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 CSC, i.e., chemoresistance and tumor-initiating properties using Chick embryo-Chorioallantoic Membrane (CE-CAM) model. Methods After obtaining IRB approval, fresh Rb tumors (n = 15) was collected and sorted using antibodies against CD44, followed by CD133. Drug resistance and gene expression were evaluated using 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 ranges from 4.8 to 28.4%, exhibiting higher drug resistance and enhanced gene expression of CSC, stemness, drug resistance, EMT & invasion, and metastasis-specific genes than tumor non-CSCs (P < 0.0001). The CSC 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 showed positive signals in the brain, which confirmed malignant round cells on histopathology and were quantitatively measured by alu-qPCR. Conclusion The Rb CSCs (CD44+/CD133-) are endowed with inherent drug resistance and tumor-initiating, as demonstrated by 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 also targets therapies.