD11c+ cells). (K) Statistical analysis of (J) (n = 3). **P < 0.01.

  • Fig. 4 In vivo PA imaging and antitumor study of AHL and ML.

    (A) Tumoral GSH detection after treatment with saline, AHL, or AHL + ML (n = 3). **P < 0.01. (B) PA images of 4T1 tumor after intravenous (i.v.) injection of AHL or AHL + ML. (C) Tumor temperature changing after treatment with saline, AHL, or AHL + ML and laser irradiation (n = 3). (D) Photothermal imaging and tumoral temperature analysis of the mice using an IR camera after treatment with saline, AHL, or AHL + ML. (E) Schematic illustration of the animal experimental design. (F) Primary tumor growth curves with the mean tumor volumes of 4T1 tumor–bearing BALB/c mice model (n = 5). ***P < 0.001. s.c., subcutaneous. (G) Primary tumor growth curves of individual mouse in different groups of 4T1 tumor–bearing BALB/c mice model. (H) The survival percentages of the tumor-bearing BALB/c mice (n = 8).

  • Fig. 5 In vivo immune activation by AHL and ML.

    (A and B) DC maturation on 4T1 tumor–bearing mice (gated on CD11+ DC cells). Cells in lymph nodes were collected on day 5 after various treatments for assessment by FCM after staining with CD11c+, CD80, and CD86. *P < 0.05 and **P < 0.01. (C and D) FCM examination of the intratumor infiltration of CD8+ T cells (gated on CD3+ T cells). ***P < 0.001. (E) Immunofluorescence images of CD8+ T cells. Scale bars, 100 μm. (F and G) Contents of tumor necrosis factor–α (TNF-α) and interferon-γ (IFN-γ) in plasma on day 5 after treatment. ***P < 0.001. (H and I) The regulatory T cell (Treg) frequencies in tumors after different treatments examined on day 5 after treatment. Data represent means ± SD (n = 5). **P < 0.01 and ***P < 0.001.

  • Fig. 6 Abscopal effect and antimetastasis effect of AHL and ML.

    (A) Schematic illustration of the animal experimental design for distant tumor. (B) Distant tumor growth curves with the mean tumor volumes of 4T1 tumor–bearing BALB/c mice model. Data represent means ± SD (n = 5). (C) Percentage of CD8+ T cells in the distant tumors. Data represent means ± SD (n = 5). *P < 0.05 and **P < 0.01. (D) Percentage of Tregs in the distant tumors. Data represent means ± SD (n = 5). *P < 0.05 and **P < 0.01. (E) Schematic illustration of the animal experimental design for antimetastatic study. (F) Representative photographs and H&E staining of lung tissues with tumor metastasis collected on day 30. Scale bars, 200 μm. (G) Quantification of pulmonary metastasis nodules in different groups of 4T1 tumor–bearing BALB/c mice. Data represent means ± SD (n = 5). **P < 0.01 and ***P < 0.001.

Supplementary Materials

  • Supplementary Materials

    GSH depletion liposome adjuvant for augmenting the photothermal immunotherapy of breast cancer

    Zhanwei Zhou, Hui Wu, Ruoxi Yang, Alan Xu, Qingyan Zhang, Jingwen Dong, Chenggen Qian, Minjie Sun

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