Chicken chorioallantoic membrane as a reliable model to evaluate osteosarcoma—an experimental approach using SaOS2 cell line

Abstract

BackgroundOsteosarcoma is the most common primary tumor that affects usually children. Due to its cellular complex and osteoid formation it is very difficult to understand the mechanism behind the progressiveness of osteosarcoma. Various animal models are available to study the issue but they are time consuming and costly. We aimed to understand the progressiveness and invasiveness of osteosarcoma induced by SaOS2 cells using chicken chorioallantoic membrane. CAM is a well-established model which allows in vivo studies of tumor induced angiogenesis and the testing of anti angiogenic molecules. However only a few reports showed the tumor forming ability of SaOS2 cells on CAM.MethodAngiogenic ability of SaOS2 cells on CAM was validated by various methods. Angiogenic ability was scored by direct visualization and scanning microscopic analysis. The sprouting ability and growth of the vessel was measured by Angioquant software under different cellular volume. The invasiveness was analyzed by histological staining. Involvement of angiogenic factors at differential stage of progressiveness was confirmed by the molecular and protein level expression analysis.ResultSaOS2 cells induces sprouting angiogenesis on CAM and shows its aggressiveness by rupturing the ectodermal layer of the CAM. Growth and development of osteosarcoma depends mainly on the activation of VEGF165, MMP2 and MMP9. CAM able to reproduce angiogenic response against the stimulation of SaOS2 cells exactly as in other animal models without inflammatory reactions.ConclusionCAM is an excellent alternative in vivo model for studying the aggressiveness and tumor progression of osteosarcoma using various angiogenic techniques in an easily, faster and affordable way. We further provided insight about the involvement of various angiogenic growth factors on the development of osteosarcoma which will enable to find the suitable therapeutic molecule for the treatment of osteosarcoma. CAM model could provide a wide space using modern techniques like micro array or in situ hybridization to have a better understanding about the progression and invasiveness of osteosarcoma cells to develop suitable therapeutic molecules.

Sarcoma treatment in the era of molecular medicine

“…In vivo , the chick chorioallantoic membrane (CAM) assay is a valuable option due to its low costs and relatively easy implementation. CAM assays have been employed to study sarcoma angiogenesis, fibroblast infiltration, tumorigenesis, tumor invasion, and metastasis in CHS, EwS, fibrosarcoma, LPS, and OS (Sys et al , 2013; Patil et al , 2014; Manjunathan & Ragunathan, 2015; Cimpean et al , 2018; Kunz et al , 2019; Perut et al , 2019; Steinestel et al , 2020). Numerous additional in vivo models of inducible or spontaneous sarcomas have been described in non‐mammalian vertebrates (e.g., zebrafish; Leacock et al , 2012; Mohseny et al , 2012; Brown et al , 2017b; Hayes & Langenau, 2017; Ignatius et al , 2018; Fleming et al , 2019) and in mammalians (e.g., mouse, rat, and dog; Cannon, 2015; Jacques et al , 2018; Castillo‐Tandazo et al , 2019; Pomella & Rota, 2020).…”

Methods to label, image, and analyze the complex structural architectures of microvascular networks

“…AngioQuant has been developed to analyze endothelial networks in vitro, with a focus on quantifying various metrics of tubule formation using bright field images . Recently, it has been adapted for use in evaluating higher resolution datasets of microvascular networks in vivo and in histological samples . Its validation is focused on quantification of in vitro experiments showing trends of changes with various metrics, but no statistical comparisons between study groups.…”

Understanding and Modeling Metastasis Biology to Improve Therapeutic Strategies for Combating Osteosarcoma Progression

“…The CAM has proven to be a useful model in studying tumor angiogenesis (90,91), tumor cell migration and invasion (92), intravasation into blood vessels (93), tumor cells in transit within the vasculature, extravasation out from blood vessels ( Figure 2B) (94)(95)(96)(97)(98), and the outgrowth of patient derived xenografts (99). In OS research, the CAM model has been used to study tumor growth of a variety of OS cell lines (100), angiogenesis (101), and metastasis to distant sites (102). The main benefits of using the chick CAM model include:…”