Transformation of lysosomes is a long-known trait of cancer cells. Lysosomes are the main degradative compartments of the cell and central in regulating the delicate balance between anabolism and catabolism. How lysosome transformation occurs in cancer cells and how this translates into cancer progression is not known, but clearly of great importance. A major obstacle has been the lack of suitable imaging methods to follow single lysosomes in migrating cancer cells. Traditional ‘bulk’ microscopy yields averaged data, which hampers to distinguish ‘transformed’ lysosomes from ‘housekeeping’ lysosomes. Moreover, cancer cell migration should be studied in 3D, which poses another challenge for microscopy. Recently, I overcame these obstacles by developing a novel correlative 3D live-cell to electron microscopy (3D- CLEM) method which I applied to 3D cancer cell cultures. This resulted in 2 intriguing observations. First, I found a direct correlation between the invasive traits of cancer cells and accumulation of peripheral lysosomes at the cellular pseudopods. Second, in contrast to common theories, I found that most peripheral lysosomes are poor in lysosomal hydrolase activity and by Electron Microscopy (EM) show a drastically altered morphology. Based on these data I hypothesize that cancer cells form a special subpopulation of lysosomes that reposition to the cell periphery as a crucial step in cancer cell invasion. The aim of this proposal is to understand the role of these transformed lysosomes and establish the molecules and pathways that regulate their transformation. To this end I will advance the 3D-CLEM approach into a high throughput method to allow quantitative subcellular imaging in 3D cell cultures. I will complement this with mass spectroscopy to unravel the molecular composition of the transformed lysosomes. Moreover, I will investigate the cellular transport mechanisms that repositions lysosomes to the invading edge. These studies will lead to novel fundamental insights in lysosome biology and can reveal thus-far unexplored targets for cancer therapies.