DanioCTC Workflow. A MBC patient undergoes diagnostic leukapheresis (DLA). CTCs are enriched from a DLA aliquot with the Parsortix system and isolated by FACS after staining. Cells are further stained by CellTracker Red for tracking in zebrafish embryos. Single CTCs are isolated by using the CellCelector and injected into 2 dpf old Tg(kdrl:EGFP) zebrafish embryos. Adapted from [27,30].

Capillaries. Self-prepared capillary for standard zebrafish embryo injections with a long taper narrowing and an opening diameter of approximately 25 µm. The CellCelector capillary tapers quickly, is rather short and has an opening diameter of 20 µm.

Adapted CellCelector setup. Representative images of the CellCelector picking area of picking a single CellTracker Red-stained CTC and the attached stereomicroscope required for CTC injections in the deposit area of the CellCelector. (1) CellCelector cell picking area and image of region of interest before and after cell picking. (2) CellCelector deposit/zebrafish injection area and brightfield image of embryo being injected.

Dissemination of MDA-MB-231 cells after injection with the standard workflow into zebrafish larvae. Depicted are the absolute MDA-MB-231 cell numbers in the head, trunk and tail at 1 and 3 dpi (n = 6). dpi: days post injection. ANOVA followed by post hoc Bonferroni test, *** 0.0001 < p < 0.001.

Dissemination of MDA-MB-231 cells spiked into a DLA sample after injection with the DanioCTC workflow. Cell localization was monitored at 1 and 3 dpi. (A) Representative image of 50 MDA-MB-231 cells injected into the DoC of a zebrafish embryo (1 hpi). (B) Representative image of the tail and CHT region at 3 dpi with localized MDA-MB-231 cells in the CHT (black arrowheads). (C) Representative image of the tail region at 3 dpi showing extravasated MDA-MB-231 cells (black arrowheads). (D) Absolute MDA-MB-231 cell numbers detected in the head, trunk and tail regions at 1 and 3 dpi (n = 11 embryos). Green label: endothelium; red label: MDA-MB 231 cells. dpi: days post injection. Kruskal–Wallis test followed by post hoc Dunn’s test, * 0.01 < p < 0.05; ** 0.001 < p < 0.01.

Dissemination of isolated CTCs of a MBC patient after injection with the DanioCTC workflow. Isolated CTCs, labeled in red, were monitored at 1 and 3 dpi and showed dissemination into the head, trunk and the tail. (A,B) Representative images of the head region of a Tg(kdrl:EGFP) positive embryo after injection of 50 CTCs (indicated by white arrowheads). (C) Representative image at 3 dpi showing a disseminated CTC in the CHT of an embryo (indicated by white arrowhead). (D) Absolute CTC numbers in the head, trunk and tail at 1 and 3 dpi (n = 9). dpi: days post injection. Kruskal–Wallis test followed by post hoc Dunn’s test. No statistically significant differences were observed between groups.