Gold nanoparticle-directed autophagy intervention for antitumor immunotherapy via inhibiting tumor-associated macrophage M2 polarization

Abstract

Tumor-associated macrophages (TAMs), one of the dominating constituents of tumor microenvironment, are important contributors to cancer progression and treatment resistance. Therefore, regulation of TAMs polarization from M2 phenotype towards M1 phenotype has emerged as a new strategy for tumor immunotherapy. Herein, we successfully initiated antitumor immunotherapy by inhibiting TAMs M2 polarization via autophagy intervention with polyethylene glycol-conjugated gold nanoparticles (PEG-AuNPs). PEG-AuNPs suppressed TAMs M2 polarization in both in vitro and in vivo models, elicited antitumor immunotherapy and inhibited subcutaneous tumor growth in mice. As demonstrated by the mRFP-GFP-LC3 assay and analyzing the autophagy-related proteins (LC3, beclin1 and P62), PEG-AuNPs induced autophagic flux inhibition in TAMs, which is attributed to the PEG-AuNPs induced lysosome alkalization and membrane permeabilization. Besides, TAMs were prone to polarize towards M2 phenotype following autophagy activation, whereas inhibition of autophagic flux could reduce the M2 polarization of TAMs. Our results revealed a mechanism underlying PEG-AuNPs induced antitumor immunotherapy, where PEG-AuNPs reduce TAMs M2 polarization via induction of lysosome dysfunction and autophagic flux inhibition. This study elucidated the biological effects of nanomaterials on TAMs polarization and provided insight into harnessing the intrinsic immunomodulation capacity of nanomaterials for effective cancer treatment.

Keywords: Autophagy flux; Cancer therapy; Gold nanoparticles; Lysosomal damage; M2 macrophage; Macrophage polarization; Nanomaterials; Tumor-associated macrophages.

Graphical abstract

Figure 1

Characterization of PEG-AuNPs. (A) The size of PEG-AuNPs was measured with dynamic light scattering (DLS). (B) TEM images of PEG-AuNPs. Scale bar = 20, 50 or 100 nm. (C) Photographs of PEG-AuNPs in aqueous suspension. (D) UV–Vis absorption spectra of PEG-AuNPs or FITC-labeled AuNPs at wavelengths from 400 to 800 nm.

Figure 2

The cellular uptake and penetration of PEG-AuNPs. (A) Quantitative ICP-AES measurement of PEG-AuNPs uptake in RAW264.7 and Hepa1-6 cells. (B) The analysis of the CD206⁺/F4/80⁺ cells was performed with flow cytometry in RAW264.7 cells cocultured with TSN for 48 h; (C) The IL-10 concentration of supernatant from RAW 264.7 cells co-cultured with TSN at different time points as measured using ELISA; (D) The levels of the M2 phenotype related genes Arg1 and Cd163 mRNA were measured using RT-qPCR in RAW264.7 cells co-cultured with TSN for 48 h; (E) Quantitative ICP-AES measurement of PEG-AuNPs uptake in RAW264.7 cells co-cultured with TSN; (F) TEM images of PEG-AuNPs uptake and penetration in TAMs. Data are expressed as mean ± SD (n = 5). ∗∗P < 0.01, ∗∗∗P < 0.001. Scale bar = 50 nm, 500 nm, 1 μm and 2 μm.

Figure 3

PEG-AuNPs suppress the polarization of macrophages to the M2 phenotype in vitro. TSN co-cultured RAW264.7 cells were treated with or without PEG-AuNPs for 48 h at concentrations of 10 and 50 μmol/L, respectively. The expression of (A) CD80 and (B) CD206 of macrophages was evaluated using flow cytometry. (C) The concentrations of IL-12 and IL-10 of macrophages were analyzed using ELISA. (D) The mRNA levels of Tnf-α, Inos, Arg1 and Cd163 of macrophages were measured using RT-qPCR. Data are expressed as mean ± SD (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 4

In vivo antitumor activity and inhibition of M2 macrophage polarization by PEG-AuNPs. (A) Tumor growth curves. Hepa1-6 cells were subcutaneously inoculated into the right flank of male BALB/c mice with or without 20 nm PEG-AuNPs, and the tumor growth was monitored for 21 days. (B) The gross images of excised tumors from the mice at the endpoint. (C) Tumor weight at the endpoint. (D) Representative immunofluorescence staining of CD206⁺ and F4/80⁺ macrophages in tumor tissues on Days seven and 14 after implantation. Scale bar = 200 μm. (E) CD206⁺/F4/80⁺ macrophages were analyzed using flow cytometry in tumor tissues on Days seven and 14 after implantation. (F) The mRNA levels of genes (M2 phenotype) measured using RT-qPCR in tumors on Days seven and 14 after implantation in mice. Data are expressed as mean ± SD (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 5

The effects of PEG-AuNPs on the immune cells and tumor cells in the tumor environment. (A) Representative flow cytometry plots and (B) quantitative analysis of mature DCs (CD11⁺CD86⁺) in tumors. (C) Representative flow cytometry plots and (D) quantitative analysis of CD3⁺CD4⁺ T cells in tumors. (E) Representative flow cytometry plots and (F) quantitative analysis of CD3⁺CD8⁺T cells in tumors. Data are expressed as mean ± SD (n = 6). (G) Representative images and (H) quantitative analysis of KI67 staining in tumors. (I) Representative image and (J) quantitative analysis of Tunel staining in tumors. Scale bar = 50 μm. Data are expressed as mean ± SD (n = 10). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 6

PEG-AuNPs block autophagic flux and cause lysosomal dysfunction in macrophages. (A) Western blotting analysis of autophagy-related proteins. (B) The images of representative immunofluorescence and quantitative analysis of the number of yellow autophagosomes (G⁺R⁺) and red autolysosomes (G⁻R⁺) of mRFP-GFP-LC3 dots in macrophages. Scale bar = 10 μmol/L. (C) Fluorescence images of LysoSensor Green DND-189 stained macrophages. Scale bar = 50 μmol/L. (D) Flow cytometry analysis of LysoSensor Green DND-189 stained macrophages. (E) Flow cytometry analysis of macrophages stained with acridine orange (AO). Data are expressed as mean ± SD (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 7

Inhibition of M2 macrophage polarization by autophagy blockade. Analysis of relative mRNA expression of Arg1, Cd163, Cd206, and Il-10 in TSN co-cultured RAW 264.7 cells incubated with Atg5 siRNA (A), chloroquine (CQ) (B), and rapamycin (Rapa) (C). Data are expressed as mean ± SD (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ns, not statistically significant.

Figure 8

The schematic diagram of the mechanism underlying PEG-AuNPs-induced antitumor immunotherapy, involving inhibiting TAMs M2 polarization via induction of lysosome dysfunction and autophagic flux inhibition. After PEG-AuNPs internalization, the autophagic flux of TAMs was blocked due to lysosomal dysfunction, as reflected by autophagosome accumulation. After then, the M2 polarization of TAMs was inhibited, resulting in potential antitumor immunotherapy.