Docetaxel-loaded M1 macrophage-derived exosomes for a safe and efficient chemoimmunotherapy of breast cancer

Abstract

The conversion of tumor-promoting M2 macrophage phenotype to tumor-suppressing M1 macrophages is a promising therapeutic approach for cancer treatment. However, the tumor normally provides an abundance of M2 macrophage stimuli, which creates an M2 macrophage-dominant immunosuppressive microenvironment. In our study, docetaxel (DTX) as chemotherapeutic modularity was loaded into M1 macrophage-derived exosomes (M1-Exo) with M1 proinflammatory nature to establish DTX-M1-Exo drug delivery system. We found that DTX-M1-Exo induced naïve M0 macrophages to polarize to M1 phenotype, while failed to repolarize to M2 macrophages upon Interleukin 4 restimulation due to impaired mitochondrial function. This suggests that DTX-M1-Exo can achieve long-term robust M1 activation in immunosuppressive tumor microenvironment. The in vivo results further confirmed that DTX-M1-Exo has a beneficial effect on macrophage infiltration and activation in the tumor tissues. Thus, DTX-M1-Exo is a novel macrophage polarization strategy via combined chemotherapy and immunotherapy to achieve great antitumor therapeutic efficacy.

Keywords: Breast cancer; Exosomes; Macrophage polarization; Mitochondrial functions; Tumor immunity.

Fig. 1

Morphological characterization of exosomes with or without drug loading: TEM images of (A) M1-Exo and (B) DTX-M1-Exo

Fig. 2

Cell cytotoxicity study of control, DTX, M0-Exo, DTX-M0-Exo, M1-Exo, and DTX-M1-Exo against 4T1 breast cancer cells. (** P < 0.01, *** P < 0.001, **** P < 0.0001 vs control)

Fig. 3

The cellular uptake behavior of the exosomal vesicles (M1-Exo or M1-Exo) toward 4T1 cancer cells after 6 h incubation. In the CLSM images, cell nuclei were stained by DAPI (blue), while the exosomes were stained by fluorescent dye PKH67 (green). The scale bar is 20 µm

Fig. 4

Macrophage phenotypes after treatment of IL-4, LPS + IFN-γ, DTX, M0-Exo, DTX-M0-Exo, M0-Exo, and DTX-M1-Exo, by measuring the expression of M1-macrophage markers (A) iNOS and (B) TNF-α, as well as M2-macrophage markers (C) Arg-1 and (D) CD206. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. the control group)

Fig. 5

Macrophage repolarization capability of IL-4-induced M2 macrophages, followed by the treatment of LPS + IFN-γ, DTX, M0-Exo, DTX-M0-Exo, M0-Exo, and DTX-M1-Exo, by measuring the expression of M1-macrophage markers (A) iNOS and (B) TNF-α, as well as M2-macrophage markers (C) Arg-1 and (D) CD206. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. the control group)

Fig. 6

Mitochondria function of macrophages with IL-4, LPS + IFN-γ, DTX, M0-Exo, DTX-M0-Exo, M0-Exo, and DTX-M1-Exo, determined by (A) mitochondrial membrane potential changes and (B) mitochondrial ROS induction. (* P < 0.05, ** P < 0.01, **** P < 0.0001)

Fig. 7

Tumor growth inhibition of breast cancer 4T1in BALB/C mice. Mice were injected i.v. with PBS control, M1-Exo, DTX, and DTX-M1-Exo at concentration of 5 mg/kg DTX: (A) Tumor volume changes. (B) Representative histological images of slices of 4T1 tumor after final treatment using (i) H&E staining (scale bar = 100 μm) and (ii) Ki67 staining (scale bar = 200 μm)

Fig. 8

The distribution of exosome formulations and macrophages in vivo. Representative fluorescent images showing nucleus (blue), PKH67-labelled exosomes (green), F480-labelled macrophages (red), and merged channels (from left to right). The scale bar denotes 20 μm

Fig. 9

In vivo biosafety assessment. A Body weight changes. B Representative histological images of H&E-stained slices of each organ two days after treatment of (i) control, (ii) DTX, (iii) M1-Exo, and (iv) DTX-M1-Exo. The scale bar is 100 μm. (n = 3)