Isolation and characterization of extracellular vesicles coming from breast cancer and macrophages.

CELESTE RODRIGUEZ BAILI; SABRINA DE LOURDES DHOOGE; CLOTILDE THÉRY; GERMÁN ALEJANDRO GIL

SALTA

Congreso; ? LV Annual - XIV PABMB.; 2019

SAIB-PABMB.

Extracellular vesicles (EVs) are lipid bilayer-enclosed extracellular structures secreted by almost all cellular lines with the capacity of transfer their cargo, playing an important role in cellular communication, growth of microenvironment and tumor progression, generating a substantial interest in the scientific community. Worldwide, breast cancer is the most frequently diagnosed malignant cancer and the main cause of mortality in women. It is known that tumor associated macrophages (TAMs) promote tumor growth; however, it is unknown how this communication is occurred, and we proposed that extracellular vesicles coming from both cellular lines are involved. We perform two different isolation techniques of Evs, coming from breast cancer cells, and from macrophages cell line. EVs were isolated by ultracentrifugation and ultrafiltration following ultracentrifugation; in both cases five million cells were seeded per plate and cultured for 24 h, after that complete medium is removed, and changed for fetal bovine serum (FBS) free medium for 24 h. Following the protocols of both methods, medium was collected and intact EVs recovered and resuspended in PBS. For the EVs characterization, were revealed by Western blotting using anti-tetraspanins Abs, a protein widely used in literature and also with other proteins of our interest. Moreover, we observed the absence of Golgi markers, assuming that there is not contamination with other vesicles. The presence of total proteins and DNA in Evs? were detected using bicinchoninic acid assay (BCA assay) and qPCR respectively. We also performed Nanotracker analysis (analysis of particles) to estimating the total amount of vesicles in each fraction. Isolation and characterization of EVs is the first step to analyze the content of the vesicles in our cells of interest for further functional assays that will allow us to determinate if they are involved in the communication between TAMs and tumoral cells, and if EVs are responsible of the endocrine resistance that some estrogen receptor positive cancers acquired with endocrine treatments.