Characterization of ascites-derived aldehyde dehydrogenase–positive ovarian cancer stem cells isolated from Leghorn chickens

Leghorn chickens are used as a preclinical model of ovarian cancer as they develop epithelial ovarian adenocarcinoma spontaneously at a very high frequency. Ovarian cancer is the most lethal disease among all gynecological malignancies in women. A small proportion of ovarian cancer stem cells are responsible for drug resistance and relapse of ovarian cancer. The objectives of this study are to isolate ovarian cancer stem cells from ascites of Leghorn chickens that spontaneously developed ovarian cancer and to determine their invasiveness, spheroid formation in three-dimensional culture devoid of extracellular matrix over several months. Ovarian cancer cells obtained from ascites were subjected to ALDEFLOUR assay that measures aldehyde dehydrogenase (ALDH) activity to separate ALDH1⁺ and ALDH1^- cells by fluorescence-activated cell sorting. The cells were cultured using serum-free media for up to 6 mo in ultra-low attachment plates. Invasiveness of ALDH1⁺ and ALDH1^- cells was determined by Matrigel invasion assay. Cellular uptake of acetylated low-density lipoprotein was evaluated. A small proportion (<4.75%) of ovarian cancer cells isolated from ascites were found to be ALDH1⁺ cells. ALDH1⁺ cells formed a greater number of spheroids and were also highly invasive in extracellular matrix compared to ALDH1^- cells. Several spheroids developed 0.1- to 1-mm-long capillary-like tubules connecting other spheroids, thus forming a complex network that underwent remodeling over several months. Cells in the spheroids incorporated acetylated low-density lipoprotein suggestive of scavenger receptor activity. In summary, ALDH1⁺ ovarian cancer stem cells isolated from ascites of chickens appear to be invasive and form spheroids with complex networks of tubules reminiscent of vascular mimicry. Understanding the structure and function of spheroids and tubular network would provide valuable insight into the biology of ovarian cancer and improve poultry health.