Figure 3.

In vitro model of delivery of tumor-derived substances to distal tumor sites via tumor-derived EVs

(A) Experimental design for the co-culture model. Briefly, HCT116-RFP cells and HCT116 cells were seeded in the lower and upper chambers, respectively. HCT116-RFP cells were treated with PBS or OBP-301 (MOI of 100) and the culture medium was changed to FBS-free medium 24 h after treatment. Cells were incubated for another 48 h and then harvested for analysis. GW4869 was added to the culture medium at 6 h prior to PBS or OBP-301 treatment to block Exo secretion. (B) EVs isolated from culture medium in the bottom chamber after PBS or OBP-301 treatment of HCT116-RFP cells were subjected to western blot analysis for RFP, E1A, and CD9. (C) EVs isolated after GW4869 treatment at the indicated doses in addition to OBP-301 treatment were subjected to western blot analysis for CD9. Relative intensities to non-treatment condition (measured by ImageJ) are provided beneath the blot. (D and E) HCT116 cells in the upper chamber after treatment of HCT116-RFP cells in the bottom chamber with OBP-301 and GW4869 were subjected to flow cytometry to measure RFP uptake (n = 3). Representative data for each treatment are shown in (D), and statistical assessments of RFP amounts in each treatment are shown in (E). ∗p < 0.05, ∗∗p < 0.01.