(158f) ?2,3 Sialylated Colon and Breast Cancer Cell Extracellular Vesicles Bind to L-Selectin Under Flow Conditions

Extracellular vesicles (EVs) are naturally occurring vesicles released by all cells, including cancer cells. EVs are involved in normal cellular processes such as intercellular communication, but EVs released from cancer cells and their role in cancer metastasis need further understanding. In particular, it is unclear whether EVs released from cancer cells express sialofucosylated glycans that can function as L-selectin ligands, similar to the L-selectin ligands on the cancer cells from which they were derived. Therefore, we hypothesize that breast and colon cancer cells and model EVs isolated from these cancer cells express α2,3 sialylated L-selectin ligands that interact with L-selectin under flow conditions. Giant plasma membrane vesicles (GPMVs, 5-20 µm diameter vesicles) were isolated from cancer cells as a model of naturally occurring large cancer EVs (i.e., large oncosomes). GPMVs were generated from the LS174T colon cancer cell line and BT-20 and SK-Br-3 breast cancer cell lines using chemical vesiculation agents (25 mM formaldehyde and 2 mM dithiothreitol). The parallel plate flow chamber adhesion assay was used to functionally assess LS174T colon cancer cells, BT-20 and SK-BR-3 breast cancer cells, and GPMVs isolated from these three cancer cell lines that were incubated with either V. cholera sialidase (VC, broad substrate specificity), C. perfringens sialidase (CP, specificity of cleavage: α2,3 > α2,6 sialylation), or left untreated. Treatment of cancer cells and GPMVs with VC and CP sialidase significantly reduced interactions (>50%) with L-selectin substrates (L-selectin hFc coated plates) at physiologically relevant wall shear stresses compared to the untreated cancer cells and GPMVs. In addition, flow cytometry was performed on cancer cells and GPMVs treated with VC or CP sialidase or left untreated to determine the expression of candidate L-selectin ligands. All three cell lines and their isolated GPMVs tested positive for cutaneous lymphocyte antigen (HECA-452 antigen) and sialyl Lewis X (sLeX, CSLEX1 mAb), with VC and CP treatment reducing expression of HECA-452 antigen and sLeX greater than 90% as measured by mean fluorescent intensity levels. These results indicate similar L-selectin ligands are expressed on cell lines and GPMVs. Furthermore, GPMVs interacted with human peripheral blood polymorphonucleocytes and mononuclear cells in an L-selectin-dependent manner, similar to the original cell lines. Altogether, these results indicate that LS174T colon cancer cells, BT-20 and SK-BR-3 breast cancer cells, and GPMVs isolated from these cancer cells express α2,3 sialylated L-selectin ligands that interact with L-selectin under flow conditions. Understanding how EVs released from cancer cells interact with cell adhesion molecules such as L-selectin may help elucidate the role of intercellular adhesion in metastasis, leading to the development of novel diagnostics, prognostics, and therapeutics.