title: An investigation into the relationship between cellular metabolism and small extracellular vesicle release in metastatic breast cancer
Creators: name: Gounis, Michail D.
Publisher: name: University of Glasgow
Year: 2022

description: Altered cellular metabolism and release of small extracellular vesicles (sEVs) contribute to acquisition of invasive phenotypes that drive tumour dissemination and metastasis, the main cause of cancer-related death. Both these processes depend on membrane trafficking events that allow communication of cancer cells with different microenvironments on the path to metastasis, and thus shape their ability to colonise and outgrow in distant organs. To investigate these processes, we have combined the MMTV-PyMT mouse model of mammary carcinoma with a syngeneic transplantation and primary tumour resection approach to generate isogenic cells from primary tumours and their corresponding lung micrometastases. Phenotypic characterisation of these cell lines showed that, despite similar growth rates, lung micrometastatic cells exhibited more mesenchymal characteristics than primary tumour-derived cells. In particular, gene set enrichment analysis (GSEA) of differentially expressed genes identified an epithelial-to-mesenchymal transition (EMT) signature in micrometastatic cells, despite the retention of some epithelial characteristics, such as E-cadherin expression. Furthermore, when plated in 3D microenvironment, cells lines from lung metastases were more invasive than those from primary tumours. Liquid chromatography-mass spectrometry (LC-MS) analysis indicated that there were significant differences between the steady-state metabolome of primary tumour and micrometastatic cells – notably that glutathione levels were decreased in micrometastatic cells and that they synthesised and secreted more proline than their primary tumour counterparts. We also used LC-MS to compare the lipidome of PyMT-derived cell lines and found that micrometastatic cells differed in lipid composition. Notably, micrometastatic cells were enriched in certain sphingolipid species, in particular (C16-, C18-) ceramide, which is known to influence sEV biogenesis. Using differential centrifugation in combination with nanoparticle tracking and Western blotting, we found that cells from lung micrometastases secreted more sEVs than their primary tumour counterparts, and these vesicles are highly enriched in the sEV markers, CD63, CD81, and TSG101. We then explored the role of ceramide in sEV release by generating neutral sphingomyelinase (nSMase) CRISPR-knockout isogenic cell lines, and demonstrated that increased sEV release from micrometastatic cells, but not from their matched primary tumour cells, was opposed following deletion of nSMase2. However, no significant differences were found in the size or number of sEVs released from nSMase1-knockout PyMT-derived cells. Furthermore, pharmacological depletion of glutathione led to upregulation of CD63 positive sEVs released from primary tumour cells in a nSMase2-dependent manner. Deletion of nSMase2 did not affect the invasive migration of PyMT-derived micrometastatic cells on fibroblast-derived ECM, but abrogated their ability to invade into a collagen-rich organotypic environment. Collectively, these data indicate that increased sEV release by micrometastatic cells contributes to the generation of microenvironment that supports invasiveness. Taken together, these data provide evidence that metabolic rewiring, and in particular glutathione and ceramide levels, influence production of sEVs in cells from mammary carcinoma-derived lung micrometastases in a way that supports a more invasive microenvironment.