Stimuli-responsive nano vehicle enhances cancer immunotherapy by coordinating mitochondria-targeted immunogenic cell death and PD-L1 blockade

Abstract

Induction of immunogenic cell death promotes antitumor immunity against cancer. However, majority of clinically-approved drugs are unable to elicit sufficient ICD. Here, our study revealed that mitochondria-targeted delivery of doxorubicin (DOX) massively amplified ICD via substantial generation of reactive oxygen species (ROS) after mitochondrial damage. The underlying mechanism behind increased ICD was further demonstrated to be ascribed to two pathways: (1) ROS elevated endoplasmic reticulum (ER) stress, leading to surface exposure of calreticulin; (2) ROS promoted release of various mitochondria-associated damage molecules including mitochondrial transcription factor A. Nevertheless, adaptive upregulation of PD-L1 was found after such ICD-inducing treatment. To overcome such immunosuppressive feedback, we developed a tumor stimuli-responsive nano vehicle to simultaneously exert mitochondrial targeted ICD induction and PD-L1 blockade. The nano vehicle was self-assembled from ICD-inducing copolymer and PD-L1 blocking copolymer, and possessed long-circulating property which contributed to better tumor accumulation and mitochondrial targeting. As a result, the nano vehicle remarkably activated antitumor immune responses and exhibited robust antitumor efficacy in both immunogenic and non-immunogenic tumor mouse models.

Keywords: Antitumor efficacy; Antitumor immune responses; Endoplasmic reticulum stress; Immunogenic cell death; Mitochondria-associated damage molecules; Mitochondrial targeting; PD-L1 blockade; Stimuli-responsive.

Graphical abstract

Scheme 1

Schematic illustration of the self-assembled nano vehicle (SNV) consisting of mitochondrial targeted ICD induction and PD-L1 blocking to activate immunity for cancer immunotherapy.

Figure 1

(A) Characterization of the HPMA copolymer-DOX conjugates. (B) Cellular uptake of B16F10 cells for 4 h by flow cytometry. (C) Analysis of mitochondrial accumulation on B16F10 cells for 4 h by CLSM and (D) flow cytometry. Blue: nucleus; Green: mitochondria; Red: DOX, Rr: Pearson correlation coefficient. Higher Rr indicates better colocalization between fluorophores. Scale bar: 10 μm. (E) ROS level of B16F10 cells after treated with PD, P-D-R8 and P-D-R8MTS for 12 h. (F) Ecto-calreticulin and (G) extracellular ATP level of B16F10 cells after treated with PD, P-D-R8 and P-D-R8MTS for 24 h. ∗∗ indicated P < 0.01 versus PD. ^## indicated P < 0.01. (H) Expression of CHOP, GRP78 and P-ELF2α on B16F10 cells treated with PD, P-D-R8 and P-D-R8MTS for 24 h. (I) TFAM expression and quantification in the extracellular supernatant by western blot. Results were mean ± SD (n = 3). ∗∗ indicated P < 0.01 versus Control group. ^# and ^## indicated P < 0.05 and P < 0.01, respectively. The equivalent DOX concentration was 10 μg/mL.

Figure 2

(A, B) PD-L1 level of B16F10 cells after treated with P-D-R8MTS for 24 h. (C) Size and zeta potential of copolymers and SNV. (D) Zeta potential variation of P-MSP-DMA as a function of incubation time at pH 7.4 or 6.5. (E) Transmission electron images of SNV incubated at pH 7.4 or 6.5 for 2 h. Scale bar: 50 nm. (F, G) PD-L1 level of B16F10 cells after treated with various copolymers and SNV for 24 h. Results were mean ± SD (n = 3). ∗ and ∗∗ indicated P < 0.05 and P < 0.01 versus control group, respectively. ^## indicated P < 0.01.

Figure 3

(A) Qualitative (by CLSM) and (B) quantitative (by flow cytometry) analysis of DOX accumulation in mitochondria of B16F10 cells for 4 h. Blue: nucleus; Green: mitochondria; Red: Dox. Scale bar: 10 μm. Results were mean ± SD (n = 3). ∗ and ∗∗ indicated P < 0.05 and P < 0.01 versus P-D-R8MTS group, respectively. ^## indicated P < 0.01. (C) ROS level and (D) MPTP opening of B16F10 cells after treated with copolymers and SNV. Results were mean ± SD (n = 3). ∗ and ∗∗ indicated P < 0.05 and P < 0.01 versus control group, respectively. ^# and ^## indicated P < 0.05 and P < 0.01, respectively.

Figure 4

(A) Illustration of ICD induction by P-D-R8MTS. P-D-R8MTS targeted mitochondria to promote substantial ROS generation. On one aspect, ROS induced apoptosis to promote release of mitochondria-associated damage molecules (TFAM and ATP). On another aspect, ROS triggered ER stress with elevated expression of marker proteins (CHOP, GRP78 and P-ELF2α), leading to surface exposure of CRT. As a consequence, P-D-R8MTS induced massive ICD. (B, C) Ecto-calreticulin expression of B16F10 cells analyzed by CLSM and flow cytometry. Blue: nucleus; Green: DOX; Red: ecto-CRT. (D) Extracellular ATP level of B16F10 cells treated with copolymers and SNV for 24 h. (E) TFAM expression in the extracellular supernatant, HMGB1 expression in whole cell lysate (C-HMGB1) and in the extracellular supernatant (R-HMGB1). (F, G) Percentage of BMDC phagocytosis towards B16F10 cells pre-treated with copolymers and SNV for 24 h. Data were analyzed by flow cytometry and CLSM. Blue: nucleus; Green: BMDCs; Red: B16F10 cells. Scale bar: 20 μm. (H) BMDCs maturation after phagocytosis of B16F10 cells treated with copolymers and SNV for 24 h. Results were mean ± SD (n = 3). ∗∗ indicated P < 0.01 versus control group. ^# and ^## indicated P < 0.05 and P < 0.01, respectively.

Figure 5

(A) Pharmacokinetics of free DOX, P-D-R8MTS and SNV after intravenous injection. At different time points, blood samples were taken and the concentration of DOX in blood was analyzed. Results were mean ± SD (n = 5). (B) Representative in vivo images of B16F10 tumors in C57/BL6 mice at 1, 6, 12 and 24 h post-injection of Cy5-labeled HPMA copolymers or SNV. (C) Representative ex vivo images and (D) corresponding semi-quantitative radiant efficiency of main organs from B16F10 tumor-bearing C57/BL6 mice at 24 h post-injection of Cy5-labeled HPMA copolymers or SNV. Accumulation of DOX in (E) tumor tissue and in (F) tumor mitochondria after intravenous injection of free DOX, P-D-R8MTS and SNV for 12 h. Results were mean ± SD (n = 3). ∗ and ∗∗ indicated P < 0.05 and P < 0.01, respectively.

Figure 6

In vivo antitumor efficacy. (A) Schematic illustration of treatment schedule and tumor growth of B16F10 tumor-bearing C57/BL6 mice (n = 5). Mice were intravenously administrated with saline, P-D-R8MTS (equivalent DOX dose 5 mg/kg), P-MSP-DMA (equivalent MSP dose 5 mg/kg) and SNV (equivalent DOX dose 5 mg/kg, MSP dose 5 mg/kg) on Days 8, 11, and 14. (B) Tumor inhibition rate of B16F10 tumors for each group harvested on Day 19 (n = 5). (C) Mice survival rate over time during the treatments (n = 8). (D) Analysis of CRT expression and (E) mature DCs infiltration and (F) PD-L1 expression after different treatments. Results were mean ± SEM (n = 5). ∗ and ∗∗ indicated P < 0.05 and P < 0.01 versus control group, respectively. ^# and ^## indicated P < 0.05 and P < 0.01, respectively.

Figure 7

(A, B) Analysis of CD3⁺CD8⁺ T cells and CD8⁺ T cells to Treg ratio after different treatments. Results were mean ± SEM (n = 5). (C) Immunofluorescence analysis of TFAM and IFN-γ expression in tumor. Blue: nucleus; Red: TFAM; Green: IFN-γ. Scale bar: 20 μm. Results were mean ± SEM (n = 5). ∗∗ indicated P < 0.01 versus control group. ^## indicated P < 0.01. (D) Schematic illustration of treatment schedule and tumor growth curves of B16F10 tumor-bearing C57/BL6 mice. Mice were intravenously administrated with saline, SNV and SNV + CD8-delpeting antibodies on Days 8, 11, and 14. Results were mean ± SEM (n = 5). ∗∗ indicated P < 0.01 versus all other group. (E) Mice survival rate over time during the treatment (n = 8).

Figure 8

(A) Qualitative analysis of DOX accumulation in mitochondria of 4T1 cells by CLSM for 4 h. Blue: nucleus; Green: mitochondria; Red: DOX. Scale bar: 10 μm. Rr: Pearson correlation coefficient. (B) ROS level of 4T1 cells after treated with copolymers and SNV for 12 h. (C) Ecto-calreticulin, (D) extracellular ATP level and (E) PD-L1 level of 4T1 cells after treated with copolymers and SNV for 24 h. Results were mean ± SD (n = 3). ∗ and ∗∗ indicated P < 0.05 and P < 0.01 versus control group, respectively. ^# and ^## indicated P < 0.05 and P < 0.01, respectively.

Figure 9

In vivo immune responses in 4T1 tumors. (A) Schematic illustration of treatment schedule in 4T1 tumor-bearing Balb/c mice. Mice were intravenously administrated with saline, P-D-R8MTS, P-MSP-DMA and SNV on Days 7, 10, 13 and 16. Immunology analysis was investigated on Day 11. (B) Analysis of CRT expression and (C) mature DCs infiltration after different treatments. (D, E) Analysis of CD3⁺CD8⁺ T cells and CD8⁺ T cells to Treg ratio after different treatments. (F) PD-L1 expression after different treatments. Results were means ± SEM (n = 5). ∗ and ∗∗ indicated P < 0.05 and P < 0.01 versus control group, respectively. ^## indicated P < 0.01.

Figure 10

In vivo antitumor efficacy in 4T1 tumors. (A) Tumor growth and (B) tumor inhibition rate of 4T1 tumor-bearing Balb/c mice during treatment (n = 5). (C) Mice survival rate over time during the treatments (n = 8). (D) Representative image and HE staining of lungs and (E) quantitative analysis of lung metastatic nodules harvested on Day 24. Red and black circles indicated metastatic nodules. Scale bar: 500 μm. Results were mean ± SEM (n = 5). ∗ and ∗∗ indicated P < 0.05 and P < 0.01 versus control group, respectively. ^# and ^## indicated P < 0.05 and P < 0.01, respectively.