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Geeta K Vemuganti, Rohini M Nair, Sucharita G, Narayana RVL, Swathi Kaliki; Chick Embryo CAM model of Retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1772.
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© ARVO (1962-2015); The Authors (2016-present)
Development of animal models for Retinoblastoma (Rb) that recapitulates the tumor phenotypically and functionally has been challenging despite advances in molecular biotechnology. This study attempts to develop an in vivo model that is easy to establish, visualize and assess the in vivo behavior of RbY79 cell line and CD133lo/hi populations using the experimental system of Chick Embryo Chorio-allantoic Membrane model (CE-CAM).
Embryonated eggs of Gramapriya breed (G.gallus), procured after IAEC approval, were incubated at 38°C/60%RH for 8 days, after which a small window was made to introduce tumor cells. Y79 cells were evaluated for CD133 marker and sorted using MACS. The 3 groups of CM-Dil labelled cells(106 each): Total Y79 (GroupA, n=5), CD133lo(GroupB, n=6) and CD133hi(GroupC, n=5) in Matrigel, were placed on abraded CAM surface of E8 embryos, incubated and sacrificed after 1 week. The CAM was dissected and evaluated by confocal microscopy for tumor cells followed by routine histology. In vivo imaging of whole embryos was done to detect distant metastasis. The fluorescence images were quantified by ImageJ(AUF) and statistical analysis by GraphPad Prism.
RbY79 cell line revealed 16.1±0.2% C133lo and 83.25±0.85% CD133hi cells which were sorted using MACS with over 90%purity and 80% viability. Survival % of the embryos were 42.5±17.5, 60.5±10.5 and 75.5±4.5 respectively for Groups A,B and C. The tumors formed pinkish-white raised wet perivascular nodules with feeder vessels on the CAM. Fluorescence intensity analysis of CAM tissues showed that GroupB (AUF=6.3*107±7.7*106) had higher localization of cells when compared to GroupA (p=0.1, AUF=3.3*107±0.2*106) and GroupC (p<0.0001,AUF=1.08*107±1.6*106). Fluorescence signal from in vivo imaging of the embryos was noted predominantly in abdominal area in all groups but GroupB embryos showed intense and more spread out signal compared to Groups A and C(p=0.8;0.5) with additional evidence of fluorescence in the cephalic region hinting at possible metastasis, as confirmed by histology.
In vivo tracking of RbY79 cell line in CE-CAM model demonstrated formation of tumor nodules in CAM with preliminary evidence of metastasis to the embryo. The study also provides proof that the CD133lo cells show higher propensity to form tumor deposits compared to CD133hi cells, suggesting they are possibly endowed with cancer stem cell like properties in Rb, which needs to be further validated.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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