Development of the chick chorioallantoic membrane (CAM) xenograft model to study metastasis and angiogenesis in autophagy deficient osteosarcoma cells

Osteosarcoma (OS) is the most common primary malignant bone cancer in children. Despite the improvement in multiagent chemotherapy, 5-year survival rate for metastatic OS is only 20% to 30%. One of the main reasons for failure in therapy is due to the activation of autophagy in metastasis and angiogenesis, leading to an unpredictable prognosis.

The Chicken Chorioallantoic Membrane (CAM) assay has emerged as a valuable tool, offering an easy, inexpensive and rapid alternative for direct visualisation of tumour development. This study aimed to design and optimise the CAM assay as a physiologically relevant model to understand OS metastasis and angiogenesis, and how the expression of cancer markers may vary in a 3D environment when autophagy is suppressed.

In this study, the CAM assay was completed in 13 embryonic development days (EDD). After several optimising the protocol for viability and tumour implantation, the experimental survival rate improved significantly to ~69% (p<0.01), and successfully formed tumours from metastatic HOS-143B cells wild type (WT), scrambled control (NC), and CRISPR/Cas9 knockout (KO) ATG7 (ATG7 is an essential regulator of the autophagy process).

The role of autophagy in the in ovo and in vitro environment was shown to be context-dependent, with metastasis and angiogenesis linked genes. In an in ovo microenvironment, metastatic gene expression (CXCR4, CCL5, EZRIN, FGF2, EGFR) was decreased by ~13.08-fold (p<0.05) on average in ATG7 CRISPR/Cas9 knocked-out HOS-143B tumours compared to WT HOS-143B tumours, and by ~9.21-fold on average compared to NC HOS-143B tumours. Interestingly, in vitro, ATG7 KO cells had a 94.1-fold (p<0.05) and a 2.56-fold (p<0.05) fold increase on MMP2 and MMP9 genes compared to WT and NC HOS-143B cells respectively. However, in the in ovo environment, a reduction of 20.17-fold (p<0.001) and 14.31-fold (p<0.05) on MMP2 and MMP9 genes was seen compared to WT and NC HOS-143B tumours, suggesting that autophagy may trigger different signalling pathways relevant to the microenvironment. When stained with haematoxylin and eosin (H&E), the highest levels of cancer cell proliferation, vascularisation, and implant attachment were seen in WT and NC HOS-143B tumours compared to ATG7 KO HOS-143B tumours. According to immunohistochemistry, levels of the angiogenesis marker-VEGFR2 in WT and NC HOS-143B cells was significantly greater by 29.16% (p<0.05) compared to ATG7 KO HOS-143B cells, which demonstrated limited vascularisation ability. Therefore, this work contributes to our understanding of osteosarcoma metastasis, but also identifies autophagy as a therapeutic target to suppress metastasis and angiogenesis, offering hope to patients and healthcare professionals.