The mouse bladder cancer cell line MB49 was obtained from the Chinese Academy of Sciences. MB49 cells were cultured in DMEM medium (GE Healthcare Life Sciences, Pittsburgh, PA, USA),supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Science, Massachusetts, USA).
Bone marrow-derived macrophages (BMDM) were isolated from the femur and tibia of adult male mice according to our previous publication. Briefly, mouse femoral tissue was isolated, and the bone marrow was flushed with 3 ml of normal saline (NS). After red blood cell lysis, bone marrow cells were suspended at 2–4×106 cells/ml in RPMI-1640 medium, supplemented with 10% FBS and 30 ng/ml GM-CSF (PEPROTECH Inc., Rocky Hill, USA). BMDM were cultured in a humidified incubator at 37°C and 5% CO2.
Isolation and identification of exosomes
MB49 cells were washed twice with PBS when its confluence reach to 90%, following cultured with serum-free RPMI 1640 medium for 24 h. The culture supernatant was collected and centrifuged at 300 g and 15,000 g for 20 min respectively, which is to remove the suspended cells and cell debris. Then the collected supernatant was subjected to ultracentrifugation at 100,000g for 70 min at 4°C (HITACHI ultracentrifuge, CS150FNX). The obtained exosome pellet was washed with PBS and concentrated after ultracentrifugation, which is to improve the purification of exosomes. The MB49-derived exosomes were verified by transmission electron microscopy (HT7800, HITACHI) and the expression of exosome specific markers CD63 and HSP90 by Western blot analysis.
RNA extraction and qRT-PCR
The total RNA of exosomes stimulated BMDM was extracted with Trizol® reagent (Invitrogen, Carlsbad, CA, USA), and reverse transcription was performed with PrimeScript™ RT Master Mix (Takara Co. Ltd.). Real-time PCR was performed on the ABI PRISM 7300HT sequence detection system (Applied Biosystems, Foster, CA, USA) using SYBR Green PCR Master Mix (Application Takara, Otsu). The relative expression level of mRNA was calculated by the 2−ΔΔCt method. The sequences of all primers are shown in the supplementary table 1. Each experiment was repeated three times.
BMDM were lysed with RIPA buffer (Beyotime institute of Biotechnology, Shanghai, China). The bicinchoninic acid protein assay (BCA) kit (Beijing Leagene Biotech co. Ltd) was used to determine the protein concentration. Total 20 µg protein were separated on SDS-PAGE gel and transferred to 0.45 µm PVDF membrane. The membrane was blocked with 5% skimmed milk at room temperature for 1 h. The primary antibodies were incubated overnight at 4˚C. The peroxidase-conjugated secondary antibody was incubated for 60 min at room temperature. The protein bands were then visualized using an enhanced chemiluminescence kit (Beyotime institute of Biotechnology) and scanned by an imaging system (Bio-Rad Laboratories, Inc. Hercules, CA, USA). The ImageJ software v1.8.0 (National Institutes of Health) was used to quantify the density measurement. The primary and secondary antibodies used in this study were listed in supplemental table 2.
To detect the uptake of MB49-derived exosomes by BMDM, the fluorescent dye azide cyanine-Cy5.5 (0.2 µM, Fanbo BioChemical, Beijing, China) was added to the exosomes at 37°C for 30 min, following the Cy5.5 labeled exosomes were centrifuged at 100,000 g, 4˚C for 70 min to remove residual dye, and then incubated with BMDM for 3 h in the dark at 37˚C. The nuclei were stained with DAPI (Beyotime institute of Biotechnology, Shanghai, China) at room temperature for 5 min, and then observed with a laser scanning confocal microscope or analyzed by flow cytometry analysis. For tracking the uptake of circulating MB49-derived exosomes in vivo, we first injected Cy5.5 labeled exosomes (1 µg in 100 µl PBS) into the mouse via the tail vein. After 12 h, the bladder was harvested and digested with 0.1 mg/ml collagenase D / DNase I (100 U/ml) (Sigma-Aldrich, Inc. St. Louis, MO, USA) solution. The Cy5.5 positive cells in CD45+F4/80+CD11b+ macrophages were analyzed by flow cytometry.
T cell co-culture experiments
MB49 cells were treated with/without 10 µM GW4869 (MCE, HY-19363) for 24 h. Afterwards, the exosomes were collected from culture supernatant by ultracentrifugation. After stimulating BMDM with exosomes for 48 h, Carboxyfluorescein succinimidyl ester (CFSE, abcam, 113853)-labeled splenic T cells were added and co-cultured for another 3 d. Then the percentage of CD4+/CD8+ T cells was detected by flow cytometry.
To detect the effects of MB49 conditional medium (CM) and MB49-derived exosomes on BMDM, the freshly isolated BMDM were stimulated with MB49 CM or MB49-derived exosomes for 48 h. Then BMDM were collected and stained with CD45, F4/80, CD206 and CD11b. For the tumor associated immune cells identification, the MB49 derived subcutaneous tumor tissues were taken and digested with 0.1 mg/ml type D collagenase (Sigma-Aldrich, Inc. St. Louis, MO, USA) for 30 min at 37°C, and the single cell suspension were filtered and stained with macrophage markers (CD45, F4/80, CD206 and CD11b). The macrophage population was detected by using flow cytometer (BD Canto, Franklin Lakes, USA), and data were analyzed by FlowJo X (Tree Star, Ashland, OR, USA) software. Each experiment was repeated three times. The used antibodies were listed in supplementary table 2.
Microarray analysis of exosomal miRNAs
To determine the miRNAs contained in MB49-derived exosomes, a microarray analysis using Agilent mouse miRNA microarray kit (Agilent Technologies) was performed (OE Biotech Company, Shanghai, China). The sample preparation,miRNAs labeling, microarray hybridization and washing processes were carried out according to the manufacturer’s protocols. Data extraction and visualization were performed using Feature extraction software (version 10.7.1.1, Agilent Technologies).
To evaluate the influence of exosomes to macrophage polarization in vivo, we established a mouse subcutaneous tumor model. Briefly, total 1 × 106 MB49 cells were transplanted subcutaneously into the right flank of a male 8–10 week-old mouse. From day 3, GW4869 (2.5 µg/g in DMSO/saline) was intraperitoneally injected in every 3 days for total 4 times. A sham control group was injected with DMSO/saline alone. Tumor volumes were calculated as 1/2 × length × width2. The mice were sacrificed on day 15 and the tumor/body weight ratio was measured. The animal experiment was approved by the Ethical Review Committee of the First Affiliated Hospital of Zhengzhou University.
The statistical analysis was performed using GraphPad Prism 8 software. Comparisons between two groups were made using t-test as appropriate. For multiple comparisons, one-way ANOVA was applied. Statistically significant differences are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001.