Development of the chick Chorioallantoic Membrane (CAM) xenograft model to study metastasis in autophagy-deficient osteosarcoma cells
Conference item
Tha Shrestha, B., Ghali, L., Roberts, S.J. and Roberts, H.C. 2024. Development of the chick Chorioallantoic Membrane (CAM) xenograft model to study metastasis in autophagy-deficient osteosarcoma cells. Bone Research Society Annual Meeting 2024. Shefield, UK 10 - 12 Jul 2024
| Title | Development of the chick Chorioallantoic Membrane (CAM) xenograft model to study metastasis in autophagy-deficient osteosarcoma cells |
|---|---|
| Authors | Tha Shrestha, B., Ghali, L., Roberts, S.J. and Roberts, H.C. |
| Abstract | Osteosarcoma (OS) is the most common type of bone cancer in children. Despite advances in chemotherapy, the survival rate for metastatic OS is only 20% to 30%. We have previously shown that the activation of autophagy may be linked to metastasis and chemoresistance, leading to an unpredictable prognosis. The Chicken Chorioallantoic Membrane (CAM) assay is an inexpensive and rapid methodology to visualise tumour development, making it a valuable tool in studying cancer metastasis. This study aimed to develop the CAM assay as a physiologically relevant model to better understand OS and how 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 optimisation steps for viability and tumour implantation, the experimental survival rate improved significantly to 69% (p<0.01), and successfully formed tumours from metastatic wild type (WT) HOS143B cells (n=10), scrambled control (SC) (n=9), and CRISPR/Cas9 knockout (KO) ATG7 HOS-143B cells (n=9). ATG7 is an essential regulator of the autophagy process. The role of autophagy in the in ovo and in vitro environment was found to be context-dependent. In the in ovo microenvironment, metastatic gene expression was downregulated in CXCR4 by 5.04-fold (p<0.05), CCL5 by 5.16-fold (p<0.05), and EZRIN by 3.75-fold in ATG7 CRISPR/Cas9 KO HOS-143B tumours compared to WT HOS-143B tumours, and CXCR4 by 1.37-fold, CCL5 by 1.59-fold, and EZRIN by 2.08-fold in ATG7 KO tumours compared to SC HOS-143B tumours. In vitro, ATG7 KO cells exhibited a 94.1-fold (p<0.05) and 2.56-fold (p<0.05) increase in 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 14.31fold (p<0.05) in 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. Immunohistochemistry demonstrated that WT and SC HOS-143B cells exhibited significantly greater levels of the angiogenesis marker VEGFR2, at 33.93% (p<0.01) and 24.40% (p<0.05), respectively, compared to ATG7 KO HOS-143B cells, which had reduced vascularisation. This study contributes to our understanding of OS metastasis and also identifies autophagy as a therapeutic target to modify metastatic associated genes/proteins. Importantly, in ovo culturing was required to fully reveal these outcomes, thus defining the importance of considering the interaction of the microenvironment and potentially vasculature in understanding OS metastasis. |
| Keywords | Osteosarcoma; Chicken Chorioallantoic Membrane assay; Metastasis; angiogenesis; microenvironment |
| Sustainable Development Goals | 3 Good health and well-being |
| Middlesex University Theme | Health & Wellbeing |
| Conference | Bone Research Society Annual Meeting 2024 |
| Publication process dates | |
| Completed | 10 Jul 2024 |
| Deposited | 10 Oct 2025 |
| Output status | Published |
| Additional information | The abstract for this oral presentation was published in the journal JBMR Plus: |
| Related Output | |
| Is documented by | https://doi.org/10.1093/jbmrpl/ziae109 |
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