Figure 2: EVs from brain metastatic cancer cells were incorporated into endothelial cells, regulating the invasion through BBB of cancer cells.

(a) Phase-contrast electron microscopy was used to visualize resuspended EVs pellets. Scale bar, 100 nm. (b) The intensity of PKH67-labelled EVs was measured by ImageJ. Error bars represent s.d., Student’s t-test, n=3. (**P<0.01). (c) EVs isolated from cancer cells were labelled using PKH67 and added to the upper chamber. Representative pictures of endothelial cells, pericytes and astrocytes are shown. Negative control (N.C.), EVs from MDA-MB-231-luc-D3H2LN (D3H2LN), EVs from BMD2a and EVs from BMD2b are shown. Scale bar, 20 μm. Bar in the panel of astrocytes represents 100 μm. (d) The value of the TEER was monitored before (day 4) and after (day 5) the addition of EVs isolated from each cell line. EVs isolated from brain metastatic cancer cells were incubated in the in vitro BBB model for 24 h. Error bars represent s.d., Student’s t-test, n=3. (**P<0.01, *P<0.05). (e) Assessment of BBB permeability determined by NaF (molecular weight=376.27). EVs from MDA-MB-231-luc-D3H1 (D3H1), D3H2LN, BMD2a or BMD2b cells and N.C. were added to the in vitro BBB model. After 24 h, NaF was added. NaF that had passed through BBB was measured by a fluorometer. Error bars represent s.d., Student’s t-test, n=3. (**P<0.01). (f) PKH26-labelled cancer cells (2 × 104 cells) were added to the in vitro BBB model. After a 48-h incubation, endothelial cells were removed, and the invading cells were counted using a fluorescence microscope. (g) In vitro BBB transmigration activity of D3H1, D3H2LN, BMD2a or BMD2b cells. The number of transmigrated cells relative to the D3H1 cell lines is plotted. Error bars represent s.d., Student’s t-test, n=3. (*P<0.05, **P<0.01). (h) In vitro BBB transmigration activity of the BMD2a treated with control siRNA (N.C.), RAB27B, nSMase2 and both siRNAs (S+R). The number of transmigrated cells relative to the control cells is plotted. Error bars represent s.d., Student’s t-test, n=3. (**P<0.01). (i) The number of transmigrated D3H1 cells relative to the control cells is plotted. Error bars represent s.d., Student’s t-test, n=3 (**P<0.01). Data are representative of at least three independent experiments each. (Fig. b,c,d,e,g,h,i).