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. 2022 Jul 18;12(12):5488-5503.
doi: 10.7150/thno.73104. eCollection 2022.

Co-delivery of phagocytosis checkpoint and STING agonist by a Trojan horse nanocapsule for orthotopic glioma immunotherapy (V体育官网入口)

Ying Zhou  1   2 , Yuxin Guo  2 , Lufei Chen  1 , Xiaoli Zhang  1 , Wei Wu  1 , Zhimin Yang  1   2 , Xuejie Li  1   2 , Yuanzhuo Wang  2   3 , Zhiyuan Hu  1   2   3   4 , Zihua Wang  1   2
Affiliations

"V体育ios版" Co-delivery of phagocytosis checkpoint and STING agonist by a Trojan horse nanocapsule for orthotopic glioma immunotherapy

Ying Zhou et al. Theranostics. .

Abstract

Rationale: Cancer immunotherapy has demonstrated significant antitumor activity in a variety of tumors; however, extensive infiltration of immunosuppressive tumor-associated macrophages (TAMs) in the glioblastoma (GBM) tumor microenvironment (TME) and the existence of the blood-brain barrier (BBB) might lead to failure of the checkpoint blockade therapy. Methods: Herein, we have developed a smart "Trojan horse" BBB-permeable nanocapsule termed "NAcp@CD47" to deliver anti-CD47 antibodies and stimulator of interferon genes (STING) agonists into GBM tissues in a stealth-like manner to reshaped the immune microenvironment by switching the phenotype of microglia and macrophages. Results: Both in vitro and in vivo studies demonstrate that NAcp@CD47 could effectively penetrate the BBB, increase the polarization of M1-phenotype TAMs, help reduce tumor immunosuppression, and inhibit the orthotopic GBM growth by phagocytosis of macrophages and microglia VSports手机版. Conclusions: Our findings indicate that the well-designed NAcp@CD47 not only enhances the phagocytosis of cancer cells but also efficiently enhance antitumor immunogenicity and reverses immune suppression to convert uninflamed "cold" tumors into "hot" tumors. .

Keywords: CD47; glioblastoma; immunotherapy; phagocytosis; tumor associated macrophages. V体育安卓版.

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Conflict of interest statement (V体育ios版)

Competing Interests: The authors have declared that no competing interest exists.

Figures (V体育平台登录)

Scheme 1
Scheme 1
Schematic illustration of the NAcp@CD47 nanocapsule for dual delivery of anti-CD47 antibodies and STING agonists for GBM immunotherapy by repolarization of macrophages and promotion of phagocytosis. (A) The process of constructing the FAPá responsive prodrug nanocapsule: anti-CD47 antibodies and CDG are co-encapsulated in NAcp@CD47 using MPC, APM and FAP-α-responsive peptide crosslinker in situ polymerization nanocapsule. (B) The mechanism of NAcp@CD47 polarization of microglia and macrophages against GBM. After intravenous injection, NAcp@CD47 can be delivered to the CNS crossing the BBB mediated by MPC, release anti-CD47 antibodies and CDG after FAP-α enzymatic degradation in the GBM microenvironment to block the phagocytosis checkpoint CD47-SIRPα and promote the production of IFNs via the STING signaling pathway. Consequently, the reprogrammed microglia (or macrophages) enhances the phagocytosis of cancer cells. Similarly, IFNs facilitate increased infiltration of immune cells and increase immunogenicity to convert “cold” tumors into “hot” tumors.
Figure 1
Figure 1
Characterization of NAcp@CD47. (A) TEM images of NAcp@CD47 (scale bar, 200 nm). (B) The average hydrodynamic size of NAcp@CD47 determined using dynamic light scattering (DLS). (C) TEM images of NAcp@CD47 after FAP-α enzymatic degradation. (D) Cell viability of GL261 glioblastoma cells and primary mouse astrocytes after incubation with various concentrations of NAcp for 24 or 48 h (n = 3, mean ± SD). (E-F) Cumulative release profiles of aCD47 (E) or CDG (F) from the NAcp@CD47 dispersed in artificial cerebrospinal fluid (ACSF) or PBS (25 ìg/mL) containing FBS with or without FAP-α enzyme at 37℃ as determined by high performance liquid chromatography (HPLC). (G) Schematic of the in vitro BBB model. (H) Average fluorescence signal of GL261 GBM cells in the in vitro BBB model after 4 h of incubation with NAcp@CD47-APC, Lipo@CD47-APC, or aCD47-APC with or without nAChR inhibitor. (I) Average fluorescence signal of GL261 GBM cells in the in vitro BBB model after 0 to 4 h of incubation with NAcp@CD47-APC, Lipo@CD47-APC, or aCD47-APC. (J) Ex vivo fluorescence imaging of brains from mice after injection of NAcp@CD47-APC, Lipo@CD47-APC, or aCD47-APC. Excitation/emission wavelengths 488/507 nm for GFP and excitation/emission wavelengths 633/670 nm for APC revealed a colocalization of GBM and NAcp@CD47 (scale bar, 2 mm). (K) In vivo fluorescence imaging of mice at indicated time points after intravenously injected with NAcp@CD47-APC, Lipo@CD47-APC, or aCD47-APC. (L-M) Quantitative analysis of in vivo (L) and ex vivo (M) fluorescence efficiency of NAcp@CD47 and control groups in the brain. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Figure 2
Figure 2
NAcp@CD47-mediated phagocytosis in vitro. (A-B) Representative fluorescence images of phagocytosis assays, in which peritoneal macrophage (A) and brain microglia (B) from eGFP transgenic mice displayed green were co-cultured with GL261 GBM cells labelled with Hoechst 33342 (blue) for 30 min in the presence of NAcp@CD47, CD47 antibody or IgG control. Scale bar, 200 ìm. Experiments were repeated three times. (C-D) Representative flow cytometric analysis images (C) and relative quantification (D) of the phagocytosis of GL261 GBM cells by peritoneal macrophage and brain microglia in the presence of NAcp@CD47, CD47 antibody or IgG control. Data are presented as mean ± SD (n = 3). Phagocytosis or phagocytized cancer cells were quantified as the percentage of double-positive cells. ***P < 0.001; ns, not significant.
Figure 3
Figure 3
NAcp@CD47-mediated antitumor immune response in subcutaneously implanted tumors in vivo. (A) Photographic illustration of tumors harvested from mice intravenously injected with PBS, CD47 antibody control, and NAcp@CD47 at the end point. Each scale of the ruler represents 1 mm. (B-C) The tumor size (B) and tumor weight (C) of the excised tumors in different groups. (D) Weight changes of mice in distinct groups. Data are presented as mean ± SD (n = 6). (E-I) Representative flow cytometric analysis images (E) and percentages (F-I) of M2-like macrophages (CD206hi in F4/80+CD11b+) and M1-like macrophages (CD86hi in F4/80+CD11b+) and cytotoxic T lymphocytes (CD3+CD8+, CTLs). (J-K) The levels of tumor necrosis factor alpha (TNF-α) (J) and interferon-gamma (IFN-γ) (K) in the blood of mice before and after various treatments detected by ELISA (n = 6). Data are presented as mean ± SD (n = 6). (L-M) Representative H&E staining images and fluorescence microscopic images (L) of TUNEL assay and CD8+ cells in excised tumors of separate groups and relative quantification (M). (N-O) Relative quantification of the staining intensity (N) of the protein expression levels (O) of inducible nitric oxide synthase (iNOS), arginase-1 (ARG1), and CD8 in excised tumors of distinct groups analyzed by western blotting. (P) The levels of interferon-beta (IFN-β) in the tumor tissues. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Figure 4
Figure 4
NAcp@CD47-mediated therapeutic effects in in vivo orthotopic syngeneic models for GBM. (A) Schematic illustrating NAcp@CD47 therapy in orthotopic syngeneic models for brain tumors with stereotaxic injection of GL261-GFP/LUC cells. (B-C) Imaging of in vivo bioluminescence in GL261 tumors in response to treatment with PBS, aCD47 control, and NAcp@CD47 on day 14, 21, and 28 after injection, as detected using an IVIS spectrum instrument (B) and by relative quantification (C). (D) Weight changes of mice in different groups. (E) Photographic illustration of brains with syngeneic tumors harvested from mice intravenously injected with PBS, aCD47 control, and NAcp@CD47 at the end point (n = 4). Scale bar, 1 mm. (F) Paraffin-embedded brain tissue sections in different groups subjected to H&E immunohistochemical staining. Scale bar, 200 mm. (G) Kaplan-Meier survival curves of mice in different groups. (H) Changes in the percentages of M1-like TAMs during therapeutic process. (I) Changes in the percentages of M2-like TAMs from tumor tissues during therapeutic process. (J) Changes in the ratio of M1/M2 from tumor tissues during therapeutic process. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Figure 5
Figure 5
NAcp@CD47-mediated antitumor immune response in in vivo orthotopic syngeneic models for GBM. (A) Immunofluorescence analysis of frozen sections stained with IBA1 (red) in GBM tumors and peri-tumoral tissues in different groups. Scale bar, 200 mm. (B, D) iNOS, CD47, ARG1, CD8, and IBA1 protein expression levels in GBM tumors and peri-tumoral tissues of groups treated with NAcp@CD47 and aCD47, as determined by western blotting (B) and relative quantification (D). (C, E) Representative fluorescence microscopic images of frozen sections stained with TUNEL, iNOS, CD47, ARG1, CD8, CD47 and FAP-α in different groups (C) and relative quantification of the staining intensity (E). (F) The levels of IFN-β in tumor tissues and peri-tumoral tissues by ELISA. Scale bar, 200 ìm. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
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