Circulating tumour-derived microvesicles in plasma of gastric cancer patients

Abstract

Cell membrane microfragments called microvesicles (MV) originating from different cells are circulating in the blood of healthy subjects and their elevated numbers are found in different diseases, including cancer. This study was designed to characterise MV present in plasma of gastric cancer patients. Since majority of MV in blood are platelets-derived (PMV), plasma samples deprived of PMV were used. In comparison to control, the number of MV in patients was significantly elevated in all stages, higher in more advanced disease. Patients' MV showed an increased membrane expression of CCR6 and HER-2/neu. The proportion of MV carrying some leucocyte determinants was low and similar in patients and control. Transmission electron microscopy showed their substantial heterogeneity in size and shape. The size determined by dynamic light scattering analysis confirmed this heterogeneity. The MV size distribution in patients was broader within the range of 10-800 nm, while in control MV showed 3-mode distribution within the range of 10-400 nm. Atomic force microscopy confirmed MV size heterogeneity with implication that larger objects represented aggregates of smaller microparticles. Patients' MV exhibited increased absolute values of zeta potential, indicating a higher surface charge. Tumour markers HER-2/neu, MAGE-1, c-MET and EMMPRIN were detected both in control and patients' samples with stronger expression in the latter. Significantly higher expression of MAGE-1 and HER-2/neu mRNA was observed in individual patients. All together, it suggests that at least some MV in plasma of gastric cancer patients are tumour-derived. However, their role in cancer requires further studies.

Fig. 1

The number of MV (mean ± SD) in a whole plasma of control and patients’ samples. b PFP of control and patients with different stages of gastric cancer. Significance in comparison to control; *P < 0.001

Fig. 2

FACS analysis of morphology and CD61 expression in different preparations of plasma. Acquisition parameters were set-up to exclude remaining platelets. a Events from plasma samples were gated into two populations according to FSC and SSC parameters (R1 and R2) (left dot plot) and analysed for CD61 expression. Data from R1 (middle) and R2 (right) are shown. b Plasma samples were centrifuged (15,000×g) and both supernatants and pellets were stained for CD61 expression. Dot-plots show CD61 staining in whole plasma (left dot plot), supernatant (middle) and pellet (right). Data from representative experiment are shown

Fig. 3

Membrane expression of HER-2/neu on MV of stage IV gastric cancer patients (n = 13) and control (n = 10); *P < 0.05

Fig. 4

Morphology and size of isolated MV from PFP. a Transmission electron microscopy of purified MV from plasma of gastric cancer patients (magnification 60,000×) and size distribution of MV in PFP from b control and c patients analysed by dynamic light scattering

Fig. 5

AFM analysis of MV. a Scan of MV taken at scan area 500 nm × 500 nm. b Marked three representative MV sizes detected by dynamic light scattering. c Scan of MV with quantified cross-section. d Complex structure of MV after cross-section

Fig. 6

Fold increase in a MAGE-1 and b HER-2/neu mRNA expression in isolated MV of patients with stage IV gastric cancer (n = 5) as compared to control subjects (n = 5); *P < 0.05