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.