Chick Embryo: A novel xenograft model for Retinoblastoma

Abstract

Purpose : Cancer Stem Cells (CSCs), also known as tumor initiating cells, are endowed with property of tumor initiation and the ability to metastasize to the distant organs. Our previous study on Rb Y79 cell lines revealed that the CD133^lo cells harbor the CSCs which was further validated by functional and gene expression studies. In this study, we aim to further investigate the in-vivo tumor forming ability and metastatic potential of CD133^lo cells in Rb Y79 cells using the Chick Embryo- Chorioallantoic Membrane (CE-CAM) model.

Methods : Rb Y79 cells were transected with EGFP and sorted for CD133 marker using magnetic cell sorter. One million total, CD133^lo and CD133^hi Rb Y79 cells were transplanted on day-7 on the CE-CAM of embryonated eggs and incubated at 38⁰C and 60% relative humidity till day-14. The tumorogenic and metastatic potential of Rb Y79 sorted and unsorted cells were evaluated using gross, confocal microscopy, in-vivo imaging and histology.

Results : CD133^lo constituted 15±0.8% of total cells and with a purity <90% after MACS sorting. The transplanted cells formed pinkish-white raised wet perivascular nodules on CAM with establishment of feeder vessels and tumor volume of Rb Y79 GFP CD133^lo cells transplanted CAM was significantly higher (40.44±7.74mm³) when compared to total (p=0.02, 5.3± 1.01mm³) and CD133^hi (p=0.01, 2.56± 0.66mm³) cells. The Confocal fluorescence intensity of CAM tissues showed that CD133^lo had higher localization of cells when compared to total Y79 (p=0.08) and CD133^hi cells (p=0.001). The in-vivo imaging revealed that the embryos had predominant fluorescent signals in the cephalic, abdominal areas as well as in the bones of hind limbs suggestive of spontaneous metastasis to the embryo, which was higher in CD133^locells as compared to total Y79 (p=0.05) and CD133^hi group (p=0.001) confirming higher metastatic potential. Histopathological studies of the CAM and embryo revealed presence of tumor nodules on the CAM and within the brain, orbital bones and interestingly in liver more so with CD133^lo CSCs and these cells were immunoreactive to human Ki-67.

Conclusions : This study highlights the successful generation of tumorigenesis and spontaneous metastasis of Rb Y79 cells in CE-CAM model. The CE-CAM model also provided evidence that the tumorogenic and metastatic potential is higher in the Rb Y79 CD133^lo population thus supporting our earlier studies that these cells are endowed with CSCs properties in Rb.

This is a 2020 ARVO Annual Meeting abstract.