Characterization of ascites-derived chicken ovarian cancer cells: evidence for the presence of a pool of highly invasive cancer stem cells
Open Access
- Author:
- Tiwari, Anupama
- Graduate Program:
- Animal Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 09, 2014
- Committee Members:
- Dr Ramesh Ramachandran, Dissertation Advisor/Co-Advisor
Alan Leslie Johnson, Committee Member
Joy Lee Pate, Committee Member
Kumble Sandeep Prabhu, Committee Member
Daniel R Hagen, Committee Member - Keywords:
- Ovarian cancer
chicken ovarian cancer cells
ovarian cancer stem cells
aquaporin-5 in chicken ovarian cancer - Abstract:
- Ovarian cancer (OC) is a highly metastatic disease and fifth leading cause of cancer-related deaths in women worldwide. Laying hens are the most appropriate animal model for human OC as chickens develop OC spontaneously, similar to women. Although the chicken model is being extensively used to study human OC by several groups across the world, the cellular and molecular biology of chicken ovarian cancer (COVCAR) cells has not been explored in the past. The objective of the first study was to isolate COVCAR cells from ascites, develop an in vitro culture system and characterize gene expression, growth pattern, and invasive property of OC cells. COVCAR cells expressed several tumor metastasis-related genes, exhibited robust growth pattern at earlier passages and were found to exhibit greater invasive properties compared to normal ovarian surface epithelial (NOSE) cells. Since advanced stages of OC are usually accompanied by the presence of ascites and greater expression of water channel protein aquaporin-5 (AQP5) which has been positively correlated with ascites volume and lymph node metastasis in OC patients, we sought to elucidate the expression of AQP5 in the cancerous chicken ovaries and in ascites-derived COVCAR cell lines. Therefore, the objective of the second study was to determine if AQP5 expression was altered in COVCAR cells and cancerous chicken ovaries compared to that in NOSE cells and normal ovaries, respectively. Both AQP5 mRNA and protein levels were found to be altered in COVCAR cells and cancerous ovaries, suggesting association of AQP5 expression with ovarian tumorigenesis in chicken. Cancer stem cells (CSCs), also known as tumor initiating cells, are reported to be responsible for tumor origin, development, metastasis, and treatment failure in several cancers including OC. High aldehyde dehydrogenase 1 (ALDH1) activity has been associated with stemness in various cell lineages including OC cells. We sought to determine if a stem cell pool with high ALDH1 activity existed in ascites-derived COVCAR cells. Therefore, the objective of the third study was to isolate stem (ALDH1+) cells from ascites-derived COVCAR cells utilizing greater ALDH1 activity and in addition, to compare ALDH1+ and ALDH1- cells for their anchorage-independent growth, invasive properties and ovosphere forming abilities in vitro. Utilizing next-generation sequencing approach, a global transcriptomic analyses was performed to determine differential expression of genes and identify biological pathway networks in ALDH1+ and ALDH1- cells. ALDH1+ cells exhibited greater sphere formation and invasive properties than ALDH1- cells. ALDH1+ cells also exhibited a distinct morphology in an anchorage-independent three dimensional culture with formation of unique intra-and inter-ovosphere channels connecting multiple ovospheres. Interestingly, ALDH1+ cells exclusively expressed genes related to self-renewal and stem cell homeostasis (WNT7B) and invasiveness (GNRHR and TNFRSF19), while ALDH1- cells expressed genes related to apoptosis (CD72). Collectively, through the studies presented herein we have developed an in vitro system for culturing COVCAR cells, developed 31 ascites-derived COVCAR cell lines, and identified altered expression of water channel protein AQP5 and presence of a CSCs pool in COVCAR cells. This study enhances our understanding of the cellular and molecular biology of COVCAR cells. These COVCAR cells can be utilized in the future to identify factors that could contribute to their invasive properties. It would also be of interest to determine angiogenic and tumorigenic potential of CSCs in an in vivo system and the effect of hypoxia on these properties of CSCs. Furthermore, CSCs and COVCAR cells developed in this study could be utilized for screening potential anti-cancer compounds in combination with in vivo cancer preventive and therapeutic approaches.