Isolation of exosomes from breast cancer cells and its role in the interaction with Tumor Associated Macrophages (TAMs).

MARÍA C. RODRIGUEZ-BAILI; GERMAN GIL

Buenos Aires

Congreso; SAIB; 2017

SAIB SAIC etc

Worldwide, breast cancer is the most frequently diagnosed malignant cancer and the main cause of mortality in women. Despite the clinical benefits that the endocrine therapies offer to breast cancer patients, a significant rate develops resistance to those therapies. It is known that tumor associated macrophages (TAMs) promote tumor growth; however it is unknown if they play a key role in the development of endocrine resistance. Lately, there has been a greater interest in the nanovesicles called exosomes that are secreted by almost all cellular lines. These exosomes play an important role in cellular communication, growth of the microenvironment and tumor progression since it would allow the transmission of proteins, miRNA, RNA between cells; yet their mechanism and biology are still unclear. Here, we study different isolation techniques of the exosomes of the MCF-7 breast cancer cell-line and macrophagues KG-1 trying to understand if TAMs induce endocrine resistance in vitro. We used two different methods: ultracentrifugation and the commercial reagent ?Isolation Exosomes Reagent (Invitrogen)?. MCF-7 cells and macrophagues were cultured until reached 70-80% of confluency and then, medium depleted of SFB exosomes was added and cultured for 48 h. Following the protocols of both methods, the supernatant was collected and the intact exosomes were obtained. To confirm the presence of the exosomes, we performed Electron Microscopy, and concordant images were obtained as previously described in bibliography. Additionally, the exosomes were detected by Western Blot with anti CD63 (a tetraspanin used for the detection of nanovesicles). Moreover, it was not observed the presence of Golgi markers, assuming that there is not contamination with other cellular vesicles. Exosomes of MCF-7 cells and macrophagues were successfully isolated by both methods and we found that the commercial reagent is simpler, faster and its results are as good as one obtained by ultracentrifugation.