BackgroundRetinoblastoma (Rb), the most common childhood intraocular malignant tumor, is reported to have cancer stem cells (CSCs) similar to other tumors. Our previous investigation in primary tumors identified the small sized cells with low CD133 (Prominin-1) and high CD44 (Hyaluronic acid receptor) expression to be putative Rb CSCs using flow cytometry (FSClo/SSClo/CD133lo/CD44hi). With this preliminary data, we have now utilized a comprehensive approach of in vitro characterization of Y79 Rb cell line following CSC enrichment using CD133 surface marker and subsequent validation to confirm the functional properties of CSCs.MethodsThe cultured Rb Y79 cells were evaluated for surface markers by flow cytometry and CD133 sorted cells (CD133lo/CD133hi) were compared for CSC characteristics by size/percentage, cell cycle assay, colony formation assay, differentiation, Matrigel transwell invasion assay, cytotoxicity assay, gene expression using microarray and validation by semi-quantitative PCR.ResultsRb Y79 cell line shared the profile (CD133, CD90, CXCR4 and ABCB1) of primary tumors except for CD44 expression. The CD133lo cells (16.1 ± 0.2%) were FSClo/SSClo, predominantly within the G0/G1 phase, formed larger and higher number of colonies with ability to differentiate to CD133hi cells, exhibited increased invasive potential in a matrigel transwell assay (p < 0.05) and were resistant to Carboplatin treatment (p < 0.001) as compared to CD133hi cells. The CD133lo cells showed higher expression of several embryonic stem cell genes (HOXB2, HOXA9, SALL1, NANOG, OCT4, LEFTY), stem cells/progenitor genes (MSI2, BMI1, PROX1, ABCB1, ABCB5, ABCG2), and metastasis related gene- MACC1, when compared to the CD133hi cells.ConclusionsThis study validates the observation from our earlier primary tumor study that CSC properties in Rb Y79 cell line are endowed within the CD133lo population, evident by their characteristics- i.e. small sized, dormant in nature, increased colony forming ability, differentiation to CD133hi cells, higher invasiveness potential, drug resistance and primitive gene expression pattern. These findings provide a proof of concept for methodological characterization of the retinoblastoma CSCs with future implications for improved diagnostic and treatment strategies.

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The study provides promising, first-of-its-kind evidence for the generation of lacrispheres from fresh HuLG, with enriched population of stem cells and secretory competent differentiated cells. The dual properties of these spheres make them a highly suitable source of transplantable cells for restoring the structure and function of damaged lacrimal gland.

Advances in animal models of retinoblastoma have accelerated research in this field, aiding in understanding tumor progression and assessing therapeutic modalities. The distinct pattern of mutations and specific location of this unique intraocular tumor have paved the way for two types of models- those based on genetic mutations, and xenograft models. Retinoblastoma gene knockouts with an additional loss of p107, p130, p53 and using promoters of Nestin, Chx10, and Pax6 genes show histological phenotypic changes close to the human form of retinoblastoma. Conditional knockout in specific layers of the developing retina has thrown light on the origin of this tumor. The use of xenograft models has overcome the obstacle of time delay in the presentation of symptoms, which remains a crucial drawback of genetic models. With the advances in molecular and imaging technologies, the current research aims to develop models that mimic all the features of retinoblastoma inclusive of its initiation, progression and metastasis. The combination of genetic and xenograft models in retinoblastoma research has and will help to pave way for better understanding of retinoblastoma tumor biology and also in designing and testing effective diagnostic and treatment modalities.

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