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.

BackgroundCancer Stem Cells (CSCs) reported in various tumors, play a crucial role in tumorigenesis and metastasis. Following the efforts to reduce, replace and refine the use of mammalian models, we aimed to establish a short-term xenograft for Retinoblastoma (Rb) to evaluate the tumorigenic and metastatic potential of CD133lo CSCs in Rb Y79 cells, using the well-established chick embryo (CE) model. MethodsTotal and CD133 sorted Rb Y79 cells, labelled with eGFP/CM-Dil tracking dye, were transplanted onto the chorioallantoic membrane (CAM) of day-7 chick embryos and incubated for 7 days. The tumor formation on CAM and metastasis to the embryos were evaluated by confocal microscopy, in-vivo imaging, and histopathology. ResultsY79 cells formed pink-white raised perivascular nodules on the CAM with CD133lo CSCs exhibiting larger nodules when compared to CD133hi cells and total Y79 (p<0.05). In-vivo imaging revealed that the labeled cells metastasized to the embryos with the fluorescent signals visible in the abdominal area, cephalus and the limbs. Histopathologic studies confirmed the presence of tumor cells on the CAM, organs of embryos transplanted with Y79 cells, more so with CD133lo CSCs. ConclusionsThis study highlights that the CE-CAM is a feasible alternative non-mammalian model for evaluating tumorigenicity and metastatic potential of Rb CSCs. The study also provides preliminary evidence that Rb Y79 CD133lo CSCs show higher propensity to form tumor nodules on the CAM and are more invasive than non CSCs, thus, supporting our earlier evidence that they are endowed with CSC properties.