Regulation of cancer stem cell metabolism and mitochondrial dynamics by extra cellular matrix Priya Gatti*1, Pritha Mukherjee2, Urmi Chatterji2, Marc Germain1 1 Département de Biologie Médicale, Université du Québec à Trois-Rivières Québec, CERMO-FC UQAM, Canada. 2Department of Zoology, University of Calcutta, India Abstract: Cancer stem cells (CSCs) are a subpopulation of cells within a tumour that have stem cell-like properties with tumorigenic capacity, making them a driver of metastasis and therapeutic resistance. Like other stem cells, CSCs are suggested to have a distinct metabolic profile, with recent reports indicating that CSCs can switch their metabolic phenotypes between glycolytic and OXPHOS-dependent pathways according to their microenvironment. The key metabolic hub, mitochondria are also altered in CSC, although different groups reported conflicting results in its structure and function. As these unrecognized pathways regulating mitochondria in CSC promoting growth and survival is unknown, we set to identify these factors. Here, we show that CSC attachment to the extra cellular matrix (ECM) drives changes in mitochondrial structure and impact their function. To study the role of the ECM in the regulation of CSC, we generated CSCs from different breast cancer cell lines. We then attached the CSC to different ECM substrates and measured mitochondrial structure and function. Specifically, CSCs generated from breast cancer cell lines in suspension showed fragmented mitochondria, but rapidly elongated their mitochondrial network upon attachment. This was associated with the oligomerization of mitochondrial inner membrane dynamin OPA1, which regulates mitochondrial fusion and cristae maintenance. Importantly, these changes did not affect cell viability, but regulated mitochondrial metabolism and function. Altogether, our results indicate that CSC mitochondrial structure and function is regulated by ECM attachment. Different microenvironment (for example cells within a solid tumour vs cells that have escaped the tumour microenvironment) could show distinct metabolic profiles independently of their genetic makeup.