Chicken chorioallantoic membrane as a reliable model to evaluate osteosarcoma—an experimental approach using SaOS2 cell line
© Manjunathan and Ragunathan. 2015
Received: 13 May 2015
Accepted: 30 May 2015
Published: 6 June 2015
Osteosarcoma 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.
Angiogenic 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.
SaOS2 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.
CAM 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.
Osteosarcoma is the most common malignant bone tumor that affects mostly children. So far tremendous advanced therapies are available for the management of osteosarcoma, but the survival rate is comparatively poor . Various animal models are available to screen tumor growth and its development with wide range of acceptable and reproducible capacity [2, 3]. But these animal models have major limitations such as, need of prolonged experimental time, expensive, mature immune system and large number of animal sacrifice associated with ethical issues . Therefore, developing a model system which have advantages over on above mentioned issues and also helpful for the better visualization of vascularization that can provide the basis behind the interaction of tumor cells with surrounding stroma with respect to metastasis progress is highly acceptable. Also the model has to be useful for a wide range of analysis like large scale micro array or array based genomic hybridization within shorter period to identify candidate gene expression to find out candidate anti angiogenic agents with clinical benefits for the treatment of osteosarcoma. An attractive option in this issue is the use of chicken chorioallantoic membrane (CAM) assay.
CAM is a well-established model in the field of angiogenesis and is widely used for the monitoring of tumor angiogenesis . CAM has many advantages such as an extensive vascularized network, very easy to access within shorter period, in expensive, easy to manage in a lab atmosphere, no ethical issue and no immune response . Various reports suggested that human tumor cells or tissues implanted on CAM are able to induce angiogenesis and this potential is utilized for the development of anti angiogenic agents to provide better therapy to avoid tumor progression [6, 7]. In 2010, Balke et al., showed that various osteosarcoma derived cells are able to induce vascularization and tumor growth on CAM membrane with morphological characterization of solid osteosarcoma . In 2011, the same research group has showed that the grafted human bone tumor giant cells are able to intersperse with chick derived capillaries to induce new vascularization . But there were not enough studies have been reported about the direct angiogenic ability of SaOS2 cell line on CAM in detail.
In this concern, we aimed to validate the angiogenic efficacy of human osteosarcoma derived SaOS2 cell line using chicken late CAM assay as a model system. The model has been used to understand the interactive and invasive character of SaOS2 cells with its surrounding stroma as an indication of its angiogenic ability which in turn favor tumor growth and metastasis. The angiogenic and tumor progressive effect of SaOS2 cells was analyzed under three different cellular volumes on CAM to have a better understanding about the progressiveness of the tumor in stepwise manner. We demonstrated that SaOS2 cells are able to induce rich vascularization at the implanted area which can be directly visualized and quantified and also provided idea about the invasiveness and progressiveness with reproducible similar key features of human osteosarcoma growth at its different stages of progression with large scale analysis. Since, CAM assay is reproducing the characterization of osteosarcoma exactly as in various in vivo animal models this assay can be useful for having wide range of experimental analysis to have a better understanding about osteosarcoma for therapeutic approach.
SaOS2 cells induces new blood vessel formation on CAM vascular bed—CAM enables direct visualization of angiogenesis
SaOS2 cells induces sprouting angiogenesis—CAM provide quantification of angiogenesis directly from visual images
SaOS2 cells changes the morphology of the CAM vascular bed—CAM allow the invading of SaOS2 cells
SaOS2 cells upregulates the transcription level of angiogenic growth factors on CAM—CAM highly response with the growth demands of SaOS2 cells
SaOS2 cells alter the micro vascular morphology of CAM vasculature—CAM modify its vascular bed based on the demand of external SaOS2 cells
SaOS2 cells increases the protein level expression of VEGF A, MMP2 and MMP9 – CAM vascular bed is reliable to evaluate the protein level expression
Osteosarcoma is the most common primary bone tumor characterized by complex mixture of cell types with aggressive local growth . The extra cellular matrix produced by osteosarcoma cells protect the tumor from apoptosis induced by external anticancer agents [3, 11]. Various animal models are available to evaluate the dynamic process of osteosarcoma progression for evaluating the most effective antitumor drugs against of osteosarcoma progression . However these animal models are very costly and time consuming. The alternative choice is to develop other assay models with highly reproducible efficacy like in other animal models. In this study we used chicken CAM assay as an alternative model to evaluate the morphological and molecular characteristic of osteosarcoma induced by SaOS2 cells in details.
CAM is an extra embryonic membrane with dense vascular network which physiologically serves as a respiratory organ of the embryo until hatches. CAM has been widely used as in vivo model system for angiogenesis research and also established as a highly reproducible model to study the aggressiveness of various tumors . Main advantages of CAM includes an extensive vasculature, ease to access, large scale screening and easily reproducible capacity with simple experimental approach . Even though CAM is an established in vivo model to evaluate the progression of various solid tumors, but there were a few reports regarding the usage of CAM for bone and soft tissue sarcoma analysis especially for osteosarcoma [13, 14, 8]. In 2010, Balke et al. reported about the ability of different osteosarcoma cell lines like MNNG-HOS, U2OS and SAOS to form vascularized solid tumors on CAM after 4 days of incubation. These cell lines are able to induce morphological changes on CAM bed which is similar like in other animal models with main advantage of no osteoid formation. In their work they showed that among these cell lines the mortality rate of embryo was significantly higher for SAOS cells due to tumor cell dissemination or by the secretion of blood coagulation factors under higher cellular volume . To avoid this issue we incubated CAM with different cellular volumes of SaOS2 cells and this experimental pattern helped to understand the time dependent progressiveness and aggressiveness of tumor cells along with an understanding about the involvement of various angiogenic factors at earlier progressive stage.
In this study we showed that human osteosarcoma derived SaOS2 cells are able to induce sprouting angiogenesis on CAM bed and this data is in concordant with earlier reports . We found that the sprouting ability of SaOS2 cells are similar for all studied volumes and is irrespective of the cellular number but the degree of angiogenic response increased with increased number of cells significantly. Advantage of using CAM is that the vascularization ability of SaOS2 cells can easily visualized directly as early as possible without much experimental method that is not possible with another animal in vivo models. The angiogenic ability of SaOS2 cells on CAM is confirmed using scanning microscope analysis which gave a clear idea about the sprouting ability of the tumor cells.
Histological evaluation of CAM indicates the invading ability of SaOS2 cells into the stroma of the CAM within 96 h of incubation to induce more vessel growth and this will help to understand the mechanism behind the aggressiveness and invasiveness of SaOS2 cells on CAM without osteoid formation like in other reports [7, 8]. CAM enables us to learn about the progressiveness and aggressiveness of the tumor cells at different cellular volumes within 96 h of incubation and this information was not reported with any of the animal models within shorter time period of tumor implantation. The benefit of this simple experiment is that it is helpful to understand the mechanism behind the anti-progressive and anti-aggressive nature of chemicals as early as possible in a therapeutic way with potential effect against osteosarcoma.
Molecular profiling of various angiogenic growth factors from in and around the CAM area where the tumor cells are implanted surprisingly reproduced data which was early shown by both in vivo animal as well as in vitro studies [3, 11, 15, 16]. In this study we found that the progression and aggressiveness of SaOS2 cells mainly depends on the activation of VEGF165, MMP2 and MMP9 at an earlier stage followed by the expression of FGF2 and NOS at later stage of progression. Usage of CAM model will provide a better understanding about the differential pattern of expression of various angiogenic growth factors under various cellular volume. The earlier finding was further confirmed by the protein level expression of VEGF165, MMP2 and MMP9 on CAM. This experimental approach using CAM insights into the role of various angiogenic factors in the progression of tumor growth and also enables to find the better therapeutic molecule based on its anti angiogenic effect against on these angiogenic factors either targeting alone or in combination.
We conformed that CAM is the robust and reliable model to study about the aggressiveness and tumor progression of osteosarcoma using various angiogenic and molecular techniques. CAM is able to produce the data’s in a promising way which was reported earlier using various in vitro and in vivo animal models. The kind of experiment enables to understand the mechanism behind the angiogenic response of SaOS2 cells. In this study we focused more on the early progressive phase of tumor growth and development by SaOS2 cells for 96 h on CAM model which enables to have better knowledge about the aggressiveness and angiogenic character of SaOS2 cells on CAM. Analyzing the angiogenic ability of SaOS2 cells under various cellular volumes on CAM enable to understand the step by step progressiveness and the involvement of various angiogenic growth factors at differential level. Further it need to use modern techniques like micro array or in situ hybridization to have a deep knowledge about the progression and invasiveness of osteosarcoma cells using CAM model to develop suitable therapeutic molecules.
Materials and methods
Human osteoblast derived SaOS2 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicilline/streptomycine, (SIGMA, Aldrich, USA) at 37 °C in a humidified 5 % CO2 incubator until it get full confluent .
Preparation of cell pellet
Prior implantation on CAM, cell suspensions were prepared by detaching cells with trypsin/EDTA (Medox, India). Cells were centrifuged at 1200 rpm for 5 min, washed twice in culture medium without FBS and suspended in DMEM alone medium at a final concentration of each 30, 60 and 120 lakhs cells per 20 μl volume .
In vivo CAM assay
Fertilized White Leghorn chicken eggs weighing 50 ± 2 g (Tamilnadu Poultry Research Station, Chennai, India) were incubated at 37 °C in a humidified atmosphere (at >60 % relative humidity) as per the protocol for the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) method. On day 3 of post incubation, 2 to 3 ml of albumin was withdrawn, using a 21 gauge needle, through a small opening at the large blunt edge of the egg to minimize adhesion of the shell membrane with CAM. A square window of 1 cm2 was opened in the egg shell at the opposite to blunt edge and sealed with paraffin film to prevent dehydration. The eggs were returned for further incubation. On embryonic day 9, gelatin sponges of size (Jhonson & Jhonson Pvt Lmtd) cut into a size of 1 mm3 were soaked with 30, 60 and 120 lakhs cells per 20 μl volume of medium were placed on the top of growing CAM under sterile condition. Control CAM was incubated with 20 μl of DMEM alone. The window was closed with a transparent adhesive tape and the eggs were returned for incubation till day 13 (for 96 h). The experimental groups were divided into 4 of each containing 40 numbers of eggs. Group1 treated with DMEM alone as control, group 2, 3 and 4 were treated with 20 μl of 30, 60 and 120 lakhs of SaOS2 cells per volume respectively. In ovo CAM was photographed at 0, 24, 48, 72 and 96 h using Cannon digital camera of 12 × 5.0 Mega Pixel (Power Shot A95) and the images were subsequently analyzed with Image J and Angioquant Toolbox, MATLAB 6.5 Software’s to measure the growth of the vessels by means of its length, size and number of vessels junctions [7, 18, 19]. The number blood vessel around the sponge was calculated from each group manually in a blind manner.
CAM incubated with SaOS2 cell line was flooded with Bouin’s fixative solution after 96 h of incubation and the treated area was removed carefully, dehydrated and embedded in paraffin wax. Vertical cross sections of 7 μm in thickness were taken using Rotary Microtome (Weswicox, Japan). After staining with haematoxylin and eosin, sections were mounted with DPX and observed using light microscope at 40× magnification for qualitative assessment. The images were recorded at 10× magnification using Nikon D70 DSLR (6.1 megapixel) camera attached with light microscope . Thickness of the CAM was measured from haematoxylin and eosin stained cross sections morphometrically with a calibrated objective at 40× magnification, using 10×10 calibrated grid at the 10× ocular. Each CAM was measured at 6 different locations from 6 serial cross sections of the same sample in micrometer and averaged to calculate mean tissue thickness (DCAM). In paraffin-embedded tissue, material shrinkage is estimated to be ~25 % relative to the fresh material. As all tissues were prepared similarly, tissue shrinkage is same for all CAM zones. Thus, shrinkage corrections are unnecessary for the comparisons of tissue thickness .
Semi-quantitative reverse transcriptase–polymerase chain reaction
Gene name and primer sequences
94 °C/1 min
59 °C/1 min
72 °C/1 min
94 °C/1 min
54 °C/1 min
72 °C/1 min
94 °C/30 s
60 °C/30 s
72 °C/1 min
94 °C/30 s
48 °C/30 s
72 °C/1 min
94 °C/1 min
57 °C/40 s
68 °C/1.5 min
94 °C/30 s
60 °C/30 s
72 °C/1 min
Scanning electron microscopic study
After 4th day of post incubation with 120 lakhs of cells/volume of SaOS2 cell line, CAM at the area of incubation was dissected out and washed with 1× PBS. The CAM was dried at room temperature without disturbances. The unfolded air dried membranes were glued onto stubs with carbon, spattered with gold (10 min, 14–17 Ma, 0.07 mbar) and observed under a Hitachi S-3400 N Variable Pressure Scanning Electron Microscope at an accelerating voltage of 15–30 kV. The images were recorded at a magnification of 200 μM .
The deparaffinised and dehydrated CAM incubated with 120 lakhs of cells/volume was allowed to undergo antigen retrieval process using Sodium Citrate (10 mM-pH 6.0) in a microwave oven for 20 min and then washed with DDH2O for 3 × 5 min in 1× PBS (pH 7.3). Normal Goat Serum Blocking Solution (2 % goat serum,1 % BSA, 0.1 % cold fish skin gelatin, 0.1 % Triton ×-100, 0.05 %, Tween- 20, 0.05 % Sodium Azide, 0.01 M PBS (pH 7.2) of 50 to 75 μl was added immediately on the sections and incubated for 1 h in a humidified chamber. After washing with 1× PBS, primary antibody of VEGF A (CALBIOCHEM, EMD), MMP2 and MMP9 (1:200 dilution) was applied on the sections and after overnight incubation rinsed with 1× PBS with 0.05 % of Tween-20. Diluted FITC and HRP conjugated secondary antibodies of 1:40 dilution was applied for 1 h according to manufacturer’s instruction (Goat ant-rabbit IgG, Bangalore Genei, India). Counterstaining with haematoxylin and eosin was performed for those sections incubated with HRP conjugated secondary antibody. Images were recorded at 40× magnification using B×51 Olympus Fluorescence Microscope at a wavelength of 515 nm with ASI FISH View 5.5 software for FITC and Light Microscope was used for HRP .
Data analysis and statistics
All the experiments were performed in triplicate (n = 3) unless otherwise specified. Data are presented as mean ± SEM and were analysed by Descriptive analysis for ± SEM and One-Way ANOVA analysis of Holm-Sidak Test for appropriate using SigmaPlot 12. P values of p = < 0.001, p = 0.001, p = 0.010 and p = 0.005 were considered for statistical significance.
We would like to thank Dr. Li Haiqing, MD, Ph.D, −Technology transfer specialist, National Cancer Institute, Rockville, USA for the kind gift of Rabbit polyclonal MMP2 antibody. We thank Dr. N. Srinivasan, Professor, Department of Endocrinology, Dr. ALM PG IBMS, University of Madras, for the kind gift of SaOS2 cell line. We thank the funding agency, University Grant commission of India for giving merit fellowship and also for supporting the department under SAP program.
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