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

转移性尤因肉瘤（EwS）是儿科癌症的主要挑战之一。EwS是侵袭性骨和软组织肿瘤，其特征是异常嵌合转录因子EWSR1-FLI1，这是染色体22和11的特异性染色体易位[t(11,22)(q24;12)]。我们正在开发细胞免疫疗法，利用嵌合抗原受体（CAR）重定向免疫细胞，通过表达细胞表面抗原来靶向EwS。为了寻找一种新的CAR - T细胞靶点，我们研究了神经节苷脂阶段特异性胚胎抗原-4 （SSEA-4）的表达。事实上，我们发现SSEA-4在17个EwS细胞系中的14个中表达，包括早期传代患者来源的细胞培养物。免疫组织学分析显示，SSEA-4在EwS患者的原发肿瘤组织样本（24%）和转移复发肿瘤组织样本（50%）中均有表达。我们观察到，与缺乏SSEA-4表面表达的亚群相比，EwS细胞系中SSEA-4高表达亚群具有更高的克隆原性、更高的增殖能力、更低的化学敏感性和更强的迁移能力。该资助旨在研究神经节苷脂SSEA-4在EwS恶性生长和迁移/转移行为中的作用和相关性。假设SSEA-4在EwS中的表面表达标志着一种与自我更新和侵袭特性相关的功能状态，我们将比较SSEA-4高表达细胞和SSEA-4阴性EwS细胞的自我更新能力、侵袭能力和体内致瘤性。SSEA-4高和SSEA-4阴性EwS细胞的蛋白质组学特征将确定SSEA-4表面表达在EwS中是否是与侵袭、迁移和转移特性相关的细胞表达特征的相关组成部分。此外，我们将通过CRISPR/Cas9基因编辑负责SSEA-4合成最后一步的ST3 β -半乳糖苷α -2,3-唾液基转移酶2 (ST3Gal2)，分析SSEA-4的表达是否决定了EwS中特定的恶性特征。最近有报道称，细胞间EWSR1-FLI1表达的异质性驱动表型变化，这种变化与“亚稳态”表型一致，这是一种复杂的功能特征，有助于进展和侵袭性。本课题将研究EWSR1-FLI1与SSEA-4在EwS细胞中的表达关系，并阐明SSEA-4在EwS细胞中的表达是否与EWSR1-FLI1的表达水平直接相关。最后，我们将在体内研究SSEA-4在EwS转移过程中的相关性，并验证消除SSEA-4表面表达可以阻止肺转移的假设。最终，我们的目标是通过消除负责侵袭性、自我更新和转移行为的肿瘤细胞亚群，开发一种有效的靶向治疗方法，例如通过car -重靶向免疫效应细胞来治疗EwS。

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.