Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane

Abstract

Background: The chick chorio-allantoic membrane (CAM) assay is a commonly used method for studying angiogenic or anti-angiogenic activities in vivo. The ease of access allows direct monitoring of tumour growth by biomicroscopy and the possibility to screen many samples in an inexpensive way. The CAM model provides a powerful tool to study effects of molecules, which interfere with physiological angiogenesis, or experimental tumours derived from cancer cell lines. We therefore screened eight osteosarcoma cell lines for their ability to form vascularized tumours on the CAM.

Findings: We implanted 3-5 million cells of human osteosarcoma lines (HOS, MG63, MNNG-HOS, OST, SAOS, SJSA1, U2OS, ZK58) on the CAM at day 10 of embryonic development. Tumour growth was monitored by in vivo biomicroscopy at different time points and tumours were fixed in paraformaldehyde seven days after cell grafting. The tissue was observed, photographed and selected cases were further analyzed using standard histology.From the eight cell lines the MNNG-HOS, U2OS and SAOS were able to form solid tumours when grafted on the CAM. The MNNG-HOS tumours showed the most reliable and consistent growth and were able to penetrate the chorionic epithelium, grow in the CAM stroma and induce a strong angiogenic response.

Conclusions: Our results show that the CAM assay is a useful tool for studying osteosarcoma growth. The model provides an excellent alternative to current rodent models and could serve as a preclinical screening assay for anticancer molecules. It might increase the speed and efficacy of the development of new drugs for the treatment of osteosarcoma.

Figure 1

Tumours of three different cell lines grown on the CAM. In vivo microscopy of tumours grown of indicated cell lines after 7 days of tumour growth. A - C: magnification 10× (scale bar 1 mm), D - E: higher magnification (40×, scale bar 250 μm) of A - C. G: Graph illustrating the mean tumour volumes calculated by the following formula: V = 4/3*π*r³ (r = 1/2 * square root of diameter 1 * diameter 2). SEM = standard error of mean.

Figure 2

Typical growth pattern of MNNG-HOS cells grafted to the CAM. A - C: In vivo microscopy (10×, scale bar 1 mm) documenting the typical growth pattern after implantation of MNNG-HOS cells, arrowheads indicate tumour boundaries. A 1 day after grafting. After 4 days a vascularized tumour becomes apparent (B). Solidification of the tumour steadily progresses until day 7 (C). Note the bleeding caused by the tumour in C. D (12.5×, scale bar 0.8 mm) and E (50×, scale bar 20 μm) are higher magnifications of C after turning the CAM upside down. Note the three smaller tumour nodules that were not visible from the upside (C). F (12.5×, scale bar 0.8 mm) and G (50×, scale bar 20 μm) is another example of a typical MNNG-HOS tumour. Note the rich vascularization of the tumour (G).

Figure 3

Standard histology of MNNG-HOS tumour. Standard histology with hematoxylin-eosin staining confirming the rich vascularisation of the tumours with capillaries originating from the CAM (arrows in A and B). The tumours also contain stromal cells with disseminated monocytes. Areas of angiogenesis (arrowheads in B and C), necrosis and haemorrhage (asterisks in C) are present. A: Macroscopic picture, B: HE 10× (scale bar 100 μm), C: HE 40× (scale bar 50 μm).