Ganglioside SSEA-4 surface expression as a marker of clonogenic, invasive and metastatic properties in Ewing sarcoma

• Applicant: Dr. Sareetha Kailayangiri
• Subject Area: Pediatric and Adolescent Medicine
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 422762709

Metastatic Ewing sarcoma (EwS) is one of the major challenges in pediatric cancer. EwS are aggressive bone and soft tissue tumors characterized by the aberrant chimeric transcription factor EWSR1-FLI1, a specific chromosomal translocation of chromosomes 22 and 11 [t(11,22)(q24;12)]. We are developing cellular immunotherapies to target EwS by their expression of cell surface antigens, using chimeric antigen receptor (CAR)-redirected immune cells. On the search for a novel CAR T cell target, we have studied expression of the ganglioside stage-specific embryonic antigen-4 (SSEA-4). Indeed, we found SSEA-4 to be expressed in 14 out of 17 EwS cell lines, including early-passage patient-derived cell cultures. Immunohistological analysis showed an expression of SSEA-4 both in primary tumor tissue samples (24%) and in metastatic relapse tumor tissue samples (50%) from EwS patients. We observed, that SSEA-4high expressing subpopulations in EwS cell lines were characterized by higher clonogenicity, higher proliferation, reduced chemosensitivity and superior migratory capacities than subpopulations lacking SSEA-4 surface expression. This grant aims to investigate the role and relevance of ganglioside SSEA-4 in malignant growth and migratory/metastatic behavior in EwS. Under the hypothesis that SSEA-4 surface expression in EwS marks a functional state that is associated with self-renewing and invasive properties, we will compare the capacity for self-renewal, invasion and in vivo tumorigenicity of SSEA-4high expressing cells with SSEA-4negative EwS cells. The proteomic profile of either SSEA-4high and SSEA-4negative EwS cells will determine whether SSEA-4 surface expression in EwS is a relevant component of a cellular expression signature associated with invasive, migratory and metastatic properties. Moreover, we will analyse whether SSEA-4 expression determines specific malignant features in EwS by CRISPR/Cas9 gene-editing of the enzyme ST3 beta-galactoside alpha-2,3-sialyltransferase 2 (ST3Gal2), which is responsible for the last step in SSEA-4 synthesis. Cell-to-cell heterogeneity of EWSR1-FLI1 expression was recently reported to drive phenotypic changes that coincidence with a suggested “metastable” phenotype, a complex functional signature which contributes to progression and aggressiveness. This grant will investigate the relationship between EWSR1-FLI1 and SSEA-4 expression in EwS cells and will clarify whether SSEA-4 expression in EwS cells is directly related to EWSR1-FLI1 expression levels. Finally, we will investigate the relevance of SSEA-4 in the metastatic process of EwS in vivo and test the hypothesis that elimination of SSEA-4 surface expression can prevent pulmonary metastasis.Ultimately, our goal is to develop an effective targeted therapy, e.g. by CAR-retargeted immune effector cells, for EwS by eliminating a subpopulation of tumor cells that is responsible for aggressiveness, self-renewal and metastatic behavior.

• DFG Programme: Research Grants