Role of extracellular vesicles in endocrine resistance of breast cancer
Celeste Rodriguez, Universidad Nacional de Cordoba, Argentina
The general objective of this project is to clarify the responsible mechanism of the intercommunication between tumoral cells and macrophages by EVs that would lead to endocrine resistance. If we were able to find the specific cardo of the vesicles coming from breast cancers that led macrophages to a Tumor-Associated Macrophages (TAM) profile that can benefit the tumor; and if we found a way to alter this uptake or their release, we could be closer to clarify the intercommunication between these cell types and the endocrine resistance we have already demonstrated in previous work.
- To isolate EVs coming from ER+ breast cancer and macrophages by Size Exclusion Chromatography (SEC)
- To test the uptake capability of Extracellular Vesicles by recipient cells in vitro
- To identify receptor or ligands molecules that could mediate intercommunication between cells by adhesion assays
Extracellular vesicles (EVs) coming from breast cancer cell lines positive for Estrogen Preceptor (ER+) and macrophages conditioned with Tumor Necrosis Factor (TNF) were successfully isolated by Tangential Flow Filtration and spin filtration followed by Size Exclusion Chromatography. We collected the EVs fractions and by Nanoparticle tracking analysis (NTA) we determined the amount of particles/mL that were used in the functional assays.
The transfection of the MCF-7 cells with the plasmid that contains CD9-EGFP-Renilla luciferase did not work as expected. We tried to obtain individual cones, but after a few passages the cells lost the plasmid. Since we wanted to observe the uptake of the EVs by our different cells, we changed the strategy and used Alexa C5 maleimide as an alternative to fluorescent lipophilic dyes, since it does not aggregate and does not diffuse between lipid membranes. We observed by flow cytometry that MCF7 cells uptake the EVs stained coming from macrophages conditioned with TNF after 2 hours of incubation. On the other hand, macrophages that were incubated with EVs from MCF7 cells uptake efficiently these EVs, and the M1 and M2 uptake them more efficiently than M0.
We have had a few difficulties in the experiment of macrophages differentiation since the antibodies for western blot and flow cytometry did not work.
In order to assess if EVs could be implicated in endocrine resistance, we treated MCF7 cells with different concentration of Tamoxifen and incubate with EVs coming from macrophages conditioned with TNF, we observed a significantly increase in the proliferation of breast cancer cells cultivated in presence of EVs after 24 hours. Also, we were able to test the ability of TNF macrophages EVs to support the adhesion of MCF-7 cells and we found that β-integrin and PD-L1 are involved in the interactions between cells and EVs.
Grant Awarded: £2,600.00