2.1 Cell lines and culture

The human breast cell line MCF-10 A, human breast cancer cell lines (MCF7, T47D, SKBR-3, MDA-MB-231 and Hs578T), human monocyte cell line THP-1 and human umbilical vein endothelial cell line HUVEC were all purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). T47D, SKBR3, MCF7, MDA-MB-231, Hs578T and HUVEC cells were cultured with DMEM medium (Procell, China) containing 10% fetal bovine serum (FBS). THP-1 cells were cultured with RPMI-1640 medium (Procell, China) with 10% FBS and 0.05 mM ß-Mercaptoethanol. MCF-10 A cells cultured with MEGM kit (Lonza Clonetics, Switzerland). All cells were cultured at 37 °C and in 5% CO2 humidified atmosphere incubator.

2.2 Hyperthermia treatment

The hyperthermia treatment conditions were as follows: temperature, 43 °C; duration, 1 h. Briefly, cells were allowed to adhere in a culture flask in advance. After reaching the logarithmic phase, the cells were incubated at 43 °C for 1 h in incubator. To reduce the time of temperature rise, we prepared a 43 °C pre-heated medium and replace the original medium. Control group cells were maintained in a 37 °C medium. After hyperthermia treatment, the cells were transferred to a 37 °C incubator. Further examinations were conducted after 6 h of recovery.

2.3 Cell counting Kit-8 (CCK-8) assay

Cells were seeded at an optimized concentration of 5000 cells/well in sterile 96-well flat-bottom culture plates. Five secondary holes were then arranged in each group. Cells were exposed to hyperthermia treatment (43 °C for 1 h), followed by a recovery period to 6 h. These groups were measured with CCK-8 reagent (APExBio, Houston, USA) and the absorption value at 450 nm was measured on 0, 24, 48, 72 h using Multiskan FC microplate Reader (Thermo Fisher Scientific, USA).

2.4 Cell apoptosis assay and cell cycle assay

After 24 h of hyperthermia treatment, cells were collected. For apoptosis assay, cells were resuspended in 300 µl of 1×binding buffer with 3 µl of Annexin V-FITC and 3 µl of PI for 15 min in the darkness. For cell cycle assay, cells were fixed with 75% ethanol at 4 °C overnight. Then, cells were collected and hydrated with PBS for 15 min, followed by resuspended in 300 µl of PI staining binding buffer (Vazyme, Nanjing, China) for 30 min in the darkness. The percentage of apoptotic cells and cell cycle was detected by using Cytoflex flow cytometer (Beckman, USA).

2.5 Exosome isolation

Exosomes were isolated using gradient centrifugation, following general protocols. After hyperthermia treatment, cells were cultured with DMEM medium containing 10% exosome-depleted FBS for 24 h. Next, the culture medium was collected and centrifuged at 300 g for 10 min, followed by centrifugation at 2000 g for 15 min and 12,000 g for 30 min in turn. The supernatant was then filtered through a 0.22 µm filter and ultracentrifuged at 100,000 g (Beckman Type 90 Ti) for 2 h twice. The exosomes samples were resuspended in PBS for analysis.

2.6 Transmission electron microscopy (TEM)

The isolated exosomes were allowed to adsorb onto a copper network for 2 min. The sample was washed with distilled water and negatively stained with uranium acetate for 2 min. The precipitate of cells was fixed with 2.5% glutaraldehyde at 4 °C overnight. All samples were gradually dehydrated in ethanol. The samples were embedded and sectioned. The section was subjected to double staining with 3% uranyl acetate-lead citrate. Transmission electron microscope (JEM-1010, JEOL, Japan) was used to observe the structure of exosomes.

2.7 Total protein extraction and Western blot (WB)

Total protein of cells and isolated exosome was extracted using phenylmethanesulfonyl fluoride (PMSF) diluted 1:100 in cell lysis buffer (Beyotime, Shanghai, China) for 30 min on ice. The protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China). After centrifuging at 12,000 ×g for 20 min, the supernatant was mixed with 5 × loading buffer (NCM Biotech, Suzhou, China) and then denatured at 99 °C for 10 min.

Equivalent concentration proteins of cells and exosomes was carefully loaded in each lane of a 10% SDS-polyacrylamide gels by electrophoresis and transferred onto a 0.45 µm PVDF membrane (Millipore, USA). Then, the membranes were blocked with QuickBlock™ Blocking Buffer for Western Blot (Beyotime, Shanghai, China) for 20 min. The membranes were incubated overnight at 4 °C with primary antibodies CD63 antibody (1:1000, Abcam, ab68418), TSG101 antibody (1:1000, Abcam, ab125011) and Calnexin antibody (1:1000, Abcam, ab133615). after washed, the membranes were incubated with species-specific secondary antibody at room temperature for 1 h. Finally, the proteins were detected by the BeyoECL Plus kit (Beyotime, Shanghai, China) under ChemiDoc™ Touch Imaging System (Bio-Rad, USA).

2.8 Nanoparticle tracking analysis (NTA)

ZetaView® PMX120 nanoparticle tracking analyzer was used to analyze the size distribution and concentration of exosomes. Exosome samples were diluted in PBS and pumped for analysis.

2.9 Cell differentiation induction

To promote the differentiation of monocytes into M0 macrophages, THP-1 cells were cultured in complete medium with 100 ng/mL PMA (Sigma-Aldrich, USA) for 48 h to promote the differentiation of monocytes into M0 macrophages. To promote the activation of M0 macrophages into M1 macrophages, the cells were incubated with preliminary medium containing 100 ng/mL LPS (Sigma-Aldrich, USA) instead of 1640 complete medium for 48 h.

2.10 Cellular uptake of exosomes

The uptake of PKH26-labeled exosomes by recipient cells was examined using confocal microscopy. The 5000 recipient cells were seeded in a confocal dish. Exosomes were labeled using the PKH26 red fluorescent labeling kit (Sigma-Aldrich, USA) following the manufacturer’s instructions. The recipient cells were co-cultured with 20 µg exosomes for 24 h. Next, the cells were washed and fixed with 4% polyformaldehyde for 30 min, followed by staining with Actin-Tracker Green (1:1000, Beyotime, China) and DAPI (1:1000, Beyotime, China) for 30 min in the dark. The uptake of the dye was examined using a Stellaris STED laser confocal microscope (LEICA, Germany).

2.11 Total RNA extraction and real-time polymerase chain reaction (RT-qPCR)

Total RNA extracted from exosomes using a Trizol ls Reagent (Thermo Fisher, USA). Total RNA extracted from breast cancer cells and macrophages using a total RNA Kit (Tian Gen, DP419). The concentration and purity of RNA were detected by a NanoDrop2000 ultraviolet spectrophotometer (Thermo Fisher, USA). The procedure of RT-qPCR is the same as prviously described [24]. All primers were shown in Supplementary Table 1.

2.12 Flow cytometry analysis of macrophages

To detect the protein markers of macrophages, cells were labeled with CD14-FITC, CD86-APC and CD163-PC7 human antibodies (Biolegend, USA). Briefly, pretreated macrophages were digested using PBS containing 2.5 mM EDTA. The digestion was stopped using 0.5% BSA in PBS. The precipitate was resuspended and incubated in 100 µL PBS with 5 µL human FcR blocking agent (Biolegend, USA) for 15 min to block Fc receptor. After washing, the cells were resuspended in 300 µL of 1× binding buffer with 3 µL CD14-FITC, 3 µL CD86-APC, and 3 µL CD163-PC7 antibodies for 30 min to stain cell surface markers. The percentage of M1/M2 macrophage cells was quantified using a Cytoflex flow cytometer (Beckman, USA).

2.13 RNA-Seq

After hyperthermia exposure, total RNA was extracted and quantified. MDA-MB-231 cells were subjected to genome-wide gene expression analysis using RNA deep sequencing. Library construction was performed by Illumina. The raw data were analyzed with R language. Resulting sequencing files were publicly available at PRJNA957775 of SRA database. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Reactome pathway analysis of differentially expressed genes (DEGs) were performed to identify the function enrichment using DAVID analysis (http://david.ncifcrf.gov).

2.14 Bioinformatics analysis

The protein-protein interaction (PPI) network was constructed using STRING (http://string-db.org) analysis. The hub genes in the PPI network were calculated by method MCC. The expression levels of hub genes in breast cancer datasets of The Cancer Genome Atlas (TCGA) database was analyzed using GEPIA online software (http://gepia.cancer-pku.cn). The correlation between the expression level of hub genes and overall survival (OS) in the datasets of breast cancer retrieved from TCGA database was analyzed using the Kaplan–Meier plotter online software (http://kmplot.com/analysis/). The correlation between the expression levels of hub genes and the degree of macrophage infiltration in breast cancer was analyzed using TIMER2.0 online software (http://timer.cistrome.org).

2.15 Cell transfection

Macrophage cells were transfected with negative control (NC) or predesigned overexpression (OE) plasmid pcDNA3.1-HSPB8 using Hieff Trans® liposomal transfection reagent (Yeasen, Shanghai, China). Meanwhile, MDA-MB-231 and Hs578T cells were transfected with NC or predesigned si-HSPB8 using Lipo8000 transfection reagent (Beyotime, Shanghai, China). Briefly, cells were planked with the density at transfection was 70% in advance. Then, prepare two tubes of mixed reagent. A: 250 µL Opti-MEM + 2.5 µg DNA/100 pmol siRNA; B: 250 µL Opti-MEM + 5 µL transfection reagent. After 5 min, we mixed two tubes and co-cultured for 20 min. Finally, the compound was added to each well for 48 h before further examination.

2.16 Immunofluorescence (IF)

The transfected macrophages were fixed with 4% polyformaldehyde for 25 min. Next, the cells were blocked with QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China) for 20 min, and then staining with anti-CD86 antibodies (1:500, Abcam, ab239075) overnight at 4 °C. Then, Cells were stained with species-specific goat anti-rabbit IgG H&L (Alexa Fluor 488) (1:2000, Abcam, ab150077) secondary antibodies for 1 h at room temperature, followed by stained with 30 µL anti-fluorescence quenching sealing agent (including DAPI) (Soleibao, Beijing, China) and covered with the cover glass. After completely drying, Thunder Imager fast high-resolution inverted fluorescence imaging system (LEICA, Germany) was used to observe the expression of CD86.

2.17 Statistical analysis

All experiments were independently performed at least in triplicates, error bars represented the mean ± SD of experiments. All assays were analyzed using t-test of means between two groups of independent samples, and using one-way ANOVAs between multiple groups. Differences were considered significant at p < 0.05, with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.