Exosomes are small vesicles which are produced by the cells and released into the surrounding space. They can transfer biomolecules into recipient cells. The main goal of the work was to study the exosome involvement in the cell transfer of hormonal resistance. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells and MCF-7 sublines resistant to SERM tamoxifen and/or biguanide metformin, which exerts its anti-proliferative effect, at least in a part, via the suppression of estrogen machinery. The exosomes were purified by differential ultracentrifugation, cell response to tamoxifen was determined by MTT test, and the level and activity of signaling proteins were determined by Western blot and reporter analysis. We found that the treatment of the parent MCF-7 cells with exosomes from the resistant cells within 14 days lead to the partial resistance of the MCF-7 cells to antiestrogen drugs. The primary resistant cells and the cells with the exosome-induced resistance were characterized with these common features: decrease in ERα activity and parallel activation of Akt and AP-1, NF-κB, and SNAIL1 transcriptional factors. In general, we evaluate the established results as the evidence of the possible exosome involvement in the transferring of the hormone/metformin resistance in breast cancer cells.

Background: Extracellular vesicles are small membrane particles which are produced by the cells and released into the surrounding space. They can transfer biomolecules into recipient cells. The typical vesicle size is 100-1000 nm, so transmission electron microscopy (TEM) is the most suitable tool for their visualization. This is especially true for the smallest vesiclesthe exosomes with the size below 200 nm. Methods: We isolated exosomes produced by MCF-7 cancer cells and the two drug-resistant MCF-7 sublines (Semina et.al., Molecules, 2018). The exosomes were isolated from the conditioned medium by ultracentrifugation, and their size was measured by nanotracking analysis (NTA) and TEM. Immunogold labelling was carried out on exosomes deposited onto carbon TEM grids. It involved the following principal steps: the vesicle deposition, sample treatment by the anti-CD9 antibodies, sample treatment by the 10 nm colloidal gold nanoparticles conjugated with protein A, staining with uranyl acetate. Washing and blocking steps were done between the principal ones. The samples were imaged using a JEM-1011 microscope at 80 kV. Fig. 1. Exosomes produced by the MCF-7 cells, labelled with colloidal gold. Results: NTA and TEM yielded similar resultsthe exosomes produced by the three studied cell lines had the almost equal size in the range from 50 to 200 nm. When imaged by TEM, most exosomes demonstrated the typical cup-shaped morphology. We used immunogold labelling to verify that the exosomes carried the CD9 marker on them (Fig. 1). We achieved the labelling specificity in the range of 8-10; this value was calculated as the ratio of the surface densities of the exosome-bound labels (colloidal gold nanoparticles) and the non-specific background ones. Conclusion: We hope that the sample preparation and imaging procedures that we used could be useful for the investigation of other extracellular vesicles.