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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Reactive oxygen species reprogram macrophages to suppress antitumor immune response through the exosomal miR-155-5p/PD-L1 pathway

Fig. 3

Exo-miR-155-5p inhibits macrophage migration and tumor infiltration. A. Macrophages were treated with Exo-con, Exo-NAC, Exo-miR-con (exosomes isolated from A2780-miR-con cells), or Exo-miR-155 (exosomes isolated from A2780-miR-155 stable cells), respectively for 48 h. The medium was then replaced and the cells were cultured for another 24 h after generating the scratches. Representative images are shown. B. Chemokines expressions in macrophages treated with Exo-con/Exo-NAC or Exo-miR-con/Exo-miR-155 were determined by RT-qPCR. C. The schematic diagram of the 3D co-culture model setup. A2780-miR-con and A2780-miR-155 stable cells were cultured to form tumor spheroid for 4 days. The same amount of macrophages were added and co-cultured for another 4 days. D. The representative images were shown after 4 days of 3D co-culture. E. The 3D co-cultures spheroids were re-suspended into single cell suspensions and analyzed by flow cytometry to determine the amounts of A2780 cells and infiltrated macrophages. F. The percentages of apoptotic A2780 cells and macrophages after co-culture were analyzed by flow cytometry. G-H. The CD14 positive infiltrated macrophages were isolated from 3D co-culture models using FACS. The expression levels of miR-155-5p (G) CXCL10, and CCL2 (H) were measured by RT-qPCR. I. A2780-miR-con and A2780-miR-155 stable cells were stained with cell tracker green before seeding onto 1% agarose concave surface for spheroid formation. Macrophages were stained by eFluor670 before adding into tumor spheroids. Representative images were taken by confocal microscope for 3D live cell imaging at 4 x magnification. The number of A2780 and infiltrated macrophages were analyzed as accumulative average fluorescent intensities. Green: A2780. Purple: macrophages. Data are representative of three independent experiments. Data = Mean ± SD. *p < 0.05, **p < 0.01

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