Tumor spheroids generate filopodia in vivo. (A–C) DCIS.COM LifeAct cells were injected as single cells into the pericardial cavity of zebrafish embryos (A and B) and imaged live using an SDC microscope 24 h postinjection. Before imaging, the embryos were anesthetized and mounted in low-melting-point agarose on glass-bottom dishes. A representative image of a DCIS.COM LifeAct spheroid growing in the zebrafish embryo pericardial cavity (20× objective, CMOS camera; bar, 100 µm; B) and a single z plane (63× water objective, CMOS camera; bars: [main] 20 µm; [inset] 5 µm) and magnification (C) are shown. (D and E) Live imaging of the DCIS.COM LifeAct spheroid, shown in B, using an SDC microscope (63× water objective, EMCCD camera, one frame per 10 s). Single z planes (D; bars: [main] 20 µm; [inset] 5 µm) and Imaris-generated 3D reconstructions (E; bars: [main] 20 µm; [inset] 5 µm) are displayed. Red insets denote magnified areas.