Tumour exosomes inhibit binding of tumour-reactive antibodies to tumour cells and reduce ADCC

Abstract

In order to grow within an immunocompetent host, tumour cells have evolved various strategies to cope with the host's immune system. These strategies include the downregulation of surface molecules and the secretion of immunosuppressive factors like IL-10 and PGE2 that impair the maturation of immune effector cells, among other mechanisms. Recently, tumour exosomes (TEX) have also been implicated in tumour-induced immune suppression as it has been shown that TEX can induce apoptosis in T lymphocytes. In this study, we extend our knowledge about immunosuppressive features of these microvesicles in that we show that TEX efficiently bind and sequester tumour-reactive antibodies and dramatically reduce their binding to tumour cells. Moreover, we demonstrate that this antibody sequestration reduces the antibody-dependent cytotoxicity by immune effector cells, which is among the most important anti-tumour reactions of the immune system and a significant activity of therapeutic antibodies. Taken together, these data point to the fact that tumour-derived exosomes interfere with the tumour-specific function of immune cells and constitute an additional mechanism how tumours escape from immune surveillance.

Fig. 1

Tumour exosomes isolated from conditioned cell culture supernatants carry HER2 and EpCAM. a Exosomes can be fractionated by sucrose gradient centrifugation and identified with cholera toxin that specifically binds to GM1. b Material isolated from fractions 3 and 4 carry the exosome markers GM1 and TSG101 as proven by dot blots. Exosomes from the breast cancer cell line BT474 also carry the tumour antigens EpCAM and HER2 whereas Mel624.38 exosomes do not. c Immune electron microscopy revealed that HER2 is associated with particles isolated from fractions 3 and 4. d FACS analysis of BT474 exosomes coupled to latex beads showed that trastuzumab binds to TEX-associated HER2 efficiently

Fig. 2

Pre-incubation with HER2+EpCAM+ TEX prevents binding of trastuzumab and C215 to HER2+EpCAM+ cancer cells. a trastuzumab and b C215 were titrated on BT474 cells to identify the antibody concentration that gives half-maximal staining. Trastuzumab c and e or C215 d and f at concentrations of 50 ng/ml were pre-incubated with 100 μg/ml HER2+EpCAM+ BT474 exosomes (c and d) or with the same amount of HER2−EpCAM− Mel624.38 exosomes (e and f). As a positive control, cells were stained with antibodies that were not pre-incubated with TEX. It became clear that pre-incubation with BT474 exosomes but not with Mel624.38 exosomes prevented trastuzumab and C215 from binding to BT474 cells. Grey histogram unstained cells. Bold line Maximal staining of trastuzumab and C215 on BT474 cells. Thin line staining of BT474 cells with pre-incubated trastuzumab. One out of the seven independent experiments is shown

Fig. 3

Sera from breast cancer patients contain particle-associated HER2. a HER2 levels present in the exosome preparations were quantified with an commercial ELISA assay in comparison with whole sera. It became clear that a part of total HER2 could be isolated by ultracentrifugation applying the protocol for the isolation of exosomes. b Average HER2 levels from the 12 patients shown in A as measured in complete serum and precipitated material. c Fractions ranging from 23 to 37% of total HER2 were precipitated from the high-HER2 sera, implicating that TEX-associated HER2 contributes by roughly one-third to the total HER2 serum levels in patients. Fractions were not calculated for low-HER2 sera that were out of the linear range of the assay

Fig. 4

Primary tumour-derived exosomes prevent binding of tumour-reactive antibodies. a and b Exosomes were isolated from the malignant ascites of two patients with ovarian cancer. C215 (left histograms) or of trastuzumab (right histograms) at 50 ng/ml each were pre-incubated with ascites exosomes (200 μg each) and used to stain BT474 cancer cells. It became clear that exosomes from malignant ascites efficiently prevented binding of the two antibodies. Grey histogram unstained BT474 cells. Bold line Maximal staining of trastuzumab on BT474 cells. Thin line staining of BT474 cells with pre-incubated trastuzumab

Fig. 5

Tumour-derived exosomes prevent ADCC initiated by trastuzumab. a Trastuzumab-promoted ADCC with PBMCs from three out of the four different donors was significantly impaired after pre-incubation of the antibody with HER+ exosomes from BT474 and SkBr3 cells when compared to pre-incubation with HER2− exosomes from MEL624.38 cells or to untreated antibody. b Titration of effector cells and BT474 exosomes demonstrated that the inhibition of trastuzumab-initiated ADCC is dose dependent. Data with PBMCs from three different donors are shown