Evaluation of Cancer Stem Cells in Patient-derived Xenografts of Retinoblastoma in Chick Embryo-Chorioallontoic Membrane Model

Abstract Background: Cancer stem cells (CSCs) in most tumors, including retinoblastoma (Rb), are associated with tumor initiation, metastasis, and drug resistance. Our previous studies identified Rb CSCs as CD44+/CD133- by flowcytometry using size and phenotype. In this study, we further evaluated two prime characteristics of CSCs, i.e., chemoresistance and tumor-initiating properties using Chick embryo-Chorioallantoic Membrane (CE-CAM) model. Methods: After IRB approval was obtained, fresh Rb tumors (n=15) were collected and sorted using antibodies against CD44, followed by CD133. Drug resistance and gene expression were evaluated using the MTT assay and qPCR. Tumor-initiation and metastatic ability were assessed using the CE-CAM assay by transplanting one million cells into a day-7 chick embryo and were evaluated by gross, confocal microscopy, IVIS spectral imaging, alu-qPCR, and histology. Results: The percentage of CSCs in Rb tumors ranged from 4.8 to 28.4%, exhibiting greater drug resistance and enhanced expression of genes related to CSCs, stemness, drug resistance, EMT and invasion, and metastasis-specific genes than tumor non-CSCs (P<0.0001). The CSCs transplanted cells formed white-colored glistening tumor nodules on the CAM and significantly higher localization of fluorescence signals than non-CSCs (p<0.0001). IVIS imaging revealed positive signals in the brain, which confirmed malignant round cells on histopathology and were quantitatively measured by alu-qPCR. Conclusion: Rb CSCs (CD44+/CD133-) are endowed with inherent drug resistance and tumor initiation potential, as demonstrated by their enhanced capacity to form tumor xenografts in the CE-CAM model with evidence of metastasis. The CE-CAM is a valuable, cost-effective pre-clinical model that evaluates the pathogenesis of tumor progression and targets therapies.