Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes

Abstract

This study was designed to determine the characteristics of tumour cell-derived microvesicles (TMV) and their interactions with human monocytes. TMV were shed spontaneously by three different human cancer cell lines but their release was significantly increased upon activation of the cells with phorbol 12-myristate 13-acetate (PMA). TMV showed the presence of several surface determinants of tumour cells, e.g. HLA class I, CD29, CD44v7/8, CD51, chemokine receptors (CCR6, CX3CR1), extracellular matrix metalloproteinase inducer (EMMPRIN), epithelial cell adhesion molecule (EpCAM), but their level of expression differed from that on cells they originated from. TMV also carried mRNA for growth factors: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8) and surface determinants (CD44H). TMV were localized at the monocytes surface following their short exposure to TMV, while at later times intracellularly. TMV transferred CCR6 and CD44v7/8 to monocytes, exerted antiapoptotic effect on monocytes and activated AKT kinase (Protein Kinase B). Thus, TMV interact with monocytes, alter their immunophenotype and biological activity. This implicates the novel mechanism by which tumour infiltrating macrophages may be affected by tumour cells not only by a direct cell to cell contact, soluble factors but also by TMV.

Fig. 1

Determination of TMV_HPC size. a Flow cytometry analysis: beads (1, 2.5 and 6 μm) were used as an internal standard. b Transmission electron microscopy of TMV released by HPC-4 cells activated by PMA

Fig. 2

Immunophenotypic characteristics of TMV_HPC. Histograms from representative experiments showing the expression of some studied determinants are presented. Dotted lines represent isotype control

Fig. 3

Expression of β actin (1), IL-8 (2), HGF (3) and VEGF (4)-mRNA by TMV_HPC detected by “real time” PCR. Negative control (5) for β actin is shown (all controls were of similar range). One representative experiment of three performed is shown

Fig. 4

Transfer of PKH26 labelled TMV_HPC to monocytes. a Confocal microscopy of monocytes exposed to TMV_HPC for 30 min, 1 h and 24 h. b Flow cytometry of monocytes exposed to TMV_HPC for 1 h and 24 h in the absence and presence of crystal violet. c Monocytes were incubated either in the medium (dotted line) or with TMV (30 μg/ml) (bold line)

Fig. 5

Expression of CCR6 and CD44v7/8 by monocytes (bold lines) following their incubation for 2 h either in the medium or with TMV_HPC (30 μg/ml). Dotted lines represent isotype control. The representative experiment of three performed is shown

Fig. 6

Chemotaxis of monocytes preincubated in the medium (a) or with TMV_HPC (b) to MIP-3α (100 ng/ml). Data are expressed as the percentage of the cells input corrected by the percentage of cells, which migrated spontaneously to the medium alone. The representative experiment of four performed is shown

Fig. 7

The effect of TMV on monocytes survival defined by MTS assay. Monocytes were cultured in serum free medium for 48 h in the presence or absence of TMV (30 μg/ml). Mean (OD at 490 nm) ± SD of six independent experiments is shown. * p<0.05; ** p<0.005

Fig. 8

The effect of TMV_HPC on spontaneous apoptosis of monocytes as measured by annexin-V binding (a) or the presence of active form of caspase-3 (b). Monocytes were incubated for 3 h or 24 h in the serum free medium (dotted line) or with TMV_HPC (bold line). Representative experiment of three performed is shown. TMV_HPC–treated monocytes at 24 h show less annexin V binding and lower expression of caspase-3

Fig. 9

Phosphorylation of AKT in unstimulated monocytes (line 1) or monocytes stimulated for 30 min with TMV_HPC (line 2), TMV_DeTa (line 3) and TMV_A549 (line 4)