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Development of three dimensional and in vivo modelling systems for the overexpression of Active-β-catenin in osteosarcoma
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- Author / Creator
- Hinton, Kristin M
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Introduction:
Osteosarcoma (OS) is a primary bone malignancy most commonly found in children and adolescents. The 5-year survival rate for OS is 60%, which decreases to 27% when distant metastases are present. Currently no tests are available to identify those patients at risk of metastatic spread at the time of diagnosis. The canonical Wnt/β-catenin signalling pathway has been implicated in many cancers including OS, and overexpression of β-catenin has been shown to correlate with metastasis in OS. Recently, active β-catenin (ABC)—the transcriptionally active form of β-catenin—has specifically been found to be elevated in the metastatic cell lines SaOS2-LM7 (LM7) and HOS-143B (143B) compared to their non-metastatic parent cell lines, SaOS2 and HOS. The present study was undertaken to develop three-dimensional (3D) cell culture and in vivo models to observe the effects of over-expression of both ABC and β-catenin in OS with the hypothesis that increased expression of ABC but not β-catenin would result in more aggressive cellular features in three-dimensional (3D) cell culture.
Methods:
OS cell lines SaOS2 and HOS underwent liposomal transfection with pEGFP-C2-ABC (SaOS2-ABC, HOS-ABC), pEGFP-C2-β-catenin (SaOS2-βcat, HOS-βcat), and an empty vector negative control (SaOS2-GFP, HOS-GFP). For each transfected cell line, untransfected parent cell line, and the metastatic daughter cell lines LM7 and 143B, 3D cell cultures (spheroids) were established in a scaffold with differing proportions of an extracellular protein-rich basement membrane matrix (BMM) and collagen and maintained for 7-14 days. Prior to fixation, low resolution images were taken of the live cells to observe the spheroid morphology and interactions. These images were analyzed to measure the invasion area (IA) and spheroid area (SA), which were used to calculate invasion length. To investigate the effects of the overexpression of ABC on OS in vivo, an orthotopic murine xenograft model was developed. The proximal tibial metaphysis of NOD/SCID mice was injected with a cell suspension containing either SaOS2, SaOS2-ABC, SaOS2-βcat, SaOS2-GFP, or LM7 cells. Mice were euthanized after 28 days, and the tibias and lungs were examined for primary and metastatic tumors.
Results
3D cultures conditions were optimized for the growth of all untransfected cells and for transiently transfected HOS spheroids. Stably transfected HOS cells formed smaller spheroids with smaller invasion areas (p<0.05). Transfected SaOS2 cells did not readily for spheroids in the 3D culture conditions. Intraosseous injection of SaOS2 and LM7 cells into the proximal tibial metaphysis did not result in primary or metastatic tumors for any cell line.
Conclusions:
We developed a 3D cell culture protocol to study the effects of ABC overexpression on OS, particularly in HOS cells. We anticipate this will allow us to further explore whether ABC plays a role in aggressive and metastatic OS that is distinct from β-catenin, which has been suggested by previous findings from our lab. Evaluating ABC levels at the time of diagnostic biopsy may be beneficial to identify patients at risk for metastasis. Additional pharmacologic and clinical investigations would confirm the viability of ABC as a prognostic marker and therapeutic target. -
- Graduation date
- Fall 2023
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.