A short-term chick embryo in vivo xenograft model to study retinoblastoma cancer stem cells

Purpose: Cancer stem cells (CSCs) reported in various tumors play a crucial role in tumorigenesis and metastasis of retinoblastoma (Rb). Following the efforts to reduce, replace, and refine the use of mammalian models, we aimed to establish a short-term xenograft for Rb to evaluate the CSC properties of CD133 - Rb Y79 cells, using the well-established chick embryo chorioallantoic membrane (CE-CAM) assay. Methods: Y79 cells were cultured, labeled with two different dyes (CM-Dil Y79 and enhanced green fluorescent protein (eGFP)) and sorted for CD133 - and CD133 + subsets. Two million cells from each of the labeled groups were transplanted onto the abraded CAM on embryonic day 7 (E7). On E14, the tumor nodule formation on CAM and spontaneous metastasis to the embryos were evaluated by confocal microscopy, in vivo imaging, and histology. Results: Y79 cells formed pink–white raised perivascular nodules with feeder vessels on the CAM with both the types of labeled CD133 - cells. CD133 - cells, when compared to CD133 + cells, demonstrated significantly larger tumor volume (40.45 ± 7.744 mm 3 vs 3.478 ± 0.69 mm 3 , P = 0.0014) and higher fluorescence intensity (CM-Dil: AUF = 6.37 × 10 7 ± 7.7 × 10 6 vs 1.08 × 10 7 ± 1.6 × 10 6 ; P < 0.0001; eGFP: AUF = 13.94 × 10 4 ± 2.54 × 10 4 vs AUF = 1.39 × 10 4 ± 0.4 × 10 4 ; P = 0.0003). The metastatic potential of CD133 - cells was also observed to be higher as noted by in vivo imaging and histopathology. Conclusion: This study highlights that CE-CAM is a feasible alternative nonmammalian model for evaluating tumorigenicity and metastatic potential of Y79 CSCs. Increased tumorigenicity and metastatic potential of CD133 - subset of tumor cells substantiate their CSC properties.