Date Published: April 15, 2019
Publisher: Public Library of Science
Author(s): Pierre Kunz, Astrid Schenker, Heiner Sähr, Burkhard Lehner, Jörg Fellenberg, Domenico Ribatti.
Survival rates of osteosarcoma patients could not be significantly improved by conventional chemotherapeutic treatment regimens since the introduction of high-dose chemotherapy 35 years ago. Therefore, there is a strong clinical need for new therapeutic targets and personalized treatment strategies, requiring reliable in vivo model systems for the identification and testing of potential new treatment approaches. Conventional in vivo rodent experiments face ethical issues, are time consuming and costly, being of particular relevance in orphan diseases like osteosarcoma. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. The CAM is a highly vascularized, non-innervated extra-embryonic membrane that is perfectly suited for the engraftment of tumor cells. However, only few reports are available for osteosarcoma and reported data are inconsistent. Therefore, the aim of this study was the adaptation and optimization of the CAM assay for its application in osteosarcoma research. Tumor take rates and volumes of osteosarcoma that developed on the CAM were analyzed after modification of several experimental parameters, including egg windowing, CAM pretreatment, inoculation technique and many more. Eight osteosarcoma cell lines were investigated. Our optimized OS-CAM-assay was finally validated against a rat animal xenograft model. Using the cell line MNNG HOS as reference we could improve the tumor take rates from 51% to 94%, the viability of the embryos from initially 40% to >80% and achieved a threefold increase of the tumor volumes. We were able to generate solid tumors from all eight osteosarcoma cell lines used in this study and could reproduce results that were obtained using an osteosarcoma rat animal model. The CAM assay can bridge the gap between in vitro cell culture and in vivo animal experiments. As reliable in vivo model for osteosarcoma research the optimized CAM assay may speed up preclinical data collection and simplifies research on potential new agents towards personalized treatment strategies. Further, in accordance with Russell’s and Burch’s “Principles of Humane Experimental Technique” the reasonable use of this model provides a refinement by minimizing pain and suffering of animals and supports a considerable reduction and/or replacement of animal experiments.
Osteosarcoma is the most common primary tumor of bone in children and young adults [1, 2]. It is characterized by a strong vascularization, cellular heterogeneity, aggressive local tumor growth and early metastasis, mainly into the lungs. Since the introduction of neoadjuvant chemotherapy about 35 years ago, the survival rate for osteosarcoma patients stagnates at approximately 60% and only 20% – 30% for patients with metastases [3–5]. Whereas personalized treatment has been translated into the clinical routine for multiple malignancies, such therapeutic approaches do not exist for osteosarcoma patients so far. The latest and largest multinational therapy-optimizing study “Euramos-1”, evaluating a multi-drug chemotherapy regimen, could not contribute to an improved survival for osteosarcoma patients . Results of the following treatment-optimizing study for osteosarcoma patients won`t be available for more than another decade, emphasizing the clinical need for new diagnostic and therapeutic approaches to improve osteosarcoma patient survival. However, their identification is substantially hampered by the limited availability of appropriate in vivo models. In contrast to in vitro models they mimic the physiological cancer environment and allow the interaction of tumor cells with the tumor-promoting stroma including endothelial cells and fibroblasts. They further allow the investigation of individualized approaches that are especially important for the treatment of such heterogeneous tumors like osteosarcoma. Yet, current animal models are time consuming, expensive and have provoked ethical concerns. In an orphan disease like osteosarcoma, where research is largely academic driven and not by pharmaceutical companies, these aspects further limit progress of new therapeutic approaches. The chicken chorioallantoic membrane (CAM) assay might be an attractive alternative to conventional animal models. The CAM assay is a well-established and highly reproducible model in the field of angiogenesis  and is also widely utilized as an in vivo system to study the aggressiveness of various tumors including prostate cancer , glioblastoma [9, 10] and colon carcinoma . During the chicken embryo development, the CAM is formed by fusion of the mesodermal layer of the allantois with the mesodermal layer of the chorion. It is highly vascularized, making it an ideal substrate for the cultivation of tumors and the study of angiogenesis. Together with the extracellular matrix proteins (ECM) the CAM mimics the physiological cancer environment. Grafting of tumors is further facilitated by a natural immunodeficiency of the CAM lacking cell-mediated immunity until day 14 . Tumor cells rapidly form three dimensional tumors, infiltrate the surrounding tissue and even metastasize to different organs of the embryo. The CAM is not innervated so that experiments are not associated with pain perception by the embryo and there is no need for ethical approval for animal experimentation. Further advantages of the CAM assay are high reproducibility, cost effectiveness and short incubation times . However, the CAM assay has several methodological challenges when used as an in vivo model in tumor research, e.g. inconsistent chick embryo viability, inconsistent and low tumor take rates and delayed or slow tumor growth. Despite these challenges, the CAM assay could be successfully adapted for different tumor entities and is now widely applied for the analysis of several tumors including neuroblastoma , squamous cell carcinoma , multiple myeloma  and glioblastoma . Unfortunately, this adaptation could not yet be achieved for osteosarcoma, resulting in a rare application of this promising in vivo model in osteosarcoma research. A PubMed search on osteosarcoma and CAM assays retrieved only 16 matches with conflicting data and little information concerning the achieved tumor take rates, tumor volumes and viability of the embryos. In addition, there are very few reports with detailed protocols [18, 19], none of them presenting a systematic adaptation and optimization of the CAM assay for osteosarcoma, as described for other entities.
In vivo models are indispensable tools for the study of osteosarcoma biology and the identification of new therapeutic targets. Especially the heterogeneity of this tumor, the interaction with the tumor microenvironment and the intratumoral vascularization are not sufficiently taken into account when in vitro assays are applied. In our effort to reduce or even replace animal experiments we aimed to investigate the suitability, reliability and reproducibility of the chicken chorioallantoic membrane assay as an in vivo tumor model for osteosarcoma research. Although the CAM assay is widely used to study angiogenesis and the biology of various types of tumors little information is available about the use of this assay for osteosarcoma research. The ability of the osteosarcoma cell line Saos-2 to induce new blood vessel formation by upregulation of angiogenic growth factors like VEGF165, FGF2, MMP2 and MMP9 has already been shown. However this study only focused on the early phase of tumor growth within the first 96 hours . Another study also demonstrated the formation of solid tumors and the induction of an angiogenic response after transplantation of different osteosarcoma cell lines onto the CAM. However, only 3 out of 8 cells lines consistently formed vascularized tumors and the mortality rate of the embryos was very high ranging from 18% – 70% . Interestingly, tumor grafts derived from sarcoma patients have been shown to retain tumor morphology, viability, and invasion potential in the chick chorioallantoic membrane model .
The CAM assay can bridge the gap between in vitro cell culture and in vivo animal experiments. As an in vivo model for osteosarcoma research it helps to investigate tumor properties and cellular mechanisms including tumor microenvironment, may speed up preclinical data collection and simplifies research on potential new agents towards personalized medicine. The reasonable use of this model provides a refinement by minimizing pain and suffering of animals and supports a considerable reduction and/or replacement of animal experiments in accordance with Russell’s and Burch’s “Principles of Humane Experimental Technique”.