DanioCTC: Injection of circulating tumor cells from metastatic breast cancer patients in zebrafish xenografts for analysis of metastasis

Circulating tumor cells (CTCs) are considered as metastatic precursor cells, and zebrafish xenografts provide an in vivo model to study cancer cell spread. Currently, the low number of patient-derived CTCs limits their analysis in animal models. We present DanioCTC, a xenograft workflow for injecting CTCs from metastatic breast cancer (MBC) patients into zebrafish embryos to study cell dissemination in vivo. The study successfully adapts existing workflows and combines diagnostic leukapheresis (DLA), the Parsortix microfluidic system, flow cytometry, and the automated cell micromanipulator CellCelector setup to enrich and isolate MBC-derived CTCs and to finally inject them into Zebrafish embryos, where their dissemination was tracked up to 3 days post-injection. MDA-MB-231 cells were used as a standard xenotransplantation control, and these cells were frequently found in the head and blood-forming regions of the tail. Using DLA aliquots spiked with MBA-MB-231 cells, the newly established DanioCTC workflow confirmed the dissemination of MDA-MB-231 cells into these regions. CTCs from an MBC patient were then enriched by DLA, Parsortix, and flow cytometry, isolated with the CellCelectorTM and xenografted into zebrafish embryos. CTCs were mainly detected in the head and trunk, unlike MDA-MB-231 cells, which were present in the head and tail. DanioCTC presents a significant breakthrough in the use of zebrafish embryos as a model to study CTC dissemination in vivo, which can be used for patient-derived CTCs instead of cell culture-derived cancer cells as a crucial step towards understanding the biology of metastatic breast cancer. Statement of significanceDanioCTC is a novel workflow to inject patient-derived CTCs into zebrafish, enabling studies on CTC dissemination and personalized treatment in vivo, therefore advancing our toolkit to fight metastatic cancer.