An Examination of Adaptive, Metabolism-Associated Cellular Reprogramming in Epithelial Ovarian Cancer Spheroids
Abstract
Epithelial ovarian cancer (EOC) is typically diagnosed at an advanced stage, for which the five-year survival rate is approximately 30%. Accordingly, EOC is the most lethal gynaecologic malignancy. The metastatic mechanism of EOC is uncommon but efficient: cells exfoliate from the primary tumour into the peritoneal space, where they form multicellular aggregates called spheroids. Natural motion of the peritoneal fluid spreads spheroids throughout the peritoneal cavity, enabling them to reattach, invade, and initiate secondary lesions at distant sites. Spheroid formation induces numerous signaling responses in constituent cells, ultimately facilitating metastasis by promoting resistance to detachment-induced cell death (anoikis). Previous studies have identified several adaptive responses that are activated in EOC spheroids and contribute to anoikis resistance, including quiescence, AMP-activated protein kinase (AMPK) signaling, and autophagy. Each of these responses is linked to altered cellular metabolism, and direct evidence of metabolic reprogramming in EOC spheroids has been uncovered in further investigations. Spatially heterogeneous environmental conditions have been shown to develop within spheroids due to their three-dimensional structure, and it is thought that these conditions may contribute to the induction of adaptive responses. However, few studies have been designed to investigate spatial regulation of these responses in spheroids. In this thesis, I have described the development of Spatial Profiling of Ratiometric Trends in Spheroids (SPoRTS) a novel image analysis tool for monitoring the spatial regulation of biological activities in spheroids. I used SPoRTS to assess spatiotemporal regulation of autophagy and mitophagy in EOC spheroids, finding disparities that prompted analysis of mitochondrial organization in these structures. This uncovered evidence of locally upregulated mitochondrial fusion in regions of spheroids with high autophagy activity and low mitophagy activity. I then demonstrated that blocking mitochondrial dynamics processes is deleterious in spheroids, and can influence spheroid aggregation. Last, I determined that ablation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKKβ)–AMPK signaling is detrimental to the growth and viability of EOC spheroids and xenografts. Altogether, I have generated a new analysis tool for the research community, elucidated the complexity of metabolic reprogramming processes during EOC metastasis, and demonstrated the potential of targeting the associated pathways therapeutically.