Extracellular vesicles mediated proinflammatory macrophage phenotype induced by radiotherapy in cervical cancer

Abstract

Background: Radiotherapy is a highly effective treatment for cervical cancer. Recent studies focused on the radiotherapy induced anti-tumor immunity. Whether tumor-derived extracellular vesicles (EVs) play roles in radiotherapy induced tumor associated macrophage (TAM) polarization remains unclear.

Materials and methods: This study analysed the phenotype of macrophages in cancer tissue and peripheral blood of cervical cancer patients using flow cytometry analysis. The role of EVs from plasma of post-irradiated patients on M2-like transformed macrophages was assessed. The M1- and M2-like macrophages were assessed by expression of cell surface markers (CCR7, CD163) and intracellular cytokines (IL-10, TNFα and iNOS). The capacity of phagocytosis was assessed by PD-1 expression and phagocytosis of pHrodo Red E. coli bioparticles.

Results: Our results demonstrated that radiotherapy of cervical cancer induced an increase in the number of TAMs and a change in their subtype from the M2-like to the M1-like phenotype (increased expression of CCR7 and decreased expression of CD163). The EVs from plasma of post-irradiated patients facilitated the M2-like to the M1-like phenotype transition (increased expression of CCR7, TNFα and iNOS, and decreased expression of CD163 and IL-10) and increased capacity of phagocytosis (decreased PD-1 expression and increased phagocytosis of pHrodo Red E. coli bioparticles).

Conclusions: Our data demonstrated that irradiation in cervical cancer patients facilitated a proinflammatory macrophage phenotype which could eventually able to mediate anti-tumor immune responses. Our findings highlight the importance of EV in the crosstalk of tumor cells and TAM upon irradiation, which potentially leading to an increased inflammatory response to cancer lesions.

Keywords: Cervical cancer; Extracellular vesicle; Macrophage; Radiotherapy.

Fig. 1

Immunohistochemical staining for tumor-associated macrophages (TAM) in cervical cancer tissue. The biopsies of cervical cancer patients were collected before and after radiotherapy. The cancer samples were fixed with formalin and embedded with paraffin. A, representative Hematoxylin-Eosin staining images. Images below were magnified 200×. B, representative images for CD68 staining. Images below were magnified 200×. C, the number of membranous CD68 positive cells was calculated in at least five randomly selected high power fields (400×). P value was calculated by Wilcoxon matched-pairs signed rank test

Fig. 2

Flow cytometry analysis for TAMs in cervical cancer tissue. Fresh biopsies of cervical cancer tissue were minced and stained immediately for phenotype analysis by flow cytometry. A, representative example of the gating strategy for macrophages isolated from viable CD45⁺ mononuclear cells of cervical cancer patients. Representative scatter diagrams (B) and histograms (C) of CD68⁺CD163⁺ TAMs in cervical cancer tissue before and after radiotherapy. Representative scatter diagrams (D) and histograms (E) of CD68⁺CCR7⁺ TAMs in cervical cancer tissue before and after radiotherapy. P value was calculated by Wilcoxon matched-pairs signed rank test

Fig. 3

Flow cytometry analysis for peripheral blood mononuclear cells of cervical cancer patients. Peripheral blood mononuclear cells were obtained by Ficoll-Plaque density gradient centrifugation from cervical cancer patients before and after radiotherapy. The cell surface marker and intracellular cytokine were stained and analyzed using flow cytometry. P value was calculated by Wilcoxon matched-pairs signed rank test. IL, interleukin; TNFα, tumor necrosis factor-α; iNOS, inducible nitric oxide synthase

Fig. 4

Identification of EVs isolated from the plasma of cervical cancer patients. The EVs were isolated from the fresh peripheral blood of cervical cancer patients using the Total Exosome Isolation Kit. A, a representative transmission electron microscopic image of EVs from plasma of post-irradiated patients was shown. EVs displayed with characteristic bilipid layer and size. B, representative nanoparticle tracking analysis of isolated EVs. C, immunofluorescence staining of characteristic EV marker (CD9) and membrane permeability to CFSE. The fluorescent stained EVs from fetal bovine serum (FBS) were also visualized as the negative control for anti-human CD9. EVs were visualized using a laser-scanning confocal microscope (TCS SP8 STED, Leica). The magnification of image below was 63×10. The total protein of EVs was separated by SDS-PAGE. D, Coomassie Blue staining was performed to demonstrate the significant differences in protein distribution. E, Western-blotting of EV markers (CD9 and TSG101) and non-EV marker (ApoA1) were performed. EV, extracellular vesicle; CFSE, carboxyfluorescein diacetate succinimidyl ester

Fig. 5

Polarization of macrophages can be facilitated by EVs from cervical cancer patients before and after radiotherapy. Peripheral blood mononuclear cells were obtained by Ficoll-Plaque density gradient centrifugation from healthy donors. Monocytes were isolated and cultured in RPMI 1640 medium. M2-polarized macrophages were obtained by IL-4+IL-13 stimulation for 48 hours. A, flow cytometry analysis the expression levels of cell surface and intracellular markers in macrophages before and after M2 polarization. M2 polarized macrophages were treated with EVs (the particle number per milliliter was 5 times higher than that in plasma) from cervical cancer patients before and after radiotherapy. B, flow cytometry analysis the expression levels of cell surface and intracellular markers. The dose effects of EVs on the expression levels of CD163 (C) and CCR7 (D) in M2 polarized macrophages were shown (n = 4). Bars presented as mean values of indicated markers. *, p < 0.05; **, p < 0.01. P value was calculated by two-tailed Mann Whitney U test

Fig. 6

EVs from cervical cancer patients after radiotherapy contribute to increased macrophage phagocytosis. Peripheral blood mononuclear cells from healthy donors were obtained and cultured in RPMI 1640 medium. M2-polarized macrophages were obtained by IL-4+IL-13 stimulation and treated with EVs from cervical cancer patients before and after radiotherapy. A, representative scatter diagrams of flow cytometry analysis the expression of PD-1 and CCR7 in macrophages (CD45⁺CD14⁺CD11b⁺) were shown. Histograms of PD-1⁺ cells in total macrophages (B) and in CCR7⁺ macrophages (C) were shown. Macrophage phagocytosis was assessed using pHrodo Red E. coli bioparticles or CFSE labeled EVs. Representative diagram (D) and histogram (E) about mean fluorescent intensity (MFI) of pHrodo in macrophages were shown. Representative scatter diagram (F) and histogram (G) of flow cytometry analysis the correlation between PD-1 expression and MFI of pHrodo in macrophages were shown. Representative diagram (H) and histogram (I) of CFSE MFI in macrophages co-cultured with EVs labeled with or without (Control) CFSE, after treated with EVs from cervical cancer patients before and after radiotherapy for 24 hours, were shown. Data were presented as mean±SD. P value was calculated by two-tailed Mann Whitney U test