ISOLATION AND CHARACTERIZATION OF EXTRACELLULAR VESICLES COMING FROM BREAST CANCER CELLS AND MACROPHAGES

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

Salta

Congreso; SAIB PABMB; 2019

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 performed two different isolation techniques of EVs, coming from breast cancer cells, and 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 24h, 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, we revealed by Western blotting anti-tetraspanins antibodies, (proteins widely used in literature) and with others 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 estimate 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.