Surface expression of the immunotherapeutic target G(D2) in osteosarcoma depends on cell confluency

BACKGROUND: Chimeric antigen receptor (CAR) T‐cell therapy of pediatric sarcomas is challenged by the paucity of targetable cell surface antigens. A candidate target in osteosarcoma (OS) is the ganglioside G(D2), but heterogeneous expression of G(D2) limits its value. AIM: We aimed to identify mechanisms that upregulate G(D2) target expression in OS. METHODS AND RESULTS: G(D2) surface expression in OS cells, studied by flow cytometry, was found to vary both among and within individual OS cell lines. Pharmacological approaches, including inhibition of the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) and modulation of the protein kinase C, failed to increase G(D2) expression. Instead, cell confluency was found to be associated with higher G(D2) expression levels both in monolayer cultures and in tumor spheroids. The sensitivity of OS cells to targeting by G(D2)‐specific CAR T cells was compared in an in vitro cytotoxicity assay. Higher cell confluencies enhanced the sensitivity of OS cells to G(D2)‐antigen specific, CAR T‐cell‐mediated in vitro cytolysis. Mechanistic studies revealed that confluency‐dependent upregulation of G(D2) expression in OS cells is mediated by increased de novo biosynthesis, through a yet unknown mechanism. CONCLUSION: Expression of G(D2) in OS cell lines is highly variable and associated with increasing cell confluency in vitro. Strategies for selective upregulation of GD2 are needed to enable effective therapeutic targeting of this antigen in OS.