Uptake of tumor-derived RNA in platelets. (A) U87 glioma-derived microvesicles were labeled with PKH67 green fluorescent dye and incubated with isolated platelets. After 15, 30, 45, and 60 minutes of incubation in the presence and absence of microvesicles, the platelets were washed and subjected to FACS analysis of PKH67 fluorescence. (B) Platelets were incubated with PKH67-labeled microvesicles (MV) from glioma or prostate cancer (PCa) patients, stained with Texas-red wheat germ agglutinin, and analyzed by confocal microscopy for PKH67-labeled microvesicle uptake. Merged images and size bars are shown. (C) RNA was isolated from RNAse-treated platelets after incubation with U87 glioma-derived microvesicles under different conditions. RT-PCR was performed to detect EGFRvIII RNA. MV/MV-EGFRvIII indicates microvesicles isolated from U87/U87-EGFRvIII cells. (D) Mice were implanted with U87-Fluc-EGFRvIII cells or no cells and imaged after 2 weeks. Shown are representative bioluminescence images and corresponding EGFRvIII RT-PCR on mouse platelets. (E) RNA was isolated from platelets from 12 healthy control subjects and 26 glioma patients (only 18 patients shown here) and subjected to RT-PCR analysis. Corresponding glioma tissue biopsies served as control. Platelet activation and heterogeneous EGFRvIII tissue distribution may have caused the possible false-negative signals. PC indicates U87-EGFRvIII RNA; NC, H2O; and nd, not determined. *Positive signal. (F) RNA was isolated from platelets from healthy control subjects (n = 10) and prostate cancer patients (n = 12) and subjected to PCA3 and GAPDH RT-PCR analysis.
