| Abstract |
Urothelial carcinoma (UC) often exhibits a “cold” tumor microenvironment with limited immune cell infiltration, leading to resistance to immune checkpoint inhibitors. Neutral sphingomyelinase (nSMase) is a key regulator of extracellular vesicle (EV) biogenesis, and pharmacological inhibition of nSMase using GW4869 has been widely used to modulate EV biogenesis. However, its impact on EV dynamics and anti-tumor immunity remains unclear. Using the syngeneic murine UC cell line, MBT-2, we assessed the effects of GW4869 on gene expression and protein secretion using RNA sequencing and enzyme-linked immunosorbent assays. Natural killer (NK) cell function was evaluated using lactate dehydrogenase cytotoxicity assays and CD107a degranulation analysis, and public datasets were analyzed to examine the clinical relevance of C-X-C motif chemokine ligand 10 (CXCL10) expression in human UC. GW4869 treatment was associated with altered EV dynamics, characterized by increased particle yields and changes in EV size distribution. Functional assays demonstrated that GW4869-treated tumor cells enhanced NK cell-mediated cytotoxicity and degranulation, and this anti-tumor effect was augmented by programmed cell death ligand 1 (PD-L1) blockade. These effects were attenuated by C-X-C motif chemokine receptor 3 (CXCR3) neutralization, suggesting the involvement of the CXCL10–CXCR3 axis. Clinically, high CXCL10 expression was associated with favorable survival outcomes and positively correlated with NK cell-associated markers in patients with UC. In conclusion, pharmacological nSMase inhibition modulates EV and secretome dynamics and promotes NK cell activation associated with the CXCL10–CXCR3 axis. These findings support further investigation of nSMase-targeted strategies for enhancing anti-tumor immunity in UC.
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