Abstract 2626: Exosomes derived from the BT-20 human breast cancer cell line express E-selectin ligands

Exosomes are small extracellular vesicles (40-150 nm in diameter) secreted by cells and initially proposed as a mechanism to eliminate cellular waste. More recently, exosomes have been reported to participate in cancer metastasis because of their ability to mediate local and distant cell-to-cell communication. Furthermore, exosomes create cancer-favored microenvironments, potentially enhancing cancer metastasis. However, it is unknown whether exosomes interact with E-selectin, an adhesion molecule expressed by vascular endothelium and shown to be involved in metastasis. Our lab has previously shown that BT-20 human breast cancer cells adhesively interact with endothelial E-selectin via several E-selectin ligands such as sialofucosylated CD44 variants, Mac-2BP, and gangliosides. Therefore, we hypothesize that exosomes secreted by the BT-20 human breast cancer cell line interact with E-selectin. To investigate this hypothesis, exosomes were generated by culturing cells in serum-depleted media and isolated by a series of ultracentrifugation steps. Molecular size analysis using dynamic light scattering revealed that the size range of the isolated particles was similar to the expected size of exosomes. To detect CD9 (an exosome marker) and E-selectin ligands, both breast cancer whole cell lysate and exosome lysate were tested by Western blot. The higher expression of CD9 in isolated particles than the whole cell lysates confirmed that the isolated particles were exosomes. When Western blots were labeled with recombinant E-selectin/Fc chimera with Ca2+, bands were detected at approximately 130 kDa, indicating the presence of E-selectin ligands. As a negative control, the divalent cation chelator EDTA was added to the primary labeling incubation. Bands were not detected in these negative control experiments, indicating no E-selectin interactions in the absence of Ca2+. Human IgG (hIgG) was also used as a negative control in Western blots and showed no signal with exosome lysates, as expected. In comparison, experiments were also performed with Hs578T breast cancer cells, a cell line that our lab has shown does not express E-selectin ligands. Exosomes isolated from Hs578T cells did not express E-selectin ligands, consistent with whole cells. Altogether, these results suggest that BT-20 human breast cancer exosomes express E-selectin ligands. Future work will focus on identifying and characterizing the exosome E-selectin ligands, as well as investigating other canc