Retinoblastoma, a childhood ocular cancer, accounts for 3% of all childhood tumors and can be fatal if left untreated owing to the rapidly growing tumor cells within the developing mutated retina(24). Although several in-vitro and in–vivo animal model systems have been explored to study the underlying mechanisms of Rb tumorigenesis and metastasis, there is a challenge in investigating the tumor in a developmental microenvironment. In this study, we demonstrate the establishment of CE-CAM xenograft model for Rb Y79 cell line with formation of tumor nodules on CAM, that exhibit spontaneous metastasis to the embryo. Our study also provides preliminary evidence for CD133lo, being the putative stem cells in Rb with higher potential to form tumor and metastases as documented by the in-vivo study using CM-Dil labeled and eGFP transduced Rb Y79 cells on chick embryo.
This novel work demonstrates that the CE model is a cost effective, time saving (procurement and maintenance), and a suitable model for visualization and examining Rb tumorigenesis and metastasis that could pave the way for exploring targeted therapy. This study provides in-vivo evidence and validates the results of our in-vitro studies that cancer stem cells properties are evident in CD133lo population of RbY79 cells(18).
Animal models using transgenic and xenograft approaches have been well-established for Rb(13)(25). Most of them include the mammalian system in order to resemble the tumor microenvironment. However, a developmental model is difficult to recapitulate in this system due to many technical difficulties(13). There is a dire need to simulate Rb tumorigenesis and metastasis within a developmental microenvironment in order to develop strategies for better therapeutic targeting. The CE-CAM, therefore, offers not only a temporal investigation but also provides a reliable tool for screening therapeutics in a short duration.
Our study demonstrated the formation of tumor nodules on CAM and spontaneous metastatic spread to the embryo using CM-Dil labeled and eGFP Y79 cells. The potential of CE-CAM model in Rb studies has been explored by Busch et al, who demonstrated the tumor forming ability of different cell lines (normal and chemoresistant) in soft agar as well as CAM and also evaluated the tumor suppressor effect of the Trefoil factor family (TFF) peptides in Rb cells using the CE- CAM model(15)(16)(25). Though the purpose of their study was towards cell line characterization and chemoresistance, the data does not provide any evidence for metastasis within the embryo. One of the possible reasons could be that the authors checked for invasion on E17/18, by which time the immune system of the embryo is observed to be fully functional and reactive(3). We concur with their observation of Y79 cells being capable of forming nodules on CAM and having the potential of spontaneous invasion within the CAM tissue. This observation is also similar to studies done on other solid tumors, such as glioblastoma, ovarian cancer, prostate cancer and uveal melanoma(7)(17).
The tumor nodules formed by the Y79 cells had presence of feeder vessels developing around indicative of tumor angiogenesis. Ribatti et al., demonstrated the presence of multiple host vessels around and within the tumor nodule in a neuroblastoma CAM xenograft model suggesting that the tumor cells were capable of inducing angiogenesis using the chorioallantoic vasculature(26). This could also serve as an important model to enhance or suppress Rb angiogenesis in tumor models due to its ease of visualization.
The spontaneous metastasis was observed in the cephalic, abdominal and limb regions of the embryo using the whole embryo in-vivo imaging and confirmed by histology in the sections of embryonic organs such as the brain, eye and liver. Rb is clinically known to metastasize to the brain by invading through optic nerve and to the bone and liver via haematogenous spread and, therefore, involvement of optic nerve, uvea and sclera are considered as one of the important prognostic factors to predict metastasis(10)(27). Our findings also concur with the study done by Palmer and co-workers who investigated spontaneous metastasis of Human Epidermoid Carcinoma cell line (HEp3) to the chick liver and lung(28). Though the imaging of CAM and embryo was possible after dissection, there was an observed signal in the beak area which did not reveal any presence of tumor cells upon histological analysis. This could be due to autoflourescence due to the density of the bone and keratin present on the beak.
A further novel aspect of our study is the use of this model to study the tumorigenic and metastatic potential of CD133lo CSCs within theY79 cell line. This is an important validating point as CD133hi subset is projected as CSC in tumors of the brain(29), while the negative cells seem to harbour CSC properties in Rb(18). It would be logical to propose that while many CSC markers could be common to most tumors, some of them could vary in each tumor specifically due to the lineage of the tumor or specific aspects of differentiation of cells in that target tissue. For instance, CD133 is a marker of photoreceptor differentiation(30), hence their precursor cells are expected to be negative for this marker, which we have documented both in primary as well as Y79 cell line(18)(31).
The CE-CAM model is an ideal short-term in-vivo system to study different subsets of tumors and identify tumor initiating cells. We also demonstrate the use of a cell tracker dye CM-Dil, with comparison to eGFP labeled cells, in order to track Rb cells in-vivo. CM-Dil dye has been successfully used earlier in Rb zebrafish orthotopic xenograft model by Chen et al. who tracked the tumor cell invasion locally and along the optic nerve(32). However, considering that CM-Dil dye intensity could reduce with proliferation of cells(33), we also assessed the same using stable eGFP labeled Y79 cells. GFP labeled tumor cells were successfully used by several investigators to track in-vivo metastases within the chick embryos in several tumors(17)(34)(35). Our analyses showed that both the cell labeling strategies can be utilized for in-vivo tracking. To the best of our knowledge, this is the first study that explores the use of in-vivo fluorescence imaging of whole embryo for identifying spontaneous metastasis in a CE-CAM Rb xenograft model.
Use of other alternate imaging technologies such as in-vivo PET/CT has been demonstrated using radiotracers by Warnock et al in a glioblastoma xenograft model for studying tumor metastasis(36). Their studies showed the PET tracer uptake over time by the tumor within the embryo and were able to measure the tumor volume with improved accuracy using CT imaging. The CE-CAM, owing to the ease of visualization through the window from the eggshell makes it extremely suitable and convenient for tracking tumor cells in-vivo.
The clinical relevance of this study is the use of an inexpensive, easy to establish in-vivo model using cell lines and patient derived xenografts for exploring existing and novel therapeutics. However, there are certain limitations of the CE model and further studies are required to understand the cellular adaptations to avian microenvironment. The incubation period of about a week may not be suitable for slow growing tumor cells and it may be difficult to observe visible metastases in such cases. We believe that validating these results with early passage Rb primary cells and other Rb cell lines- WERI-Rb1, etc., in the future, would add more value to this in-vivo model. Use of CM-Dil dye for tracking rapidly growing cells may be a challenge owing to its loss with each successive tumor generations, which could possibly under represent the tumor load of the tissue. Parallel confirmation with GFP labeled Y79 cells helped overcome this limitation by demonstrating similar results in tumor formation and metastasis. Quantitative studies using human specific gene expression assays could compliment these assays in future(28). Though the imaging of CAM and embryo was possible after dissection, the serial imaging of the entire live egg including the shell would be ideal for which different kinds of fluorescent dyes and standardization of the software settings may be required, which we hope to address in our future studies.