Development of the chick chorioallantoic membrane (CAM) model to assess tumour growth and drug response in osteosarcoma and bladder cancer

The prognosis for cancer patients with metastasis remains generally low, prompting research into understanding the different metastatic pathways and testing novel chemotherapeutical agents. While the traditional in vivo mouse models have been widely used in research, they are often expensive and time- intensive, leading researchers to explore alternative models. This study aims to optimize and apply the chick chorioallantoic membrane (CAM) assay, an innovative, cost-effective in ovo model that allows visualisation of tumour growth in an in vivo-like environment while minimising animal suffering. While CAM studies have shown promise in osteosarcoma research, their application in bladder cancer (BC) remain limited. This study examines the growth patterns of two BC cell lines (T24 and SCaBER) alongside an osteosarcoma cell line (HOS-143B) using the CAM model. Additionally, BC cell behaviour was assessed in a bone-like environment, simulated using a NuOSS scaffold, and compared to a non-bone-like extracellular matrix. Furthermore, a STAT3 inhibitor (STAT3-IN-1) will be applied to HOS-143B cells to evaluate its effects on tumour growth and behaviour in an in vivo-like setting.

In this study, the CAM assay was conducted over a 13-day period, resulting in tumour formation for all three cell lines when using the NuOSS scaffold with a cell density of 2x10⁶ cells per egg. Haematoxylin and eosin (H&E) staining confirmed tumour growth in all cell lines. HOS-143B exhibited the most robust proliferation, characterised by basophilic nuclei, acidophilic cytoplasm and vascularisation. In contrast, BC cell lines demonstrated slower proliferation and localised invasion, likely due to the bone scaffold not being their native environment but only a site of metastasis. In STAT3-IN-1 treated HOS-143B samples, histological analysis revealed reduced tumour cells proliferation and vascularisation, supporting the inhibitor’s anti-angiogenic effects. In contrast, the use of Matrigel as a scaffold posed significant challenges, with dispersal of the gel within the CAM limiting visualisation and extraction, resulting in a lack of consistent tumour formation.

This study demonstrates the CAM assay's efficacy in assessing tumour growth and drug responses, with the NuOSS scaffold emerging as particularly effective for establishing tumours. Despite its sensitivity to environmental variability and the labour-intensive nature of the technique, the CAM assay offers significant advantages. Selecting an appropriate scaffold is especially crucial, as it supports cell growth and closely replicates the native tumour environment. These findings emphasize the CAM model's potential as a valuable tool in cancer research, providing meaningful insights into the proliferative and invasive behaviours of cancer cells across varied microenvironments.