Establishment of a protocol for the isolation of MV from peripheral blood

we asked if it is possible to detect T-MV in peripheral blood of cancer patients without any previous sorting steps. In order to investigate this question, we first aimed to define which MV populations are found in the blood of healthy individuals and then compare their distribution to samples from cancer patients.

For the analysis of different MV populations in peripheral blood and to ensure that the previously established tumor marker proteins (see 3.3.1) are not expressed on MV from other cell types, we further started to identify markers which are specific for distinct benign MV populations. While CD62E has already been described as a marker for endothelial cell-derived MV (Dignat-George & Boulanger, 2011) and CD235a as specifically expressed on red blood cell-derived MV (Setty et al, 2000), we characterized P-MV and Mϕ-MV to define markers for these two MV populations.

Fig. 38: Characterization of P-MV

P-MV (5 µg) were stained for different tumor and blood cell markers and analyzed by flow cytometry.

Representative histogram overlays are shown with the specific fluorescent signal in green and the corresponding isotype control in grey.

As shown in Fig. 38 and Fig. 39, both MV populations were negative for expression of the red blood cell marker CD235a, the endothelial cell marker CD62E as well as the tumor markers EGFR and MUC1. In contrast, they expressed considerable amounts of EMMPRIN which indicates that EMMPRIN expression alone cannot be used for the flow cytometric identification of T-MV in peripheral blood. Furthermore, P-MV characteristically displayed high levels of the cell adhesion molecule CD62P and low expression of CD45, while CD11c, a marker for myeloid cells, was not present. On the contrary, Mϕ-MV carried low amounts of CD11c on their surface and showed a strong expression of CD45. The P-MV marker CD62P was not detectable on Mϕ-MV. In summary, the results show that CD45, CD62P and CD11c together with the established markers CD62E and CD235a can be used to define subsets of benign MV present in blood. Moreover, all of these markers were negative on malignant breast cancer-derived T-MV as confirmed by flow cytometry (Fig. 40).

Fig. 39: Characterization of Mϕ-MV

Mϕ-MV (5 µg) were stained for different tumor and blood cell markers and analyzed by flow cytometry.

Representative histogram overlays are shown with the specific fluorescent signal in green and the corresponding isotype control in grey.

Fig. 40: Blood cell markers on T-MV

T-MV (5 µg) were stained for the established blood cell marker proteins and analyzed by flow cytometry.

Representative histogram overlays are shown with the specific fluorescent signal in green and the corresponding isotype control in grey.

Regarding methodological issues, the choice of the anticoagulant which is used during collection of the blood samples can have significant effects on subsequently isolated EV.

While some publications show a substantial loss of MV in EDTA-anticoagulated blood compared to sodium heparin-treated blood samples (Jayachandran et al, 2012), others discourage the use of heparin-based anticoagulants (Witwer et al, 2013) because heparin was shown to interact with MV (Maguire et al, 2012) and is able to activate platelets (Krauel et al, 2012). For this reason, we analyzed which anticoagulant is most suitable for isolation of total EV from cancer patients.

Peripheral blood was drawn from five cancer patients and either anticoagulated with EDTA or Lithium-heparin (Li-Hep). Serum or plasma samples were stored at -20°C for <14 days and then thawed for EV isolation (see 2.2.2.1.3) which resulted in visible agglutination in all samples derived from Lithium-heparin-anticoagulated blood. This could neither be prevented by EV isolation directly after blood withdrawal nor by addition of new heparin after each centrifugation step (personal observation). Correspondingly, comparison of EV counts elucidated that MV yields were significantly lower when lithium-heparin had been used as anticoagulant, while Exo, which also tended to aggregate after ultracentrifugation, were slightly increased. In line with this, the number of CD62P- and EMMPRIN-positive MV was decreased in lithium-heparin anticoagulated samples, although this trend did not reach statistical significance. Since both of these proteins had been found in high levels on P-MV

(see Fig. 38), these findings seem to support the results which demonstrated an activating effect of heparin on platelets, as well as their MV, thereby inducing coagulation. Interestingly, the percentage of CD11c-positive MV was elevated in samples with Lithium-heparin as anticoagulant (8,0%) compared to EDTA-coagulated samples (1,2%). However, the significance of this observation remains unclear.

Fig. 41: Comparison of EDTA and heparin as anticoagulants for patient samples

A-B, EV isolated from EDTA- or Lithium-Heparin (Li-Hep)-anticoagulated blood were quantified by Lowry assays (A) (*p<0,05) and MV further characterized for selected blood and tumor cell markers by flow cytometry (B) (means±SD, n=5, *p<0,05, n.d. = not detectable). C-D, EDTA-anticoagulated blood from six cancer patients was either directly or after storage at -20°C for <14 days used for EV isolation. EV yields were determined by Lowry assays (C) and MV analyzed regarding expression of selected markers (D) (flow cytometry, means±SD, n=6, *p<0,05). Lines display the mean of the samples.

After having shown that EDTA is more suitable as anticoagulant for patient samples, we asked whether storage of serum samples at -20°C influences EV counts or distribution of MV-associated markers. Therefore, EDTA-anticoagulated blood was drawn from six cancer patients and serum samples either directly subjected to EV isolation or frozen at -20°C for

<14 days previous to ultracentrifugation. As shown in Fig. 41C+D, freezing of serum had no significant effects on MV or Exo counts or expression of any of the selected markers for tumor or blood cell-derived MV except a slight decrease in the number of CD62P-positive MV.