Figure 7.

Metastatic oral cancer-derived MEVs induced macrophages M2 polarization. (a–f) Immunohistochemistry (IHC) of oral cancer patient-derived specimens. (a) Representative IHC showing HSP90α and HSP90β in stage I vs. stage IV oral cancers. Tu, tumour. St, stroma. --- borders between tumour and stroma. (b–e) Rate of (b,c) HSP90α+ or (d,e) HSP90β+ cancer cells or stromal cells. The rates were calculated by counting immuno-positive cells per total cell number in each random field in (b,d) tumour areas or (c,e) 100 mm² stromal areas. Five random fields each in 8 oral cancer cases. N = 40. (f) IHC for HSP90β in stage I vs. stage IV oral cancer. Arrowheads, HSP90β-positive infiltrating tumour-associated macrophages (TAM). (g–j) Macrophage M2 polarity was altered by MEV and T-MEV. THP-1 cells were stimulated with PMA for the differentiation to macrophages and then treated with MEV, T-MEV (25 μg/mL) or PBS. (g) Representative images of THP-1 cells treated with or without PMA for 24 h. (h) Western blotting of extracellular and intracellular MMP-9 in THP-1 cells with or without PMA treatment. (i,j) Immunocytochemistry of macrophage markers with or without MEV or T-MEV. (i) Rate of CD206/CD68 double-positive M2-like macrophages. N = 9. (j) Representative staining images of CD206 (green) and CD68 (red). Blue, DAPI. Scale bars, 100 μm.