Monitoring of tumor growth and vascularization with repetitive ultrasonography in the chicken chorioallantoic-membrane-assay

Abstract

The chorioallantoic-membrane (CAM)-assay is an established model for in vivo tumor research. Contrary to rodent-xenograft-models, the CAM-assay does not require breeding of immunodeficient strains due to native immunodeficiency. This allows xenografts to grow on the non-innervated CAM without pain or impairment for the embryo. Considering multidirectional tumor growth, limited monitoring capability of tumor size is the main methodological limitation of the CAM-assay for tumor research. Enclosure of the tumor by the radiopaque eggshell and the small structural size only allows monitoring from above and challenges established imaging techniques. We report the eligibility of ultrasonography for repetitive visualization of tumor growth and vascularization in the CAM-assay. After tumor ingrowth, ultrasonography was repetitively performed in ovo using a commercial ultrasonographic scanner. Finally, the tumor was excised and histologically analyzed. Tumor growth and angiogenesis were successfully monitored and findings in ultrasonographic imaging significantly correlated with results obtained in histological analysis. Ultrasonography is cost efficient and widely available. Tumor imaging in ovo enables the longitudinal monitoring of tumoral development, yet allowing high quantitative output due to the CAM-assays simple and cheap methodology. Thus, this methodological novelty improves reproducibility in the field of in vivo tumor experimentation emphasizing the CAM-assay as an alternative to rodent-xenograft-models.

Utilisation of Chick Embryo Chorioallantoic Membrane as a Model Platform for Imaging-Navigated Biomedical Research

“…It is suitable to visualise tumour growth and vascularisation in the CAM assay. By using a commercial ultrasonographic scanner, tumour growth and angiogenesis were successfully monitored, and results of ultrasound images were significantly correlated with those of histological analysis from the excised tumour [ 155 ].…”

“…Osteosarcoma cells were implanted onto the CAM at ED 9 [ 153 ], and urological cancers cells were implanted onto the CAM on ED 10 [ 154 ]. Although HuH7 liver cancer cells were successfully implanted on the CAM since ED 7 [ 155 ], we still recommend EDs 8–10 as a good time course for tumour implantation.…”

Current Strategies in Assessment of Nanotoxicity: Alternatives to In Vivo Animal Testing

“…The use of non-mammalian alternative models may be an ideal strategy to overcome the ethical concerns related to the traditional animal models in the safety assessment of NPs. To assess the hazards of engineered NPs, non-mammalian models such as [ 109 ] Caenorhabditis elegans ( C. elegans ) [ 110 ], Drosophila ( Drosophila melanogaster ) [ 111 ], African clawed frog ( Xenopus laevis ) [ 112 ], chicken chorioallantoic membrane ( Gallus gallus ) [ 113 ], and zebrafish ( Danio rerio ) [ 114 ] could be considered as reliable approaches to improve the reliability of assessments of the toxicity of NPs.…”

The Chorioallantoic Membrane Assay in Nanotoxicological Research—An Alternative for In Vivo Experimentation

“…The chorioallantoic membrane (CAM) assay in a non-innervated, highly vascularized, extraembryonic membrane [57,58]. The CAM can be considered to be placental equivalent to mammals and has been established itself as a versatile alternative to conventional rodent experimentation and meets the criteria of the 3R principle [57][58][59][60]. As chicken embryos are living vertebrates with a circulatory system and organic functions, this model can serve as a suitable substitute for various in vivo experimentation (Figure 1) [57,60,61].…”

“…The CAM can be considered to be placental equivalent to mammals and has been established itself as a versatile alternative to conventional rodent experimentation and meets the criteria of the 3R principle [57][58][59][60]. As chicken embryos are living vertebrates with a circulatory system and organic functions, this model can serve as a suitable substitute for various in vivo experimentation (Figure 1) [57,60,61]. Since Luepke identified the CAM assay as an alternative in vivo method to evaluate mucosa irritation [61], the assay has substituted the Draize Eye Irritation Test for various substances [62][63][64][65].…”