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The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma
Published on: October 9, 2015
An Improved In Vivo Methodology to Visualise Tumour Induced Changes in Vasculature Using the Chick Chorionic
Naside Mangir1,2, Ahtasham Raza1, John W Haycock1
1Department of Materials Science Engineering, Kroto Research Institute, University of Sheffield, Sheffield, U.K.
Background/Aim:
Decreasing the vascularity of a tumour has proven to be an effective strategy to suppress tumour growth and metastasis. Anti-angiogenic therapies have revolutionized the treatment of advanced-stage cancers, however there is still demand for further improvement. This necessitates new experimental models that will allow researchers to reliably study aspects of angiogenesis. The aim of this study was to demonstrate an in vivo technique in which the highly vascular and accessible chorioallantoic membrane (CAM) of the chick embryo is used to study tumour-induced changes in the macro and microvessels.
Materials And Methods:
Two cancer cell lines (human melanoma (C8161) and human prostate cancer (PC3)) were selected as model cells. Human dermal fibroblasts were used as a control. One million cells were labelled with green fluorescent protein and implanted on the CAM of the chick embryo at embryonic development day (EDD) 7 and angiogenesis was evaluated at EDDs 10, 12 and 14. A fluorescently-tagged lectin (lens culinaris agglutinin (LCA)) was injected intravenously into the chick embryo to label endothelial cells. The LCA is known to label the luminal surface of endothelial cells, or dextrans, in the CAM vasculature. Macrovessels were imaged by a hand-held digital microscope and images were processed for quantification. Microvessels were evaluated by confocal microscopy. Tumour invasion was assessed by histological and optical sectioning.
Results:
Tumour cells (C8161 and PC3) produced quantifiable increases in the total area covered by blood vessels, compared to fibroblasts when assessed by digital microscopy. Tumour invasion could be demonstrated by both histological and optical sectioning. The most significant changes in tumour vasculature observed were in the microvascular structures adjacent to the tumour cells, which showed an increase in the endothelial cell coverage. Additionally, tumour intravasation and tumour thrombus formation could be detected in the areas adjacent to tumour cells. The fragility of tumour blood vessels could be demonstrated when tumour cells seeded on a synthetic scaffold were grown on CAM.
Conclusion:
We report on a modification to a well-studied CAM in vivo assay, which can be effectively used to study tumour induced changes in macro and microvasculature.
Insights
This study presents a novel chick embryo chorioallantoic membrane (CAM) assay for studying tumor-induced vascular changes. The method effectively visualizes tumor angiogenesis, invasion, and microvessel alterations in vivo.
Area of Science:
- Oncology
- Developmental Biology
- Vascular Biology
Background:
- Anti-angiogenic therapies are crucial for advanced cancers, but improved treatments are needed.
- New experimental models are required to reliably study angiogenesis.
- The chick embryo chorioallantoic membrane (CAM) is a highly vascular and accessible in vivo model.
Purpose of the Study:
- To demonstrate an in vivo technique using the CAM to study tumor-induced changes in macro and microvasculature.
- To evaluate tumor angiogenesis and vascular dynamics in a living system.
- To provide a reliable model for anti-angiogenic therapy research.
Main Methods:
- Human melanoma (C8161) and prostate cancer (PC3) cells were implanted on the CAM of chick embryos.
- Green fluorescent protein (GFP)-labeled cells and fluorescently tagged lens culinaris agglutinin (LCA) were used for imaging.
- Macrovessels were imaged with digital microscopy, and microvessels were assessed by confocal microscopy.
- Tumor invasion was evaluated using histological and optical sectioning.
Main Results:
- Tumor cells significantly increased blood vessel area compared to control fibroblasts.
- Tumor invasion, intravasation, and thrombus formation were observed.
- Microvascular changes, including increased endothelial cell coverage, were prominent near tumor cells.
- Tumor blood vessel fragility was demonstrated when cells were grown on a synthetic scaffold.
Conclusions:
- A modified CAM in vivo assay effectively visualizes tumor-induced changes in macro and microvasculature.
- This model allows for the study of tumor angiogenesis, invasion, and vascular dynamics.
- The CAM assay offers a valuable tool for research in anti-angiogenic therapies and cancer biology.
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