Elsevier

The Lancet

Volume 360, Issue 9329, 27 July 2002, Pages 295-305
The Lancet

Mechanisms of Disease
Malignant effusions and immunogenic tumour-derived exosomes

https://doi.org/10.1016/S0140-6736(02)09552-1Get rights and content

Summary

Background

Exosomes derived from tumours are small vesicles released in vitro by tumour cell lines in culture supematants. To assess the role of these exosomes in vivo, we examined malignant effusions for their presence. We also investigated whether these exosomes could induce production of tumour-specific T cells when pulsed with dendritic cells.

Methods

We isolated exosomes by ultracentrifugation on sucrose and D2O gradients of 11 malignant effusions. We characterised exosomes with Western blot analyses, immunoelectron microscopy, and in-vitro stimulations of autologous T lymphocytes.

Findings

Malignant effusions accumulate high numbers of membrane vesicles that have a mean diameter of 80 nm (SD 30). These vesicles have antigen-presenting molecules (MHC class-I heat-shock proteins), tetraspanins (CD81), and tumour antigens (Her2/Neu, Mart1, TRP, gp100). These criteria, including their morphological characteristics, indicate the similarities between these vesicles and exosomes. Exosomes from patients with melanoma deliver Mart1 tumour antigens to dendritic cells derived from monocytes (MD-DCs) for cross presentation to clones of cytotoxic T lymphocytes specific to Mart1. In seven of nine patients with cancer, lymphocytes specific to the tumour could be efficiently expanded from peripheral blood cells by pulsing autologous MD-DCs with autologous ascitis exosomes. In one patient tested, we successfully expanded a restricted T-cell repertoire, which could not be recovered carcinomatosis nodules.

Interpretation

Exosomes derived from tumours accumulate in ascites from patients with cancer. Ascitis exosomes are a natural and new source of tumour-rejection antigens, opening up new avenues for immunisation against cancers.

Introduction

Results of previous work1 have shown that a protective immune response against non-immunogenic tumours could be generated in mice and that immunogenicity might not occur in a patient because the tumour cannot activate the immune system rather than because tumour antigens are absent. Tumour antigens are also present in non-immunogenic tumours in human beings, and generate an immune response when injected with adjuvants. Activated dendritic cells derived from monocytes (MD-DC) that are pulsed with tumour peptides have antitumour effects in patients with melanoma,2 prostate cancer,3 lymphoma,4 and renal cancer.5 However, immunisation strategies for ovarian or breast cancers have not been effective for several reasons. First, most tumour antigens have been identified in tumours that are immunogenic (eg, melanoma). Second, although many investigators have tried to identify tumour antigens, they have not been able to specify which tumour antigens are tumour rejection antigens—ie, those resulting in an immune response from cytotoxic T lymphocytes. Third, most reports about a source of relevant tumour antigens have dealt with antigens that were extracted or derived from dying tumour cells and that needed an ex-vivo step of tumour manipulation (eg, irradiated tumour cells,6 fused tumour cells,7 apoptotic bodies or necrotic debris,8 and extracted heat-shock protein9).

exosomes are released in vitro by many types of cells, including tumour cell lines10 and antigen presenting cells.11, 12 These membrane vesicles have a different pattern of proteins compared with plasma membranes and are enriched in molecules involved in antigen presentation (eg, MHC class I and II molecules, costimulatory and tetraspanin molecules, heat-shock protein chaperons) and potentially in cell targeting (CD11b, ICAM, tetraspanins, lactadherin). Exosomes derived from tumour cell lines that are loaded on dendritic cells transfer shared tumour antigens, thus stimulating T cells in an MHC class I dependent way. These T cells allow cross-protection against syngeneic and allogeneic tumours in mice.9 We aimed to isolate and characterise exosomes from malignant effusions, and assessed the immunogenicity of these exosomes.

Section snippets

Patients

We included patients if they presented with peritoneal or pleural carcinomatosis associated with ascitis or pleural effusion and had tumour cells in the biological fluid. All patients gave oral informed consent, and the internal review board of Institut Gustave Roussy, Villejuif, France, and the local ethical committee approved the protocol. Ascites were removed either at the first debulking operation or examination under anaesthetic, or under local anaesthesia because of symptoms. We excluded

Results

We assessed malignant effusions for presence of exosomes in three patients with papillary adenocarcinoma, one with clear-cell adenocarcinoma, two with adenocarcinoma, two with ductal adenocarcinoma, one with mesothelioma, and two with melanoma (table 1). As assessed by whole mount immunoelectronmicroscopy studies, the ultracentrifugation pellets of the material floating at a density of 1·14–1·18 g/mL in a 30% sucrose/D2O gradient contained many membrane vesicles (figure 2). 95% of these

Discussion

We were able to isolate numerous exosomes from tumours in vivo. Exosomes were originally described in supernatants of propagated ex-vivo cultures and were shown to be antigenic in vitro and immunogenic in mice with tumours.10, 11, 12 In our study, 11 patients had exosomes that could be purified from their ascitic fluid and that constituted a source of tumour antigens that could be transferred to MD-DCs. These MD-DCs then caused differentiation and expansion of tumour-specific cytotoxic T

GLOSSARY

apoptotic bodies
Apoptotic bodies are remnants of cells in the early stages of cell death induced by apoptosis. They express annexin V but do not stain with propidium iodide and are harvested by low-speed centrifugation in vitro.
chaperons
Chaperons, also called heat-shock proteins, are intracellular proteins that bind and transport peptides between subcellular compartments. Gp96 is a specific heat-shock protein that occurs in the endoplasmic reticulum but not late endosomes (ie, exosomes).
exosomes

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