Ferroptosis-dependent breast cancer cell-derived exosomes inhibit migration and invasion of breast cancer cells by suppressing M2 macrophage polarization

Cell culture and polarization of M1 and M2 macrophages

Human acute monocytic leukemia cells, THP-1 cells, mouse macrophages, RAW 264.7 cells, human MDA-MB-231 cells, and mouse 4T1 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). THP-1 cells, RAW 264.7 cells, human MDA-MB-231 cells, and mouse 4T1 cells were cultured in RPMI-1640 medium (Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (Gibco, Waltham, MA, USA) at 37 °C in an incubator of 5% CO2 and 95% air. To induce THP-1 cells differentiate into macrophages, THP-1 cells were incubated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 48 h. To obtain ferroptosis-induced breast cancer cells, MDA-MB-231 cells and 4T1 cells were cultured with ferroptosis inducer, erastin (10 µM) for 24 h.

PMA-induced THP-1 cells and RAW 264.7 cells were cultured with five ng/mL lipopolysaccharide (Sigma) and 100 U/mL IFN- γ (Sigma) for 24 h for M1 polarization. To obtain M2 macrophages, PMA-induced THP-1 cells and RAW 264.7 cells were cultured with 10 ng/mL IL-4 (Sigma) for 24 h.

Cell Counting Kit-8 (CCK8) assay

The viability of MDA-MB-231 cells and 4T1 cells treated with erastin was evaluated via CCK-8 assay. Cells were seeded in 96-well plates with 2 × 10³ cells/well and cultured in a 5%CO2 incubator at 37 °C. CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) was added after culturing for 24, 48, or 72 h, and the OD 450 was measured by using a spectrophotometer (Infinite M1000; TECAN, Männedorf, Switzerland).

Evaluation of reactive ferrous iron (Fe²⁺)

The Iron Colorimetric Assay Kit (#E1042; APPLYGEN) were used to detect the concentration of Fe²⁺ in the MDA-MB-231 cells and 4T1 cells treated with erastin. The absorbance of cells was detected by a spectrophotometer at 550 nm (Thermo Fisher Scientific, Waltham, MA, USA).

Measurement of reactive oxygen species (ROS)

To detect the production of ROS in the MDA-MB-231 cells and 4T1 cells treated with erastin, cells were incubated in 10 µmol/L 2,7-dichlorofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, St. Louis, MO, USA) in an incubator at 37 °C for 30 min. The sample was mixed upside down every 3-5 min so that the probe was in full contact with the cells. Then, cells were washed with serum-free cell culture medium for 3 times and 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher) was used for nuclear staining. Laser confocal microscopy (Nikon, Tokyo, Japan) were used to observe the expression of ROS.

Isolation and identification of exosomes

Exosomes were isolated from negative control (NC) or ferroptosis-induced MDA-MB-231 cells and 4T1 cells using ultracentrifugation. In brief, NC or ferroptosis-induced MDA-MB-231 cells and 4T1 cells were cultured in exosome-depleted medium for 72 h. The cell medium was gathered and centrifuged at 300 g at 4 °C for 10 min. Then media were filtered using a 0.22 µm filter. The media was ultracentrifuged at 120000 g for 70 min at 4 °C and exosomes were obtained. The exosome solution was resuspended in 30 µl of PBS. Whereafter, the isolated exosomes were detected by transmission electron microscopy (TEM) and specific exosome markers. Exosomes were fixed in 1% buffered glutaraldehyde for 10 min. The 20 µL of exosomes were added to formvar carbon-coated 300-mesh copper electron microscopy grids (Electron Microscopy Sciences, Hatfield, PA, USA), and permitted to stand for 5 min. Subsequently, exosomes were redyed using 2% uranyl oxalate at room temperature. After the grids were washed three times with PBS, the exosomes were analyzed using TEM (JEM-1200EX; TEM, Tokyo, Japan). Three positive markers (CD63, CD9, and Alix) and one negative marker (Fibronectin) were used for identification of exosomes via western blot analysis.

Macrophages internalize breast cancer cell derived-exosomes induced by ferroptosis

NC or ferroptosis-induced MDA-MB-231 cell and 4T1 cell derived-exosomes were labelled with the fluorochrome DiI (Beyotime, Jiangsu, China) according to the manufacturer’s protocol. PMA-induced THP-1 cells and RAW 264.7 cells were incubated with DMEM containing corresponding DiI-labeled exosomes for 24 h. After washing the cells, DAPI were used to stain the cell nuclei. Finally, the results were analyzed under a fluorescence microscopy.

Western blot (WB) analysis

The NC or ferroptosis-induced MDA-MB-231 cells and 4T1 cells, these cell derived-exosomes, and PMA-induced THP-1 cells and RAW 264.7 cells treated with these exosomes were lysed with RIPA. A protein assay kit was used to detect the concentration of total proteins. Equal protein (50 µg) was loaded on a 10% SDS gel and transferred to polyvinylidene fluoride membranes. Afterward, membranes were blocked in 5% milk in TBS-T for 2 h. Primary antibodies against CD63 (1:1000; Abcam, Cambridge, UK), CD9 (1:2000, Proteintech, USA), Alix (1:1000; Proteintech, Rosemont, IL, USA), Fibronectin (1:10000; Proteintech), IL-1β (1:1000; Santa Cruz, USA),

Arg-1 (1:1000; Proteintech), CD86 (1:1000; Invitrogen, Waltham, MA, USA), CD206 (1:1000; Abcam, UK) and GAPDH (1:10000; Proteintech) were utilized to incubate membranes at 4 °C overnight. Membranes were washed and incubated with goat anti-mouse IgG-HRP secondary antibody (1:1000; Beyotime) for 2 h. Enhanced chemiluminescence reagent was used to visualize the bands and the bands were analyzed by Image J.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted from PMA-induced THP-1 cells and RAW 264.7 cells treated with corresponding NC or ferroptosis-induced MDA-MB-231 cell and 4T1 cell derived-exosomes using Trizol reagent (Invitrogen) and the concentration was measured by a Nanodrop ND-1000 spectrophotometer (Tiangen Biotech Co., Ltd., Beijing, China). Reverse transcription was performed with the Maxima First Strand cDNA Synthesis Kit (Promega, USA) for RT-PCR. qRT-PCR assay was performed using ABI QuantStudio 6 Flex system (Applied Biosystems, Foster City, CA, USA). The qRT-PCR reaction conditions were: 95 °C for 10 min, 40 cycles with 95 °C for 15 s and 60 °C for 60s followed by melting curves of the reaction. GAPDH was used as an internal reference gene. The relative mRNA expression levels of Arg-1, CD206, CD163, iNOS, and IL-1β were calculated by the 2^−ΔΔCt method. The sequences of all gene primers were shown in Table S1.

Flow cytometry

PMA-induced THP-1 cells and RAW 264.7 cells were incubated with phycoerythrin (PE)-conjugated CD86 (Abcam, UK) and CD206 (Proteintech) at 4 °C for 30 min in the dark. Subsequently, cells were incubated with goat anti-rabit IgG-HRP secondary antibody (Abcam) for 30 min in the dark. Afterwards, flow cytometry analysis was performed in a FACSCalibur™ flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Finally, the data were analyzed by Flowjo 7.6.1 (FlowJo, Ashland, OR, USA).

Enzyme-linked immunosorbent assay (ELISA)

The level of Arg-1 in PMA-induced THP-1 cells and RAW 264.7 cells treated with corresponding NC or ferroptosis-induced MDA-MB-231 cell and 4T1 cell derived-exosomes was detected utilizing Arg-1 ELISA Kit in accordance with the manufacturer’s instructions.

Immunofluorescence

PMA-induced THP-1 cells and RAW 264.7 cells treated with corresponding NC or ferroptosis-induced MDA-MB-231 cell and 4T1 cell derived-exosomes were seeded on cell slides and incubated overnight. Next, the cells were fixed in 4% paraformaldehyde at room temperature for 30min, permeabilized with 0.5% Triton X-100, and blocked with 1% bovine serum albumin (GIBCO, Waltham, MA, USA). The cells were incubated overnight at 4 °C with primary antibody against CD86 (1:100; Invitrogen) and Arg-1 (1:500; Proteintech). After washing with PBS, the cells were incubated with goat anti-mouse IgG-HRP secondary antibody (1:500, Abcam) for 1 h at room temperature. DAPI (Abcam) was used to stain cell nucleus. After washing 3 time with PBS, the sections were performed for photograph under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Autophagy PCR array

RNA of PMA-induced THP-1 cells treated with NC or ferroptosis-induced MDA-MB-231 cell derived-exosomes was extracted using the RNeasy min kit (Qiagen, Hilden, Germany). cDNA was synthesized by using the RT2 First Strand kit (Qiagen). Afterwards, the PCR reaction was performed as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15s and 60 °C for 1min. All samples were analyzed using the 2-ΔΔCt method with GAPDH as an internal control for normalization. The protein–protein network was constructed using Cytoscape V3.6.0.

Transwell assay

PMA-induced THP-1 cells and RAW 264.7 cells treated with corresponding NC or ferroptosis-induced MDA-MB-231 cell and 4T1 cell derived-exosomes, and macrophage supernatants were collected. Subsequently, MDA-MB-231 cells and 4T1 cells were incubated with the supernatants. The migration and invasion of MDA-MB-231 cells and 4T1 cells were investigated using the Transwell system (Corning, NY, USA). For cell migration assay, breast cells were seeded in the upper chambers of 24-well Transwell plates with serum-free DMEM. DMEM with 10% FBS was then added into the lower chambers. After incubation for 24 h at 37 °C, cells were washed with PBS, fixed in 4% formaldehyde for 15 min at room temperature and stained using 1% crystal violet solution (Beyotime) for 20 min. Finally, migratory cells were counted under a microscope. To detect the invasion ability, breast cancer cells were seeded in Transwell plates pre-coated with Matrigel matrix (BD Biosciences) and cell invasion assay was performed according to the same experimental procedures as described above.

Statistical analysis

Data were analyzed by SPSS 24.0 and presented as mean ± SD. T-test was used to analyze the difference between two groups, and one-way analysis of variance (ANOVA) was used to determine the statistical significance of more than two groups. p < 0.05 was considered statistically significant.

Ferroptosis was induced by erastin in breast cancer cells

To induce ferroptosis in MDA-MB-231 cells and 4T1 cells, we treated them with erastin, a ferroptosis inducer, and verified ferroptosis by CCK-8, iron assay kit, and immunofluorescence assay. The results showed that erastin resulted in a significant decrease of MDA-MB-231 cell and 4T1 cell viability compared with the NC group (Figs. 1A, 1B). The level of Fe2+ and ROS were dramatically enhanced in MDA-MB-231 cells and 4T1 cells induced by erastin relative to the NC group (Figs. 1C–1F). Collectively, ferroptosis was induced in breast cancer cells by using erastin.

Macrophages internalize ferroptosis-induced breast cancer cell-derived exosomes

To investigate the role of exosomes produced by ferroptosis-mediated MDA-MB-231 cells and 4T1 cells, we first isolated exosomes from normal and ferroptosis-induced MDA-MB-231 cells and 4T1 cells. TEM images indicated that the morphology of both exosomes exhibited a near spherical shape with a diameter between 50 and 200 nm (Figs. 2A, 2B). WB showed that the protein expression of exosome markers, Alix, CD9, and CD63, was increased in NC and ferroptosis-induced MDA-MB-231 cell and 4T1 cell-derived exosomes compared with corresponding cancer cells (Figs. 2C, 2D). Fluorescence microscopy showed that red fluorescence was found in PMA-induced THP-1 cells and RAW 264.7 cells that had been incubated with MDA-MB-231 cell and 4T1 cell-derived exosomes induced by ferroptosis, indicating that exosomes were internalized by macrophages (Figs. 2E, 2F).

Ferroptosis-induced cancer cell-derived exosomes inhibit M2 polarization of macrophage

To study the effect of breast cancer cell-derived exosomes induced by ferroptosis on the phenotype of macrophages, PMA-induced THP-1 cells and RAW 264.7 cells were incubated with erastin-treated MDA-MB-231 cell and 4T1 cell-derived exosomes, respectively. WB revealed that the protein expression of M2 marker, Arg-1 was dramatically reduced, while M1 markers, CD86 and IL-1β protein expression was significantly enhanced in macrophages incubated with exosomes derived from ferroptosis-induced MDA-MB-231 cells and 4T1 cells compared with negative control exosomes (Figs. 3A, 3B). In addition, qRT-PCR analysis showed that M2 markers, CD206 and CD163 mRNA expression was significantly decreased, while M1 markers, iNOS and IL-1 β mRNA expression was markedly increased in PMA-induced THP-1 cells and RAW 264.7 cells treated with MDA-MB-231 cell and 4T1 cell-derived exosomes induced by ferroptosis relative to macrophages treated with negative control exosomes (Figs. 3C, 3D). Flow cytometric analysis uncovered that the number of CD86⁺ cells was markedly enhanced, while CD206⁺ cells was obviously diminished in above macrophages incubated with ferroptosis-induced MDA-MB-231 cells and 4T1 cell-derived exosomes (Figs. 3E–3H, Fig. S1). The immunofluorescence test showed that the fluorescent expression of CD86 was remarkably increased, whereas Arg-1 was obviously reduced in above macrophages treated with ferroptosis-induced MDA-MB-231 cell and 4T1 cell-derived exosomes (Figs. 4A, 4B). In addition, the protein expression of CD206 was remarkably enhanced in M2 macrophages, while reduced in M2 macrophages incubated by ferroptosis-induced MDA-MB-231 cell and 4T1 cell-derived exosomes (Figs. 5A, 5B). ELISA results showed that M2 macrophages exhibited a significant increase in Arg-1 level, while M2 macrophages incubated by ferroptosis-induced MDA-MB-231 cell and 4T1 cell-derived exosomes exhibited an opposite result (Figs. 5C, 5D). These above data demonstrated that breast cancer cell-derived exosomes induced by ferroptosis inhibited polarization of M2 macrophages.

Ferroptosis-induced cancer cell-derived exosomes regulate autophagy

Evidence suggests that autophagy interacts with ferroptosis (Zhou et al., 2019). Studies have shown that autophagy regulates macrophage M2 polarization (Xu et al., 2021a). Therefore, we investigated whether autophagy is involved in the regulation of macrophage M2 polarization by ferroptosis-induced exosomes. We used autophagy PCR array to analyze the expression of autophagy-related genes in macrophages incubated by exosomes and normal macrophages. It was discovered that a total of 84 autophagy-related genes including TREM2, PIK3C3, MALT1, SQSTM1, ULK1, ATG9B, GSDMA, GSDMC, NLRP7, NLRP9,and SATA3 were screened and there were differences in autophagy-related gene expression between the two groups (Table 1, Fig. 6A). In addition, to construct the gene network regulated by exosomes, we analyzed protein interactions of genes regulated by these exosomes. We discovered that SQSTM1 interacted with PRKC1, and NLRP9 interacted with NLRP7 (Fig. 6B). These results indicated that ferroptosis-induced cancer cell-derived exosomes might inhibited M2 polarization of macrophage by regulating autophagy.

Macrophages incubated with ferroptosis-induced exosomes inhibit breast cancer cell progression

To investigate the effect of macrophages incubated with ferroptosis-mediated cancer cell exosomes on cell phenotype of breast cancer, culture supernatants of PMA-induced THP-1 cells and RAW 264.7 cells treated with MDA-MB-231 cell and 4T1 cell-derived exosomes induced by ferroptosis were used to incubate MDA-MB-231 cells and 4T1 cells. Transwell assay showed that PMA-induced THP-1 cells and RAW 264.7 cells incubated by ferroptosis-induced cancer cell-derived exosomes significantly suppressed the migration and invasion of MDA-MB-231 cells and 4T1 cells compared with PMA-induced THP-1 cells and RAW 264.7 cells incubated by control exosomes (Figs. 7A–7D). Collectively, ferroptosis-induced breast cancer cell-derived exosomes inhibited M2 macrophage polarization, which further inhibited breast cancer cell migration and invasion (Fig. S2).