Peptide vaccine-conjugated mesoporous carriers synergize with immunogenic cell death and PD-L1 blockade for amplified immunotherapy of metastatic spinal

Abstract

The clinical treatment of metastatic spinal tumor remains a huge challenge owing to the intrinsic limitations of the existing methods. Programmed cell death protein 1 (PD1)/programmed cell death ligand 1 (PD-L1) pathway blockade has been explored as a promising immunotherapeutic strategy; however, their inhibition has a low response rate, leading to the minimal cytotoxic T cell infiltration. To ameliorate the immunosuppressive microenvironment of intractable tumor and further boost the efficacy of immunotherapy, we report an all-round mesoporous nanocarrier composed of an upconverting nanoparticle core and a large-pore mesoporous silica shell (UCMS) that is simultaneously loaded with photosensitizer molecules, the IDO-derived peptide vaccine AL-9, and PD-L1 inhibitor. The IDO-derived peptide can be recognized by the dendritic cells and presented to CD8⁺ cytotoxic T cells, thereby enhancing the immune response and promoting the killing of the IDO-expressed tumor cells. Meanwhile, the near-infrared (NIR) activated photodynamic therapy (PDT) could induce immunogenic cell death (ICD), which promotes the effector T-cell infiltration. By combining the PDT-elicited ICD, peptide vaccine and immune checkpoint blockade, the designed UCMS@Pep-aPDL1 successfully potentiated local and systemic antitumor immunity and reduced the progression of metastatic foci, demonstrating a synergistic strategy for cancer immunotherapy.

Keywords: Immunogenic cell death; Peptide vaccine; Photodynamic therapy; Programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) blockades; Spine metastasis.

Fig. 1

Schematic illustration of the preparation of functional nanocarrier for photodynamic and peptide vaccine-amplified immunotherapy of spinal tumor

Fig. 2

TEM images of (A) UCNPs (inset shows the corresponding high-magnification image; scale bar = 10 nm), B UCNPs@SiO₂, and (C) UCMS. D High-angle annular dark field (HAADF) scanning TEM image and element mapping of the prepared UCMS. E Nitrogen adsorption isotherm of the prepared UCMS. F Zeta potential and (G) size distributions of UCMS, UCMS@Pep, and UCMS@Pep-aPDL1

Fig. 3

In vitro photon conversion and cellular cytotoxicity of UCMS@Pep. A UV-vis spectrum of UCMS@Pep-RB (red line) and up-conversion emission spectrum of UCMS@Pep under excitation at 980 nm (green color). B Effects of irradiation time on the fluorescence intensity change of SOSG at 525 nm for different samples. C Relative viability of LLC cells incubated with different concentrations of blank nanoparticles, UCMS@Pep alone, and UCMS@Pep with NIR laser. D Results of calcein-AM & PI staining that was used to discriminate between live and dead cells (a: Control, b: NIR laser, c: UCMS@Pep, d: UCMS@Pep-RB + NIR laser, scale bar = 100 μm). (E Flow cytogram showing the results of the apoptosis assay based on the annexin V-FITC and propidium iodide (PI) staining of LLC cells after different treatments. F Corresponding inverted fluorescence microscopy images of DCFH-DA probe–stained LLC cells used to evaluate the overall intracellular ROS generation (scale bar = 50 μm). (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3)

Fig. 4

Intracellular ICD marker and DCs maturation of UCMS@Pep in vitro. A CRT exposure and (B) HMGB1 analysis by immunofluorescence using CLSM (scale bar = 25 μm). Release of (C) ATP and (D) HMGB1 detected by ELISA kits for different interventions. E Schematic of the trans-well DCs maturation system. F Flow cytometry results for mature DCs after different treatments. Levels of cytokines (G) TNF-α and (H) IL-12 in the culture medium. (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3)

Fig. 5

Biosafety in vivo of UCMS@Pep-aPDL1. A Results of routine blood count in mice treated with PBS and UCMS@Pep-aPDL1 (n ≥ 3); B Biochemistry indexes in mice treated with PBS and UCMS@Pep-aPDL1 (n ≥ 3); C H&E staining results obtained for the major organs of mice subjected to different treatments (Scale bar = 100 μm)

Fig. 6

In vivo amplified anti-tumor effect of UCMS@Pep-aPDL1. A Schematics of the program used for spine metastasis tumor mode and IDO-based peptide tumor vaccine-mediated synergetic cancer treatment. Time-dependent (B) body weight and (C) tumor volume of mice in different groups. D Tumor progression in different groups evaluated by IVIS. E Tumor specimens extracted after the mice had been sacrificed. F H&E-stained histological images, G TUNEL-stained pathological changes and Ki-67-stained cellular proliferation in tumor tissues (scale bars = 100 μm). (*P < 0.05, **P < 0.01, ***P < 0.001, n > 3)

Fig. 7

In vivo immune system enhancement by UCMS@Pep-aPDL1. A–C Flow cytometric analyses of DCs, CD4 and CD8 T cells and Treg cells in the tumor tissues of mice immunized using different interventions. Secretion levels of (D) TNF-α, E IFN-γ, and F IL-12 in the serum of peripheral blood from treated mice. G Infiltration of CD4 and CD8 T cells in tumor sites detected by CLSM (scale bar = 50 μm). H Expression of CRT, HMGB1, and the infiltration of cytokines TNF-α and IL-12 in tumor sites evaluated by CLSM (scale bar = 50 μm). (*P < 0.05, **P < 0.01, ***P < 0.001, n > 3)