Exosome mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231 relies on ITGB3

Introduction: Tumors are complex systems where cell-cell communication between intra-tumoral and extra-tumoral cells play a crucial role. Extracellular vesicles (EVs), membrane-limited vesicles secreted by normal and malignant cells, have been proposed to act as mediators of intercellular communication in physiological and pathological scenarios. It is now well established that integrins are constantly endocytosed and recycled back to the plasma membrane through multiple routes. Accumulating evidence clearly indicates that endocytosis and recycling of integrins plays a crucial role during cancer progression, invasion, and metastasis. Here we describe a fundamental link between endocytosis mediated integrin trafficking and the integrin-mediated uptake of exosomes. Objectives: As the integrins are crucial for the role of exosomes in cancer progression and particularly in metastasis we were aiming at delineating the role of ITGB3 in exosome-mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231. Methodology: Using stable shRNA of ITGB3 (shITGB3) in the triple-negative breast cancer cell line MDA.MB.231, EVs were isolated by ultracentrifugation and characterized by Western blot analysis to determine the protein composition and by NTA and CryoEM to measure the amount of EVs. An LC-MS/MS-based survey was performed, to determine the differences in the protein composition of EVs after knock-down of ITGB3. Uptake of fluorescently labeled exosomes into recipient cells was measured by flow cytometry. The colony forming capacity induced by conditioned medium was used to assess the functional impact on the role of ITGB3 in intercellular communication. Results: Analysis of the EVs fraction of shITGB3 cell revealed a striking decrease of classical markers of exosomes as judged by western blot analysis of proteins such as TSG101, CD81, but not others like Flotillin-1. LC-MS/MS analysis of shITGB3-MDA.MB.231 derived-exosomes furthermore revealed clear defects in protein complexes required for exosome formation, namely the ESCRT complex and the Syndecan-syntenin-ALIX complex. Both complexes are required for the formation of endosomal intraluminal vesicles that get released as exosomes. On the other hand, NTA analysis and CryoEM showed the overall amount of EVs was increased. In line with this, the uptake of fluorescently labeled EVs was strongly impaired in shITGB3 cells. By the use of small molecule inhibitors, we can furthermore demonstrate that this defect linked to endocytosis of EVs. Conclusions: Here we demonstrate that ITGB3 plays a crucial role in controlling the composition of different vesicle populations in the extracellular space, linked to the endocytosis of integrins and interference with this mechanism results in defects in the uptake of EVs and impedes the generation of exosomes. The functional effects are discussed Citation Format: Pedro Fuentes Varela, Marta Sese, Pedro Jesus Guijarro, Marta Emperador, Hector Peinado, Stefan Hummer, Santiago Ramon y Cajal. Exosome mediated intercellular communication in the triple negative breast cancer cell line MB.MDA.231 relies on ITGB3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1897.