Identifikation von Exosomen im malignen Pleuraerguss und Aszites gynäkologischer Patientinnen

Zusammenfassung

Fragestellung: Exosomen aus malignem Aszites und Pleuraerguss bieten möglicherweise eine natürliche und neue Quelle der Gewinnung von Exosomen und stellen somit eine Erfolg versprechende Alternative zu tumorzellbasierten Vakzinen dar. Exosomen sind Membranvesikel endosomalen Urprungs mit einer Größe von 60 - 100 nm und einer Dichte von 1,21 g/ml. Die Bildung dieser Membranvesikel durch Tumorzellen konnte in erster Linie in vitro gezeigt werden. Ziel unserer Untersuchung war der Nachweis von Exosomen in vivo im malignen Aszites/Pleuraerguss beim Mammakarzinom und gynäkologischen Tumoren. Methode: Maligner Aszites von gynäkologischen Tumoren und maligner Pleuraerguss beim Mammakarzinom wurde bei acht Patientinnen mittels differenzieller Zentrifugation in fünf Schritten angereichert, gefolgt von einer Dichtezentrifugation mit kontinuierlichem Sukrosegradienten in einer Ultrazentrifuge. Die Charakterisierung und Identifizierung der Exosomen erfolgte mittels SDS-PAGE mit Coomassie-blue-Proteinfärbung, Westernblot-Analyse und Elektronenmikroskopie. Ergebnisse: Exosomen konnten im malignen Pleuraerguss und malignen Aszites gynäkologischer Patientinnen elektronenmikroskopisch nachgewiesen werden. Schlussfolgerung: Wir berichten erstmalig über den Nachweis von Exosomen aus malignem Aszites beim Endometrium- und Pankreaskarzinom. Exosomen bieten möglicherweise eine natürliche und neue pluripotente Quelle für Tumorantigene und eröffnen somit einen neuen Weg der Immunstrategie.

Abstract

Purpose: Tumor derived exosomes isolated from malignant effusions could serve as a new source of tumor-rejection antigens, and thus may lead to a new way to tumor immunotherapy. Exosomes are membrane vesicles of 60 to 100 nm in diameter with a density of 1.21 g/ml, of endocytic origin, and are released in vitro by tumor cell lines in culture supernatants. In vivo and in vitro experiments suggest that exosomes are involved in the transfer of tumor antigens to antigen presenting cells. The aim of this study was the verification of the presence of exosomes in malignant effusions of gynecological cancer patients and their subsequent isolation and characterization. Methods: The isolation of exosomes was performed by ultracentrifugation on sucrose gradients of eight malignant effusions of patients with breast cancer, ovarian cancer, endometrial cancer and pancreatic cancer. The characterization of the exosomes was performed using SDS-PAGE with Coomassie blue staining, Western blot analysis and electron microscopy. Results: In malignant effusions of gynecological patients membrane vesicles with a diameter of 60 - 100 nm could be detected. These isolated membrane vesicles have antigen-presenting molecules (MHC class-I) on their surface. These criteria and the morphological parameters (electron microscopy) indicate the similarities between these vesicles and the exosomes. Conclusion: Exosomes accumulate in malignant effusions of patients with cancer. Importantly, we were able to identify exosomes from malignant effusions of patients with endometrial and pancreatic cancer, which has not been described heretofore. Further investigations using these exosomes have to be performed to analyze the induction of the tumor specifity of T cells when pulsed with dendritic cells.

Schlüsselwörter

Exosomen - Immuntherapie - Aszites - Pleuraerguss - Endometriumkarzinom - Pankreaskarzinom

Key words

exosomes - malignant effusions - immunotherapy - endometrial cancer - pancreatic cancer

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