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

Jieru Zhou, Xiaoduan Li, Xiaoli Wu, Ting Zhang, Qinyi Zhu, Xinjing Wang, Husheng Wang, Kai Wang, Yingying Lin and Xipeng Wang
Jieru Zhou
1Department of Obstetrics and Gynaecology, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
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Xiaoduan Li
2Department of Gynecology, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China.
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Xiaoli Wu
1Department of Obstetrics and Gynaecology, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
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Ting Zhang
3Center for Reproductive Medicine, Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Renji Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai, China.
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Qinyi Zhu
2Department of Gynecology, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China.
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Xinjing Wang
1Department of Obstetrics and Gynaecology, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
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Husheng Wang
1Department of Obstetrics and Gynaecology, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
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Kai Wang
2Department of Gynecology, Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai, China.
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Yingying Lin
4Department of Neurosurgery, RenJi Hospital, Shanghai JiaoTong University School of Medicine, Shanghai, China.
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  • For correspondence: wangxipeng@xinhuamed.com.cn yylin@sibs.ac.cn
Xipeng Wang
1Department of Obstetrics and Gynaecology, XinHua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
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  • For correspondence: wangxipeng@xinhuamed.com.cn yylin@sibs.ac.cn
DOI: 10.1158/2326-6066.CIR-17-0479 Published December 2018
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  • Figure 1.
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    Figure 1.

    Distribution of Tregs and Th17 cells in EOC patients. A and B, Tregs in benign ovarian tumor tissue and in EOC tissue were stained with DAPI (blue, nuclei) and with antibodies targeting CD4 (green) and FoxP3 (red). Th17 cells in benign ovarian tumor tissue and in EOC tissue were stained with DAPI (blue, nuclei) and with antibodies targeting CD4 (green) and IL17 (pink). The mean number of Tregs and Th17 cells was quantified in five high-powered fields (HPF) for each section. C and D, Tregs in benign peritoneum tissue and in EOC peritoneum tissue were stained with DAPI (blue, nuclei) and with antibodies targeting CD4 (green) and FoxP3 (red). Th17 cells in benign peritoneum tissue and in EOC peritoneum tissue were stained with DAPI (blue, nuclei) and with antibodies targeting CD4 (green) and IL17 (pink). E, The Treg/Th17 ratio in EOC tissues (n = 124) compared with benign ovarian tumor tissues (n = 26). F, The Treg/Th17 ratio in EOC peritoneal tissue (n = 15) compared with benign peritoneal tissue (n = 6). G, Treg/Th17 ratio in the SOC (n = 72) and NSOC groups (n = 52). Scale bar, 50 μm. Results were analyzed via Mann–Whitney test; P < 0.05 was considered statistically significant.

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    Figure 2.

    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.

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    Figure 3.

    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.

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    Figure 4.

    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.

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    Figure 5.

    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.

  • Figure 6.
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    Figure 6.

    The imbalance in Treg/Th17 cells was associated with EOC grade and patient survival. A, The ROC curve reflecting the sensitivity and specificity of the Treg/Th17 ratio in EOC patients. B, EOC patient survival based on the Treg/Th17 ratio cutoff of 1.02. C, The relationship between Treg/Th17 ratio and OS among 467 EOC patients in the TCGA database. D, The relationship between miR-29a-3p and miR-21-5p expression and OS among 467 EOC patients in the TCGA database. E, Illustration of our conclusions. TAM-derived exosomes transfer miR-29a-3p and miR-21-5p to synergistically induce a Treg/Th17 cell imbalance through directly targeting of STAT3 in CD4+ T cells.

Tables

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  • Table 1.

    Association between the Treg/Th17 ratio and clinicopathologic variables in EOC patients

    Ratio < 1.02Ratio ≥ 1.02TotalP
    n (%)n (%)
    Age
     <509 (17.0)44 (83.0)530.278
     ≥5019 (26.8)52 (73.2)71
    Histologic subtype
     SOC14 (19.4)58 (80.6)720.386
     NSOC14 (26.9)38 (73.1)52
    Histologic grade
     Low grade11 (55.0)9 (45.0)20<0.001
     High grade14 (14.1)85 (85.9)99

Additional Files

  • Figures
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  • Supplementary Data

    • Supplementary Figures - 1.This file contains supplemental figure S1-S8. It includes the supplemental data about the clinical samples, the supplemental data about the in vivo experiments, the supplemental data of EOC patients in TCGA database, the flow cytometric analysis of cytokines in Tregs and Th17 cells, and so on.
    • Supplementary Tables - 2. This file contains supplemental table S1-S5. It shows univariate/multivariable analysis of OS in EOC patients, the absolute counts of Treg/Th17 cells in flow cytometric analysis, and the proportion of cytokines from Tregs/Th17 cells in flow cytometric analysis.
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Cancer Immunology Research: 6 (12)
December 2018
Volume 6, Issue 12
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Exosomes Released from Tumor-Associated Macrophages Transfer miRNAs That Induce a Treg/Th17 Cell Imbalance in Epithelial Ovarian Cancer
Jieru Zhou, Xiaoduan Li, Xiaoli Wu, Ting Zhang, Qinyi Zhu, Xinjing Wang, Husheng Wang, Kai Wang, Yingying Lin and Xipeng Wang
Cancer Immunol Res December 1 2018 (6) (12) 1578-1592; DOI: 10.1158/2326-6066.CIR-17-0479

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Exosomes Released from Tumor-Associated Macrophages Transfer miRNAs That Induce a Treg/Th17 Cell Imbalance in Epithelial Ovarian Cancer
Jieru Zhou, Xiaoduan Li, Xiaoli Wu, Ting Zhang, Qinyi Zhu, Xinjing Wang, Husheng Wang, Kai Wang, Yingying Lin and Xipeng Wang
Cancer Immunol Res December 1 2018 (6) (12) 1578-1592; DOI: 10.1158/2326-6066.CIR-17-0479
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