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

神经节苷脂 SSEA-4 表面表达作为尤文肉瘤克隆形成、侵袭和转移特性的标志

• 批准号: 422762709
• 负责人: Dr. Sareetha Kailayangiri
• 金额: --
• 依托单位: Klinik für Kinder- und Jugendmedizin - Pädiatrische Hämatologie und Onkologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/422762709?language=en
• 关键词: Ganglioside; SSEA; surface; expression; marker

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.

转移性尤文肉瘤（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，其特征在于异常嵌合转录因子EWSR 1-FLI 1，染色体22和11的特异性染色体易位[t（11，22）（q24;12）]。我们正在开发通过表达细胞表面抗原来靶向EwS的细胞免疫疗法，使用嵌合抗原受体（CAR）重定向的免疫细胞。在寻找新的CAR T细胞靶点时，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，包括早期传代的患者来源的细胞培养物。免疫组织学分析显示SSEA-4在EwS患者的原发性肿瘤组织样品（24%）和转移性复发肿瘤组织样品（50%）中均有表达。我们观察到，在EwS细胞系中，SSEA-4高表达亚群的特征在于比缺乏SSEA-4表面表达的亚群具有更高的克隆形成性、更高的增殖、降低的化学敏感性和上级迁移能力。该基金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高表达细胞与SSEA-4阴性EwS细胞的自我更新、侵袭和体内致瘤性的能力。SSEA-4高和SSEA-4阴性EwS细胞的蛋白质组谱将确定EwS中SSEA-4表面表达是否是与侵袭性、迁移性和转移性相关的细胞表达特征的相关组分。此外，我们将分析SSEA-4的表达是否决定了EwS的特定恶性特征，通过CRISPR/Cas9基因编辑酶ST 3 β-半乳糖苷α-2，3-唾液酸转移酶2（ST 3Gal 2），这是负责SSEA-4合成的最后一步。最近报道，EWSR 1-FLI 1表达的细胞间异质性驱动与所建议的“亚稳态”表型一致的表型变化，这是一种有助于进展和侵袭性的复杂功能特征。该基金将研究EwS细胞中EWSR 1-FLI 1和SSEA-4表达之间的关系，并将澄清EwS细胞中SSEA-4的表达是否与EWSR 1-FLI 1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表面表达可以预防肺转移的假设。最终，我们的目标是开发一种有效的靶向治疗，例如通过CAR重靶向免疫效应细胞，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来治疗EwS。