Exosomes Released from Tumor-Associated Macrophages Transfer miRNAs That Induce a Treg/Th17 Cell Imbalance in Epithelial Ovarian Cancer

Effect of the Treg/Th17 imbalance on ovarian cancer in vivo. A, Trichostatin A (TSA, 5 nmol/L) was used to induce an imbalance in Treg/Th17. Results were from three independent experiments; t test. B, The purity of CD4⁺ cells was nearly 90%, as detected by flow cytometry. Image shown on the left is the negative control. Results were from three independent experiments. C, At 6 weeks of age, C57BL/6 mice were intraperitoneally injected with ID8-Luc-pur cells (day 1). For the long-term experiments, additional CD4⁺ T cells were injected beginning (day 15). Each group received 0.1 mL of PBS containing 1 × 10⁶ T cells by i.p. injection once a week. The Treg/Th17-low ratio group was prestimulated with 10 μg/mL anti-CD3 and 5 μg/mL anti-CD28. The Treg/Th17-high ratio group was cultured with 5 nmol/L/mL TSA for an additional 3 days. A bioluminescence imaging system was used to observe the tumor flux (cps) on day 2 and the following weeks. Days 1 to 8 were recorded as the first week. The tumor fluorescence intensity (cps) was analyzed in the three groups after injection of ID8-Luc-pur cells at 11 weeks (n = 15 mice/per group); t test. D, Fluorescence intensity (cps) in the three groups after injection of ID8-Luc-pur cells at 2–10 weeks (every 2 weeks); n = 15 mice/per group; t test. E, Kaplan–Meier curve showing the cumulative survival rate of the three groups (n = 15 mice/group); log-rank test. F, Typical images of the three groups at week 11. G, Hematoxylin–eosin (H&E) staining of the peritoneum, mesentery, and diaphragm tissue from sacrificed mice. P < 0.05 was considered statistically significant.

M2 macrophages induce an imbalance in Tregs and Th17 cells via exosome delivery. A, CD4⁺ T cells were cocultured with control, M1, or M2 macrophages for 3 days, and the proportion of Th17 cells and Tregs was detected by flow-cytometric analysis. Results were from 10 independent experiments. B, The Treg/Th17 ratio in the three groups, n = 10/group; t test. C, Transmission electron microscopy image of exosomes collected from the supernatants of M2 macrophages. Results were from three independent experiments. Scale bar, 100 nm. D, Western blot analysis of CD9, CD 63, CD81, and Tsg 101 in exosomes using THP-1 cells as a control. Results were from three independent experiments. E, Exosomes secreted by M2 macrophages were labeled using SYTO RNASelect Green Fluorescent Cell Stain (RNA was shown as bright green fluorescence) and a red fluorescent membrane stain and cocultured with T cells for 24 hours. The T cells were collected for confocal microscopy to detect M2 macrophage–derived exosomes in T cells. RNA in exosomes (top left), exosome membranes (top right), T cells under a light microscope (bottom left), and merged image (bottom right). Results were from two independent experiments. F, T cells were cocultured with control or with exosomes from M1 or M2 macrophages for 3 days. The proportions of Th17 and Tregs were determined by flow-cytometric analysis, and the Treg/Th17 ratio was calculated, n = 3/group; t test. P< 0.05 was considered statistically significant.

Hsa-miR-29a-3p and hsa-miR-21-5p from TAM-secreted exosomes induce a Treg/Th17 imbalance and alters cytokines by targeting STAT3. A, Heat map of miRNAs expressed in THP-1 cells and THP-1–induced M2 macrophages. B, MiRNAs related to T-cell differentiation. C, The expression of five upregulated miRNAs in primary monocytes and M2 macrophages. Three duplicate wells/per group. Results were from three independent experiments; t test. D, Hsa-miR-21-5p and hsa-miR-29a-3p mimics were transfected to CD4⁺ T cells for 3 days. The proportion of Th17 and Tregs was detected by flow cytometry. The Treg/Th17 ratio was calculated. Results were from three independent experiments; t test. E, Western blot analysis was used to detect STAT3 protein in Tregs (the top images) and Th17 cells (the bottom images) after CD4⁺ T cells were transfected with NC, hsa-miR-21-5p mimic, hsa-miR-29a-3p mimic, both mimics, or both inhibitors. Results were from three independent experiments. F, ImageJ analyzed gray value of protein bands from different groups in Tregs and Th17 cells. n = 3/group, t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. G, RT-PCR was used to detect the STAT3 mRNA in Tregs and Th17 cells after native CD4⁺ T cells were transfected with NC, hsa-miR-21-5p mimic, hsa-miR-29a-3p mimic, both mimics, or both inhibitors. Three duplicate wells/per group. Results were from three independent experiments; t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. H, Prediction of the binding site of hsa-miR-21-5p and hsa-miR-29a-3p in the STAT3 gene sequence by the target scan. I, Luciferase assay revealing binding of hsa-miR-29a-3p and hsa-miR-21 to the STAT3 3′UTR. n = 3/group; t test. J and K, Expression of cytokines TGFβ, IL10, IL4 in Tregs, and TNFα or IL6 in Th17 cells after native CD4⁺ T cells were transfected with NC, hsa-miR-21-5p mimic, hsa-miR-29a-3p mimic, both mimics, or both inhibitors. Results were from three independent experiments; t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Effect of mmu-miR-29a-3p and mmu-miR-21 on the growth and metastasis of ovarian cancer in vivo. A, For T cells that received NC, mmu-miR-21-5p mimic, mmu-miR-29a-3p mimic, both mimics, or both inhibitors after 48 hours, the relative STAT3 expression (fold change) in the five groups was examined using real-time PCR; three duplicate wells/per group. Results were from three independent experiments; t test. B, After the T cells were transfected with NC, mmu-miR-21-5p mimic, mmu-miR-29a-3p mimic, both mimics, or both inhibitors for 72 hours, STAT3 was detected via Western blotting in three independent experiments. C, The tumor fluorescence intensity (cps) in the five groups after injection of ID8-Luc-pur cells for 10 weeks; n = 10 mice/per group, t test. D, At 6 weeks of age, C57BL/6 mice were intraperitoneally injected with ID8-Luc-pur cells (day 1). For long-term experiments, additional T-cell injections were initiated (day 15). Each group received 0.1 mL of PBS containing 1 × 10⁶ CD4⁺ T cells via i.p. injection once a week. The T cells were transfected with NC, mmu-miR-21-5p mimic, mmu-miR-29a-3p mimic, both mimics, or both inhibitors for 72 hours. A bioluminescence imaging system was used to observe tumor flux (cps) on day 2 and each following week. Days 1 to 8 were recorded as the first week. Tumor fluorescence intensity (cps) in the 5 groups after injecting ID8-Luc-pur cells for 2 to 10 weeks (every 2 weeks); n = 10 mice/per group, t test. E, Kaplan–Meier curve showing the cumulative survival rate of the five groups; n = 10 mice/per group; log-rank test. F, Typical images of mice in the five groups are presented for week 10. P < 0.05 was considered statistically significant.