Hypoxia promotes T241 tumor cell invasion, dissemination and metastasis. (A and D) DiI-labeled T241 tumor cells were injected into the perivitelline space of 48 h post-fertilization embryos and tumor cell invasion, dissemination and metastasis were detected under normoxia and hypoxia using fluorescent microscopy at day 3 post-injection. Arrows indicate primary tumors. Yellow arrowheads indicate pericardium edema. White arrowheads indicate disseminated tumor foci. (Scale bar, 500 μm.) (B and E) High-resolution micrographs of A and D, respectively. (Scale bar, 100 μm.) (C and F) Representative 3-D micrographs of confocal images of tumors (red) and tumoral as well as peritumoral vasculatures (green). Yellow signals show the intratumoral microvessels overlapping with tumor cells. Dashed lines encircle invasive fronts of T241 tumors under hypoxia. (Scale bar, 10 μm.) (G) Quantification of tumor volume (n = 13/group). (H) Quantification of numbers of disseminated tumor foci (n = 13/group). (I) Quantification of tumor vessel density relative to tumor sizes (n = 7/group). Data are represented as mean ± SEM.

Dissemination and metastasis of human tumor cells in zebrafish embryos. (A and C) Highly metastatic human MDA MB-231 breast and low metastatic human OVCAR 8 ovarian cancer cells were implanted into 48 h post-fertilization zebrafish embryos. Tumor cell dissemination and metastasis were detected at day 6 post-injection. Arrows indicate primary tumors and arrowheads indicate disseminated tumor foci. (Scale bar, 500 μm.) (B and D) High-resolution micrographs of A and C, respectively to visualize single metastatic tumor cells in the trunk regions. (Scale bar, 100 μm.)

Invasion, dissemination and metastasis of T241-VEGF tumors. (A and D) DiI-labeled T241-vector and T241-VEGF tumor cells were implanted in the perivitelline space and tumor cell invasion and dissemination were examined at day 6 post-injection. Arrows indicate primary tumors. Yellow arrowheads indicate pericardium edema. White arrowheads indicate disseminated tumor foci. (Scale bar, 500 μm.) (B and E) High-resolution micrographs of A and D, respectively to visualize single metastatic tumor cells. (Scale bar, 100 μm.) (C and F) Representative 3-D micrographs of confocal images of tumors (red) and tumor vasculatures (green). Dashed lines encircle invasive fronts of T241-VEGF tumors. (Scale bar, 10 μm.) (G) Quantification of tumor volume (n = 14/group). (H) Quantification of numbers of disseminated tumor foci (n = 14/group). (I) Averages of maximal distances of metastatic foci (n = 14/group). (J) Quantification of tumor vessel density relative to tumor sizes (n = 7/group). Data are represented as mean ± SEM.

Inhibition of tumor cell invasion, dissemination and metastasis by sunitinib. (A) Representative zebrafish embryos treated with or without 0.5 and 1.0 μM sunitinib. Arrows indicate primary tumors and arrowheads indicate disseminated and metastatic tumor cells in the distal parts of the fish body. (Scale bar, 500 μm.) (B) Representative 3-D micrographs of confocal images of tumors (red) and tumor vasculatures (green) in sunitinib-treated and non-treated groups. (Scale bar, 10 μm.) (C) Quantification of tumor volume (n = 10/group). (D) Quantification of disseminated tumor foci (n = 10/group). (E) Averages of maximal distances of metastatic foci (n = 10/group). (F) Quantification of tumor vessel density relative to tumor sizes (n = 7/group). Data are represented as mean ± SEM.

Inhibition of tumor cell invasion, dissemination and metastasis by VEGFR2 morpholinos. (A and D) VEGFR2 specific morpholinos and control morpholinos were injected into the blastoma of 1 h post-fertilization at 1–4-cell stages. DiI-labeled T241-VEGF tumor cells were implanted in the perivitelline space of 48 h post-fertilization embryos and tumor cell invasion, dissemination and metastasis were detected at days 0 and 6 post-injection. White arrowheads indicate disseminated tumor foci. (Scale bar, 500 μm.) (B and E) High-resolution micrographs of A and D, respectively to visualize single metastatic tumor cells in the trunk regions. (Scale bar, 100 μm.) (C and F) Representative 3-D micrographs of confocal images of tumors (red) and tumor vasculatures (green). (Scale bar, 10 μm.) (G) Quantification of tumor volume (n = 12/group). (H) Quantification of numbers of disseminated tumor foci (n = 12/group). (I) Averages of maximal distances of metastatic foci (n = 12/group). (J) Quantification of tumor vessel density relative to tumor size (n = 7/group). Data are represented as mean ± SEM.

Sunitinib inhibits hypoxia-induced invasion, dissemination and metastasis of T241 tumors. (A and D) DiI-labeled T241 tumor cells were implanted in the perivitelline space of 48 h post-fertilization embryos and shortly after injection zebrafish embryos were placed into a hypoxic chamber containing 7.5% air saturation, immediately followed by treatment with 1.0 μM sunitinib. Tumor cell invasion, dissemination and metastasis were detected at day 3 post-injection. Arrows indicate primary tumors and arrowheads indicate disseminated tumor foci. (Scale bar, 500 μm.) (B and E) High-resolution micrographs of A and D, respectively to visualize single metastatic tumor cells in the trunk regions. (Scale bar, 100 μm.) (C and F) Representative 3-D micrographs of confocal images of tumors (red) and tumor vasculatures (green). Dashed lines encircle invasive fronts of T241 tumors. (Scale bar, 10 μm.) (G) Quantification of numbers of disseminated tumor foci (n = 11/group). (H) Quantification of tumor vessel density relative to tumor sizes (n = 7/group). Data are represented as mean ± SEM.

Schematic diagram of zebrafish metastasis model under normoxia and hypoxia. Mammalian tumor cells were labeled with DiI dye in vitro and approximately 300 tumor cells in 5 nL DMEM are injected into the perivitelline space of each 48 h post-fertilization Tg(fli-1:EGFP) transgenic zebrafish embryo. Tumor cell invasion, dissemination, metastasis and angiogenesis were monitored in vivo in living fish body at different time points under normoxia and hypoxia (7.5% air saturation-the maximal tolerated oxygen tension) using fluorescent microscopy.

Hypoxia promotes LLC tumor cell invasion, dissemination and metastasis. (A and D) DiI-labeled LLC-vector tumor cells were injected into the perivitelline space of 48 h post-fertilization embryos and tumor cell invasion, dissemination, and metastasis were detected under normoxia and hypoxia using fluorescent microscopy at day 3 post-injection. Arrows in A and D indicate primary tumors. Arrowheads indicate disseminated tumor foci. (Scale bar, 500 μm.) (B and E) High-resolution micrographs of A and D, respectively. (Scale bar,100 μm.) (C and F) 3-D micrographs of confocal images of tumors (red) and tumoral as well as peritumoral vasculatures (green). (Scale bar, 10 μm.) (G) Quantification of tumor volume (n = 11/group). (H) Quantification of numbers of disseminated tumor foci (n = 11/group). (I) Quantification of maximal distances of metastasis (n = 11/group). (J) Quantification of tumor vessel density relative to tumor sizes (n = 7/group). Data are represented as mean ± SEM.

Time-course analysis of invasion, dissemination and metastasis of T241-vector and T241-VEGF tumors. T241-vector and T241-VEGF tumor cells were implanted into the perivitelline space of each developing zebrafish embryo and dissemination and metastasis of DiI-labeled tumor cells were monitored by fluorescent microscopy on days 0, 2, 4, and 6. White arrowheads indicate disseminated tumor foci and yellow arrowheads indicate edema in the pericardium. (Scale bar, 500 μm.)

Invasion, dissemination and metastasis of LLC-VEGF tumors. (A and D) DiI-labeled LLC-vector and LLC-VEGF tumor cells were implanted in the perivitelline space and tumor cell invasion and dissemination were examined at day 6 post-injection. Arrows indicate primary tumors. Yellow arrowheads in (D) indicate pericardium edema. White arrowheads indicate disseminated tumor foci. (Scale bar, 500 μm.) (B and E) High-resolution micrographs of A and D, respectively to visualize single metastatic tumor cells. (Scale bar, 100μm.) (C and F) Representative 3-D micrographs of confocal images of tumors (red) and tumor vasculatures (green). (Scale bar,10 μm.) (G) Quantification of tumor volume (n = 28/group). (H) Quantification of numbers of disseminated tumor foci (n = 28/group). (I) Averages of maximal distances of metastatic foci (n = 28/group). (J) Quantification of tumor vessel density relative to tumor sizes (n = 7/group). Data are represented as mean ± SEM.

Inhibition of LLC-VEGF tumor cell invasion, dissemination and metastasis by sunitinib. (A) Representative zebrafish embryos treated with or without 1.0 μM sunitinib. Arrows indicate primary tumors and white arrowheads indicate disseminated and metastatic tumor cells in the distal parts of the fish body. Yellow arrowheads indicate pericardium edema. (Scale bar, 500 μm.) (B) Quantification of disseminated tumor foci (n = 12/group). (C) Averages of maximal distances of metastatic foci (n = 12/group). Data are represented as mean ± SEM.