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. 2019 Jul 26;10(1):3349.
doi: 10.1038/s41467-019-11269-8.

Targeting photodynamic and photothermal therapy to the endoplasmic reticulum enhances immunogenic cancer cell death

Wei Li  1 , Jie Yang  1 , Lihua Luo  1 , Mengshi Jiang  1 , Bing Qin  1 , Hang Yin  1 , Chunqi Zhu  1 , Xiaoling Yuan  1 , Junlei Zhang  1 , Zhenyu Luo  1 , Yongzhong Du  1 , Qingpo Li  1 , Yan Lou  2 , Yunqing Qiu  2 , Jian You  3
Affiliations

Targeting photodynamic and photothermal therapy to the endoplasmic reticulum enhances immunogenic cancer cell death

Wei Li et al. Nat Commun. .

Abstract

Immunogenic cell death (ICD)-associated immunogenicity can be evoked through reactive oxygen species (ROS) produced via endoplasmic reticulum (ER) stress. In this study, we generate a double ER-targeting strategy to realize photodynamic therapy (PDT) photothermal therapy (PTT) immunotherapy. This nanosystem consists of ER-targeting pardaxin (FAL) peptides modified-, indocyanine green (ICG) conjugated- hollow gold nanospheres (FAL-ICG-HAuNS), together with an oxygen-delivering hemoglobin (Hb) liposome (FAL-Hb lipo), designed to reverse hypoxia. Compared with non-targeting nanosystems, the ER-targeting naosystem induces robust ER stress and calreticulin (CRT) exposure on the cell surface under near-infrared (NIR) light irradiation. CRT, a marker for ICD, acts as an 'eat me' signal to stimulate the antigen presenting function of dendritic cells. As a result, a series of immunological responses are activated, including CD8+ T cell proliferation and cytotoxic cytokine secretion. In conclusion, ER-targeting PDT-PTT promoted ICD-associated immunotherapy through direct ROS-based ER stress and exhibited enhanced anti-tumour efficacy.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Characterization. a Representative transmission electron microscopic (TEM) images of ICG-HAuNS and FAL-ICG-HAuNS. Scale bars, 100 nm. b Ultraviolet–visible (UV-vis) absorption spectra of ICG-HAuNS and FAL-ICG-HAuNS (indocyanine green (ICG): 10 μg/mL; hollow gold nanospheres (HAuNS): 20 μg/mL). c Representative TEM images of Hb-lipo and FAL-Hb-lipo. Scale bars, 100 nm. d UV-vis absorption spectra of Hb-lipo and FAL-Hb-lipo (hemoglobin (Hb): 80 μg/mL)
Fig. 2
Fig. 2
Lysosome and endoplasmic reticulum (ER) co-localization. a Representative fluorescent images of lysosomal co-localization of ICG-HAuNS, FAL-ICG-HAuNS, Hb-lipo, or FAL-Hb-lipo under normoxia and hypoxia, respectively. Scale bars, 10 μm, n = 3. b Enlarged images of a (red boxes). Yellow arrows indicate “merge”; green arrows indicate “lysosome”; red arrows indicate “ICG/DID”. Scale bars, 5 μm, n = 3. c Representative fluorescent images of ER co-localization of ICG-HAuNS, FAL-ICG-HAuNS, Hb-lipo, or FAL-Hb-lipo under normoxia and hypoxia, respectively. Scale bars, 10 μm, n = 3. d Enlarged images of c (red boxes). Yellow arrows indicate “merge”; green arrows indicate “ER”; red arrows indicate “ICG/DID”. Scale bars, 5 μm, n = 3
Fig. 3
Fig. 3
Reactive oxygen species (ROS) generation and antitumor effect in vitro. a ROS generation in free indocyanine green (ICG; 10 μg/mL), hollow gold nanospheres (HAuNS; 20 μg/mL), ICG-HAuNS (ICG: 10 μg/mL; HAuNS: 20 μg/mL), or ICG-HAuNS plus Hb-lipo (ICG: 10 μg/mL; HAuNS: 20 μg/mL; Hb: 20 μg/mL) treatment groups under normoxia detected with Singlet Oxygen Sensor Green (SOSG). Laser power: 1 W/cm2, 10 min, n = 3. b ROS generation in free ICG (10 μg/mL), HAuNS (20 μg/mL), ICG-HAuNS (ICG: 10 μg/mL; HAuNS: 20 μg/mL), or ICG-HAuNS plus Hb-lipo (ICG: 10 μg/mL; HAuNS: 20 μg/mL; Hb: 20 μg/mL) treatment groups under hypoxia detected with SOSG. Laser power: 1 W/cm2, 10 min, n = 5. c, d CT-26 cell viability after incubation with free ICG, HAuNS, ICG-HAuNS, FAL-ICG-HAuNS, Hb-lipo, FAL-Hb-lipo, ICG-HAuNS plus Hb-lipo, FAL-ICG-HAuNS plus Hb-lipo, or FAL-ICG-HAuNS plus FAL-Hb-lipo (equivalent ICG: 0.5, 5, 25 μg/mL; equivalent HAuNS: 1, 10, 50 μg/mL; equivalent Hb: 1, 10, 50 μg/mL) for 48 h under normoxia (c) or hypoxia (d), n = 5. All data were analyzed with one-way analysis of variance test. All error bars are expressed as ±SD. “NS” indicates “not significant”
Fig. 4
Fig. 4
Specific endoplasmic reticulum stress and calreticulin (CRT) exposure. a Western blot analysis of caspase-3 and CHOP (C/EBP-homologous protein-10) proteins expressed in CT-26 cells after treatment with free indocyanine green (ICG; 20 μg/mL), ICG-HAuNS (ICG: 20 μg/mL; hollow gold nanospheres (HAuNS): 40 μg/mL), FAL-ICG-HAuNS (ICG: 20 μg/mL; HAuNS: 40 μg/mL), ICG-HAuNS (ICG: 20 μg/mL; HAuNS: 40 μg/mL) plus Hb-lipo (40 μg/mL), or FAL-ICG-HAuNS (ICG: 20 μg/mL; HAuNS: 40 μg/mL) plus FAL-Hb-lipo (40 μg/mL) for 12 h. Laser power: 2 W/cm2, 2 min, n = 3. b Quantification of cleaved caspase-3 and CHOP proteins normalized by β-actin, respectively. c–f CRT exposure (c, d) and total amount of CRT (e, f) after treatment with ICG-HAuNS or FAL-ICG-HAuNS followed by laser irradiation (1 W/cm2 or 3 W/cm2, 2 min), n = 3. Ex: 488 nm. Scale bars, 50 μm. g Representative fluorescent imaging on CRT exposure with (1 W/cm2, 2 min) or without laser irradiation. Tocopherol (Vitamin E) was employed as a reactive oxygen species scavenger. Ex: 488 nm. Scale bars, 20 μm, n = 3. All data were analyzed with one-way analysis of variance test. All error bars are expressed as ±SD. “NS” indicates “not significant”
Fig. 5
Fig. 5
Biodistribution and antitumor effect in CT-26 tumor model. a Real-time in vivo fluorescent imaging in mice after they were intravenously injected with ICG-HAuNS (indocyanine green (ICG): 4 mg/kg; hollow gold nanospheres (HAuNS): 8 mg/kg), FAL-ICG-HAuNS (ICG: 4 mg/kg; HAuNS: 8 mg/kg), DiD@Hb-lipo (Hb: 20 mg/kg), or DiD@FAL-Hb-lipo (Hb: 20 mg/kg). Ex: 649 nm for DiD; 735 nm for ICG. b CT-26 tumor growth curves after treatment with saline, ICG-HAuNS (ICG: 0.5 mg/kg and HAuNS: 1 mg/kg per injection), FAL-ICG-HAuNS (ICG: 0.5 mg/kg and HAuNS: 1 mg/kg per injection), ICG-HAuNS (ICG: 0.5 mg/kg and HAuNS: 1 mg/kg per injection) plus Hb-lipo group (20 mg/kg per injection), or FAL-ICG-HAuNS (ICG: 0.5 mg/kg and HAuNS: 1 mg/kg per injection) plus FAL-Hb-lipo (20 mg/kg per injection). Laser power: 1 W/cm2, 2 min, n = 6. c Kaplan–Meier plots on animal survival, n = 6. d Representative Ki-67 and hematoxylin and eosin staining results in each group. Scale bars, 300 μm. All data were analyzed with one-way analysis of variance test. All error bars are expressed as ±SD
Fig. 6
Fig. 6
Immunogenic cell death induced by specific endoplasmic reticulum stress in CT-26 tumor model. Laser power: 1 W/cm2, 2 min. a Western blot analysis of caspase-3 and CHOP (C/EBP-homologous protein-10) proteins expressed in CT-26 tumors after drug administration and laser irradiation, n = 3. b Quantification of cleaved caspase-3 and CHOP proteins normalized by β-actin, respectively. c Representative fluorescent images of calreticulin expression in CT-26 tumor slices. Scale bars, 50 μm, n = 3. d Representative images of dendritic cell maturation in tumors and lymph nodes analyzed with flow cytometry, n = 3. e Representative fluorescent images of CD8 makers in tumor slices after different treatments. Scale bars, 50 μm, n = 3. All data were analyzed with one-way analysis of variance test. All error bars are expressed as ±SD
Fig. 7
Fig. 7
Relief of tumor hypoxia microenvironment in CT-26 model. Laser power: 1 W/cm2, 2 min. a Representative hypoxia fluorescent photomicrographs of tumors in each group. Scale bars, 200 μm. b Mean fluorescence intensity in the tumor slices (n = 3). All data were analyzed with one-way analysis of variance test. All error bars are expressed as ±SD. “NS” indicates “not significant”
Fig. 8
Fig. 8
Antitumor efficacy in B16 tumor model. a Growth curves of B16 tumors after treatment with saline, chemotherapy (Taxotere: 10 mg/kg per injection), FAL-HAuNS, ICG-HAuNS, FAL-ICG-HAuNS, ICG-HAuNS plus Hb-lipo, or FAL-ICG-HAuNS plus FAL-Hb-lipo (ICG: 0.5 mg/kg, HAuNS: 1 mg/kg, Hb: 20 mg/kg per injection). Taxotere, ICG-HAuNS, or FAL-ICG-HAuNS were injected on days 0, 2, and 4. Hb-lipo and FAL-Hb-lipo were injected on days 1, 3, and 5. Tumors (except the chemotherapy group) were exposed to laser irradiation (1 W/cm2, 2 min) on days 1, 3, and 5. n = 6. b Body weight change curves, n = 6. c Average tumor weights in different groups at the end of treatment. d Representative hematoxylin and eosin and Ki-67 staining photographs in each group. Scale bars, 100 μm. All data were analyzed with one-way analysis of variance test (*P ≤ 0.05; **P ≤ 0.01). All error bars are expressed as ±SD
Fig. 9
Fig. 9
Immunogenic cell death induced by specific endoplasmic reticulum stress in B16 tumor model. a Immunofluorescent staining of calreticulin expression, CD8+ T cells, interferon (IFN)-γ, or Foxp3+ T cells in tumor sections at the end of the treatments. Scale bars, 50 μm. b Dendritic cells (DCs) maturation in lymph nodes analyzed with flow cytometry, n = 6. c Quantification of mature DCs based on the results in b, n = 6. d IFN-γ, tumor necrosis factor-α, interleukin (IL)-6, and IL-10 levels in tumor detected with the enzyme-linked immunosorbent assay, n = 6. e Representative flow cytometric plots of regulatory T cells (CD3+/CD4+/Foxp3+) and activated CD8+ T cells (CD3+/CD8+/CD44+ or CD3+/CD8+/INF-γ) in the spleens after various treatments. f The growth curves of B16 tumors after treatment with saline or FAL-ICG-HAuNS plus FAL-Hb-lipo (ICG: 0.5 mg/kg, HAuNS: 1 mg/kg, Hb: 20 mg/kg per injection). FAL-ICG-HAuNS was injected on days 0, 2, and 4. FAL-Hb-lipo was injected on days 1, 3, and 5. Tumors (except the chemotherapy group) were exposed to laser irradiation (1 W/cm2, 2 min) on days 1, 3, and 5. As for CD4 or CD8 depletion group, either CD4+ or CD8+ T cells were depleted in vivo with anti-CD8 or anti-CD4 antibody (intraperitoneal, 100 μg/mice per injection on days −3, 0, 2, and 4), n = 6. g Representative flow cytometric plots of CD8+ and CD4+ T cell populations in the spleen after depletion. All data were analyzed with one-way analysis of variance test (*P ≤ 0.05; **P ≤ 0.01). All error bars are expressed as ±SD
Fig. 10
Fig. 10
The antitumor mechanism of FAL-ICG-HAuNS plus FAL-Hb-lipo. Schematic illustration of enhanced immunogenic cancer cell death and anticancer effect induced by endoplasmic reticulum-targeting photothermal/photodynamic therapy
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