Characterisation of prostate cancer sialome re-engineering via CMAH transfection reveals a bystander effect that propagates Neu5Gc presentation to neighbouring cells

Sialic acids are a family of nine-carbon -keto sugars that play essential roles in human health and disease. In mammals, they are found in two main forms: N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), with interactions between Neu5Ac-containing glycans and Siglec receptors on immune cells increasingly recognised as glyco-immune checkpoints, promoting immunosuppression. However, humans do not synthesise Neu5Gc due pseudogenisation of the CMAH gene which encodes the cytidine monophospho-N-acetylneuraminic acid hydroxylase enzyme responsible for CMP-Neu5Ac conversion into CMP-Neu5Gc, which then serves as the donor substrate for sialyltransferases. Here, we investigated the effects of re-engineering tumour cell-surface glycans in a prostate cancer cell model by expressing rat CMAH, thereby enabling the conversion of CMP-Neu5Ac to CMP-Neu5Gc. LNCaP cells transfected with the rat CMAH gene predominantly incorporated Neu5Gc into mucin-associated O-glycans implicated in immune suppression. Treatment with sialidase significantly reduced Neu5Gc expression, indicating that Neu5Gc was presented on cell-surface glycans, while cell-tracing experiments demonstrated the transfer of Neu5Gc to neighbouring cells, revealing a potential bystander effect capable of propagating Neu5Gc expression within the tumour microenvironment.