Nanoparticle-mediated celastrol ER targeting delivery amplify immunogenic cell death in melanoma

Abstract

Introduction: Chemoimmunotherapy, which benefits from the combination of chemotherapy and immunotherapy, has emerged as a promising strategy in cancer treatment. However, effectively inducing a robust immune response remains challenging due to the limited responsiveness across patients. Endoplasmic reticulum (ER) stress is essential for activating intracellular signaling pathways associated with immunogenic cell death (ICD), targeting drugs to ER might enhance ER stress and improve ICD-related immunotherapy.

Objectives: To improve the immune response of Chemoimmunotherapy.

Methods: ER targeting nanoparticles TSE-CEL/NP were constructed to enhance immunogenic cancer cell death. Flow cytometry, confocal microscope, TEM and immunofluorescence were used to evaluate the ER targeting effect and immunogenic tumor cell death in vitro on B16F10 tumor cells. Unilateral and bilateral tumor models were constructed to investigate the efficacy of anti-tumor and immunotherapy in vivo. Lung metastasis B16F10 melanoma tumor-bearing mice were used to assess the anti-metastasis efficacy.

Results: TSE-CEL/NP could specially accumulate in ER, thereby induce ER stress. High ER stress trigger the exposure of CRT, the extracellular release of HMGB1 and ATP. These danger signals subsequently promote the recruitment and maturation of dendritic cells (DCs), which in turn increase the proliferation of cytotoxic T lymphocytes (CD8⁺ T cells), ultimately resulted in an improved immunotherapy efficacy against melanoma. Invivo experiments showed that TSE-CEL/NP exhibits excellent antitumor efficacy and triggers a strong immune response.

Conclusion: Our findings demonstrated that celastrol ER targeting delivery could amplify immunogenic cell death in melanoma, which provide experimental basis for melanoma immunotherapy.

Keywords: Chemoimmunotherapy; Endoplasmic reticulum (ER) stress; Endoplasmic reticulum targeting; Immunogenic cell death.

Graphical abstract

Fig. 1

Schematic illustration. (A)The preparation of the endoplasmic reticulum targeted nanoparticle (TSE-CEL/NP). (B) TSE-CEL/NP specially accumulates in ER, inducing ER stress and thereby enhancing immunogenic death of tumor cells, ultimately resulting in an improved immunotherapy efficacy against melanoma.

Fig. 2

Characterization of nanoparticles. (A) Scheme illustration of preparation process of TSE-CEL/NP. TEM images and Size distribution of (B) blank NP, (C) CEL/NP and (D) TSE-CEL/NP, Scale bar: 100 nm. (E) The particle states of the NPs in DI water. (F) Different time points size change of blank NP, CEL/NP and TSE-CEL/NP in PBS (pH 7.4). (G) Different time points PDI change of blank NP, CEL/NP and TSE-CEL/NP in PBS (pH 7.4). (H) Hemolysis of red blood cells after treating with different concentrations of NP, CEL/NP and TSE-CEL/NP. (I) The hemolysis ratio of different group. (J) Celastrol release from nanoparticles at different time points. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Cellular uptake and ER-targeting ability of nanoparticles. (A) Microscopy images of B16F10 after free C6 or NPs treatment for 2 h, green fluorescence indicating free C6 or nanoparticles; and blue fluorescence was the nuclear staining with DAPI. Scale bars: 25 μm. (B-C) Mean fluorescence intensity of the B16F10 cells after free C6 or NPs treatment for 2 h, detected by flow cytometry. (D) Confocal images of B16F10 after NP or TSE-NP treatment, green fluorescence indicating nanoparticles; red fluorescence indicating the lysosome (LysoTracker); and blue fluorescence was the nuclear staining with DAPI. Scale bars: 20 μm. (E–F). Line-scan colocalization analysis showing the lysosome localization of NP and TSE-NP. (G) Confocal images of B16F10 showing ER localization of NP and TSE-NP, green fluorescence indicating nanoparticles; red fluorescence indicating ER (ER Tracker); and blue fluorescence was the nuclear staining with DAPI. Scale bars: 20 μm. (H–I). Line-scan colocalization analysis showing the ER localization of NP and TSE-NP. (J) TEM images of B16F10 showing ER localization of NP and TSE-NP. (The data are presented as the mean ± SD. **P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

TSE-CEL/NP induced the strongest ER stress and ICD in B16F10 cells. (A) Protein levels of p-IRE1α, ATF6 and p-eIF2α in B16F10 cells after treating with different CEL formulations, detected by western blot. (B-D) mRNA of ATF4, GRP78 and XBP1 expression in B16F10 cells after treating with different CEL formulations. (E) Confocal microscope images of ROS expression on B16F10 cells after treating with different CEL formulations. (F-G) MFI of ROS after iterating with different CEL formulations. (H-I) Confocal microscope images of CRT exposure on the cell surface of B16F10 cells, and HMGB1 release from nucleus in B16F10 cells after treating with different CEL formulations. (J) The semi-quantitative analysis of CRT fluorescence intensity. (K) The semi-quantitative analysis of HMGB1 fluorescence intensity. (L) mRNA of HMGB1 expression in B16F10 cells after treating with different CEL formulations. (M) ELISA results of HMGB1 release from B16F10 cells after treating with different CEL formulations. (N) ATP secretion in B16F10 cells after treating with different CEL formulations. (The data are presented as the mean ± SD. **P < 0.01, *P < 0.05).

Fig. 5

CEL/NPs and TSE-CEL/NPs enhanced the cytotoxicity on B16F10 cells. (A) The cytotoxicity of the empty nanoparticles without CEL on B16F10 cells. (B) The cytotoxicity of the CEL, CEL/NPs and TSE-CEL/NPs on B16F10 cells. (C) Microscopy images of calcein-AM/PI-stained B16F10 cells after treating with different drug formulations for 24 h. (D) The mortality ratio semi-quantitative analysis of (C). (E-F) Apoptosis of B16F10 cells after treatment with CEL, CEL/NPs and TSE-CEL/NPs for 24 h. (The data are presented as the mean ± SD. **P < 0.01, *P < 0.05).

Fig. 6

TSE-CEL/NP exhibited strongest in vivo anti-tumor efficacy. (A) Workflow for treatments of B16F10 tumor-bearing mice. (B) The picture of harvested tumors. (C–G) The tumor volume change curve of mice after treating with saline, free CEL, CEL/NP, TSE-CEL/NP. (H) Tumors weight of mice at the end of experiment. (I) Tumors inhibition rate of each treatment. (J) The body weight change curve of mice after treating with saline, free CEL, CEL/NP, TSE-CEL/NP. (K) Microscopy images of the tumor sections after H&E, TUNEL and Ki67 staining. Scale bars: 50 μm, TUNEL and Ki67 scale bars: 50 μm. (The data are presented as the mean ± SD. **P < 0.01, *P < 0.05).

Fig. 7

TSE-CEL/NP triggered a potent immunological response in vivo by amplifying ICD. (A) CRT exposure and HMGB1 release in tumor tissue (scale bars: 100 µm). (B) The proportion change of immature DCs in tumor tissues. (C) The proportion change of mature DCs in tumor tissues. (D-F) The proportion change of CD4⁺ T and CD8⁺ T cells in tumor tissues. (G) The proportion change of IFN-γ⁺ CD8⁺ T cells in tumor tissues. (H) The proportion change of TNF-α⁺ CD8⁺ T cells in tumor tissues. (The data are presented as the mean ± SD. **P < 0.01, *P < 0.05).

Fig. 8

TSE-CEL/NP produced a robust systemic antitumor efficacy to inhibit distant tumors. (A) Workflow for anti-tumor activity in bilateral B16F10 melanoma tumor-bearing mice. i.t. means intratumoral injection. (B-F) The tumor volume change curve of mice after treating with saline, free CEL, CEL/NP, TSE-CEL/NP. (G-J) The proportion of CD8⁺ T and CD4⁺ T cells in tumor tissue post-treatment. (K) The proportion of IFN-γ⁺ CD8⁺ T cells in distant tumors post-treatment. (L) The proportion of TNF-α⁺ CD8⁺ T cells in distant tumors post-treatment. (The data are presented as the mean ± SD. **P < 0.01, *P < 0.05).

Fig. 9

TSE-CEL/NP potently prevents the lung metastasis in vivo. (A) Workflow for anti-metastasis efficacy in lung metastasis B16F10 melanoma tumor model. (B) The picture of harvested lungs. (C) The weight of harvested lungs. (D) The lung metastasis inhibition rate of mice after treating with saline, free CEL, CEL/NP, TSE-CEL/NP. (E) The body weight change curve of mice after treating with saline, free CEL, CEL/NP, TSE-CEL/NP. (F) Microscopy images of the lung tissues after H&E staining. Scale bar: 250 μm. (The data are presented as the mean ± SD. **P < 0.01, *P < 0.05).

Fig. 10

In vivo biosafety evaluation. The blood routine parameters and biochemical indicators of C57 mice after treating with free drugs or nanoparticles. (A) mean platelet volume (MPV), (B) neutrophils (NEUT), (C) lymphocytes (LYM), (D) Red blood cells (RBC), (E) hemoglobin (HGB), (F) platelets (PLT), (G) lactate dehydrogenase (LDH), (H) creative kinase (CK), (I) urea (UREA), (J) creatinine (CREA), (K) aspartate aminotransferase (AST), (L) alanine aminotransferase (ALT). (M) Representative H&E staining images of collected organs from each group, scale bars: 150 μm. (The data are presented as the mean ± SD. *P < 0.05, **P < 0.01 vs PBS control). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)