Fig. 3
Increasing Tregs in tumor are related to increasing TGF-β1EV in TME. A Schematic diagram of tumor model. Tumor model was established by injecting 5 × 104 4T1 cells subcutaneously into the fourth mammary pad of BALB/c mice. Tumor-bearing mice were treated with 10 Gy radiation 14 days after injection of tumor cells. Tumor tissue were collected and fixed for IHC 2 or 14 days after radiation. B IHC image of TGF-β1 in tumor according to A. Scale bar, 100 µm. C TGF-β1 positivity analysis in B. For each group, n = 9. Analysis was performed by Aperio ImageScope, version 12.4. D Schematic diagram of tumor model. Tumor model was established by injecting 5 × 104 4T1 cells subcutaneously into the fourth mammary pad of BALB/c mice. Radiation (10 Gy) was performed 14 days after injection of tumor cells. Peripheral blood was collected and TGF-β1 was analyzed by ELISA. E TGF-β1 protein concentration in peripheral blood in D. F TGF-β1 protein concentration in EVs secreted by tumor pieces with or without radiotherapy. Tumor-bearing mice were treated with 10 Gy radiation. 20 days after radiotherapy, Tumor tissues were collected and cultured in vitro for 48 h. Culture medium was collected for EV isolation and ELISA was performed for analysis of TGF-β1. G Percentage of Treg in CD4+cells. Naïve spleen cells from Foxp3-GFP mice were treated by anti-CD3/CD28 functional antibody and EVs secreted by 4T1 cells with or without pretreatment of radiation for 72 h and percentage of CD4+CD25+ cells were analyzed by flow cytometry. H Confocal imaging of tumor slides injected with fluorescence labeled EVs or free TGF-β1. 500 µg of PHK fluorescence labeled EVs or FITC-labeled free TGF-β1 were intratumorally injected. After 1 h or 24 h, frozen sections were operated and detected using confocal scanned imaging. I Histogram of fluorescence photons in confocal imaging slides of (H). *p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.0001. C–F were analyzed by t-test. G was analyzed by 2-way ANOVA