Safe and effective targeting of cancer with chimeric antigen receptor (CAR) engineered T cells relies on the presence of adequate tumor-associated surface antigens. We have found that Ewing sarcomas (EwS) express ganglioside GD2 and are recognized by T cells engineered with GD2-specific CARs. In an extended series of 97 EwS patient samples, 52 were GD2pos by immunohistochemistry. Based on reports that GD2 in breast cancer defines a malignant population with stem cell characteristics, we hypothesized that GD2 expression in EwS is associated with high capacity to self-renew and reinitiate tumor growth. Among 15 individual EwS cell lines with variable surface expression of GD2 by flow cytometry, GD2 expression levels were not associated with the capacities to proliferate and expand in vitro, form colonies in semi-solid medium, nor with their chemosensitivity, assessed by cell viabilities in the presence of increasing concentrations of the cytotoxic drug doxorubicin. Subpopulations with GD2hi phenotype selected from GD2pos and from GD2low EwS cell lines by cell sorting maintained their GD2hi and GD2low expression status during subsequent cultures over several weeks. The two subpopulations did not have different in vitro growth, colony-formation capacity, or chemosensitivity. In xenografting experiments, GD2hi and GD2low subpopulations of two EwS cell lines initiated tumors with comparable efficacies. To obtain direct evidence that GD2 surface expression is irrelevant for the biology of EwS cells, we performed genetic knockdown of the GD3S gene which drives biosynthesis of GD2 by CRISPR/Cas9 gene editing. GD3S gene editing resulted in effective elimination of GD2 surface expression in the GD2hi EwS cell lines TC-71, VH-64 and A4573. The knockdown did not affect the capacity of the cells to proliferate, form colonies in soft agar in vitro, nor their chemosensitivity compared to wild-type EwS cells from the individual cell lines. We conclude that GD2 expression in EwS cells, other than reported in breast cancer, is not associated with distinct functional features. Specifically, GD2 does not affect the growth characteristics, clono- and tumorigenicity and chemosensitivity of the tumor cells. Elimination of GD2pos subpopulations from heterogeneous tumors by CAR T cell targeting is therefore unlikely to eradicate the disease and will have to be combined with additional targeting strategies.
Citation Format: Sareetha Kailayangiri, Bianca Altvater, Silke Jamitzky, Stefanie Lesch, Jan-Henrik Mikesch, Sonja Schelhaas, Schaefers Michael, Jutta Meltzer, Nicole Farwick, Wolfgang Hartmann, Eva Wardelmann, Petra Fischer, Jendrik Hardes, Claudia Rossig. Expression of ganglioside GD2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4631.