Alefeld Emily, Haase André, Van Meenen Dario, Budeus Bettina, Dräger Oliver, Miroschnikov Natalia, Ting Saskia, Kanber Deniz, Biewald Eva, Bechrakis Nikolaos, Dünker Nicole, Busch Maike Anna
Institute for Anatomy II, Department of Neuroanatomy, Center for Translational Neuro- and Behavioral Sciences (C-TNBS), University of Duisburg-Essen, Medical Faculty, Essen, Germany.
Institute for Cell Biology, University Hospital Essen, Essen, Germany.
Cell Death Dis. 2024 Dec 18;15(12):905. doi: 10.1038/s41419-024-07285-2.
Retinoblastoma (RB) is an intraocular tumor arising from retinal cone progenitor cells affecting young children. In the last couple of years, RB treatment evolved towards eye preserving therapies. Therefore, investigating intratumoral differences and the RB tumor microenvironment (TME), regulating tumorigenesis and metastasis, is crucial. How RB cells and their TME are involved in tumor development needs to be elucidated using in vitro models including RB derived stromal cells. In the study presented, we established primary RB derived tumor and stromal cell cultures and compared them by RNAseq analysis to identify their gene expression signatures. RB tumor cells cultivated in serum containing medium were more differentiated compared to RB tumor cells grown in serum-free medium displaying a stem cell like phenotype. In addition, we identified differentially expressed genes for RB tumor and stromal derived cells. Furthermore, we immortalized cells of a RB1 mutated, MYCN amplified and trefoil factor family peptid 1 (TFF1) positive RB tumor and RB derived non-tumor stromal tissue. We characterized both immortalized cell lines using a human oncology proteome array, immunofluorescence staining of different markers and in vitro cell growth analyses. Tumor formation of the immortalized RB tumor cell line was investigated in a chicken chorioallantoic membrane (CAM) model. Our studies revealed that the RB stromal derived cell line comprises tumor associated macrophages (TAMs), glia and cancer associated fibroblasts (CAFs), we were able to successfully separate via magnetic cell separation (MACS). For co-cultivation studies, we established a 3D spheroid model with RB tumor and RB derived stromal cells. In summary, we established an in vitro model system to investigate the interaction of RB tumor cells with their TME. Our findings contribute to a better understanding of the relationship between RB tumor malignancy and its TME and will facilitate the development of effective treatment options for eye preserving therapies.
视网膜母细胞瘤（RB）是一种起源于视网膜锥体细胞祖细胞的眼内肿瘤，主要影响幼儿。在过去几年中，RB的治疗逐渐向保眼疗法发展。因此，研究肿瘤内差异以及调节肿瘤发生和转移的RB肿瘤微环境（TME）至关重要。需要使用包括RB衍生基质细胞在内的体外模型来阐明RB细胞及其TME如何参与肿瘤发展。在本研究中，我们建立了原发性RB衍生的肿瘤和基质细胞培养物，并通过RNA测序分析对它们进行比较，以确定它们的基因表达特征。与在无血清培养基中生长的具有干细胞样表型的RB肿瘤细胞相比，在含血清培养基中培养的RB肿瘤细胞分化程度更高。此外，我们确定了RB肿瘤和基质衍生细胞的差异表达基因。此外，我们使一株RB1突变、MYCN扩增且三叶因子家族肽1（TFF1）阳性的RB肿瘤细胞以及RB衍生的非肿瘤基质组织的细胞永生化。我们使用人类肿瘤蛋白质组阵列、不同标志物的免疫荧光染色和体外细胞生长分析对这两种永生化细胞系进行了表征。在鸡胚绒毛尿囊膜（CAM）模型中研究了永生化RB肿瘤细胞系的肿瘤形成。我们的研究表明，RB基质衍生细胞系包含肿瘤相关巨噬细胞（TAM）、神经胶质细胞和癌症相关成纤维细胞（CAF），我们能够通过磁性细胞分选（MACS）成功分离它们。对于共培养研究，我们建立了一个包含RB肿瘤细胞和RB衍生基质细胞的3D球体模型。总之，我们建立了一个体外模型系统来研究RB肿瘤细胞与其TME之间的相互作用。我们的研究结果有助于更好地理解RB肿瘤恶性程度与其TME之间的关系，并将促进保眼疗法有效治疗方案的开发。
