N-Linked glycosylation profiles of therapeutic induced senescent (TIS) triple negative breast cancer cells (TNBC) and their extracellular vesicle (EV) progeny (2024)

N-Linked glycosylation profiles of therapeutic induced senescent (TIS) triple negative breast cancer cells (TNBC) and their extracellular vesicle (EV) progeny†

Emma L.Kavanagh,^a MelindaHalasz, ^bc PaulDowling, ^d JoWithers,^e SinéadLindsay,^a Michaela J.Higgins,^f Jane A.Irwin, ^g Pauline M.Rudd,^e RadkaSaldova ‡*^aeand AmandaMcCann ‡*^ab

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Triple negative breast cancer (TNBC) has poor clinical outcomes and limited treatment options. Chemotherapy, while killing some cancer cells, can result in therapeutic-induced-senescent (TIS) cells. Senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Recently, N- and O-linked glycosylation alterations have been associated with senescence. We aimed to profile the N-linked glycans of whole cells, membrane, cytoplasm and EVs harvested from TIS TNBC cells and to compare these to results from non-senescent cells. TIS was induced in the Cal51 TNBC cells using the chemotherapeutic agent paclitaxel (PTX). Ultra-performance liquid chromatography (UPLC) analysis of exoglycosidase digested N-linked glycans was carried out on TIS compared to non-treated control cells. LC-Mass spectrometry (MS) analysis of the N-linked glycans and lectin blotting of samples was carried out to confirm the UPLC results. Significant differences were found in the N-glycan profile of the Cal51 membrane, cytoplasm and EV progeny of TIS compared to non-senescent cells. Protein mass spectrometry showed that the TIS cells contain different glycan modifying enzymes. The lectin, calnexin demonstrated a lower kDa size (∼58 kDa) in TIS compared to control cells (∼90 kDa) while Galectin 3 demonstrated potential proteolytic cleavage with 32 kDa and ∼22 kDa bands evident in TIS compared to non-senescent control cells with a major 32 kDa band only. TIS CAL51 cells also demonstrated a reduced adhesion to collagen I compared to control non-senescent cells. This study has shown that therapeutic-induced-senescent TNBC cells and their EV progeny, display differential N-glycan moieties compared to non-senescent Cal51 cells and their resultant EV progeny. For the future, N-glycan moieties on cancer senescent cells and their EV progeny hold potential for (i) the monitoring of treatment response as a liquid biopsy, and (ii) cancer senescent cell targeting with lectin therapies.