A ZEBRAFISH XENOGRAFT PLATFORM FOR SEMI-QUANTITATIVE ANALYSIS OF INDIVIDUAL STEPS OF THE METASTATIC CASCADE

Metastatic disease represents the main cause of most cancer-related deaths, yet this process remains one of the most enigmatic aspects of cancer biology. Metastatic progression includes multiple distinct phases, such as invasion, intra- and extravasation, seeding at distant sites, micro-metastasis formation and metastatic outgrowth. Only a deep understanding of each of these steps may offer novel targets to be exploited in a clinical setting. Emerging evidence suggests that metastatization is not a genetically selected trait, but the result of phenotypic adaptation to the unfavorable microenvironmental conditions which cancer cells are exposed to (e.g. hypoxia or low nutrients in the primary site or bloodstream). Functional genomic and differential expression screenings allowed to identify numerous potential metastasis genes, that need to be prioritized and validated with appropriate pre-clinical in vivo systems, ideally able to recapitulate and explore the entire metastatic cascade. Mouse models present several critical limitations, including the difficulty to visualize and quantify the metastatic phases, except in sophisticated experimental contexts. Therefore, I propose xenotransplantation of human cancer cells in zebrafish larvae as a system to evaluate primary tumor implantation and growth and to quantitatively dissect metastatic progression, with the purpose of mapping gene functions in the multistep cascade. First, I set up a robust protocol for subcutaneous and intravenous transplantation of fluorescently labeled cells in zebrafish larvae, using the metastatic breast cancer (BC) cell line MDA-MB-231 and non-tumorigenic MCF10A mammary cells as a control. MDA-MB-231 cells survive, proliferate and disseminate in the zebrafish host, developing tumors with conserved histological features and originating extravascular metastases in just 4 days. Using a zebrafish strain with fluorescent vasculature and visually following each fish by live microscopy, I optimized a set of analytical tools to quantitatively investigate incidence, numerosity, size and proliferative index of metastases, as well as measuring frequency of metastasis initiating cells and the severity of metastatic progression. Secondly, I challenged the zebrafish platform by transplanting MDA-MB-231 cells separately interfered for 6 genetic targets, previously identified in our laboratory, as potential novel mediators of BC metastasis. I successfully validated the contribution of 5 out of 6 of these genes in BC metastatization, attributing a specific role in the metastatic cascade for each of them. The identification and in vivo validation of this stage-specific determinants of metastasis offers potential new targets to halt metastatic disease, to be further explored in future pre-clinical and clinical studies, paving the way to novel and effective anti-metastatic therapeutic approaches.