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Supplemental Information

Assessing the morphological Recovery of VEGF-depleted Cells by Cancer Cell-cultured Medium

Twenty-four h after the siRNA transfection, culture medium was replaced with fresh medium (left) or cultured medium in which normal RFP-HeLa cells were cultured for 36 h (right), and then siRNA-treated cells were cultured for another 24 h. Phase contrast images show the RFP-HeLa cells in the polymer-bottom dishes 48 h after the siRNA transfection. Bar, 20 μm.

DOI: 10.7287/peerj.preprints.500v1/supp-1

Effects of Sunitinib Treatment on Cancer Cell Properties and Extravasation

(A) Phase contrast images of control and sunitinib-treated RFP-HeLa cells. (B) Quantitative evaluation of chemotactic migration in control and sunitinib-treated RFP-HeLa cells (mean ± SD, n=3). (C) Confocal microscopic images of control and sunitinib-treated RFP-Hep2 cells showing the distributions of vinculin (green) and DNA (blue). (D) Representative images after 11 h observation of RFP-HeLa cells that formed severe emboli with and without sunitinib treatment. Red colors in the bottom images show the area of extravasated RFP-HeLa cells. Bars, 20 μm (A, C), 40 μm (D).

DOI: 10.7287/peerj.preprints.500v1/supp-2

Cancer Cell Invasion-type Extravasation

Invasion-type extravasation of RFP-HeLa cells that formed severe emboli in the caudal artery of the zebrafish larva. Number, elapsed time in minutes.

DOI: 10.7287/peerj.preprints.500v1/supp-3

Endothelial Covering-type Extravasation

Endothelial covering-type extravasation of RFP-HeLa cells that formed severe emboli in the caudal artery of the zebrafish larva. Number, elapsed time in minutes.

DOI: 10.7287/peerj.preprints.500v1/supp-4

3D image of embolous-forming cancer cells and covering endothelial cells

3D image was created with confocal microscopic images (47 slices, step size: 1 mm) taken at 10 hour postadministration.

DOI: 10.7287/peerj.preprints.500v1/supp-5

Control siRNA-treated Cancer Cells in culture

Phase contrast images of RFP-HeLa cells treated with control siRNA in the polymer-bottom dish. Number, elapsed time in minutes.

DOI: 10.7287/peerj.preprints.500v1/supp-6

VEGF-depleted Cancer Cells in culture

Phase contrast images of RFP-HeLa cells treated with siRNA against VEGF in the polymer-bottom dish. Number, elapsed time in minutes.

DOI: 10.7287/peerj.preprints.500v1/supp-7

Extravasation of VEGF-depleted Cancer Cells

Extravasation of the VEGF-depleted RFP-HeLa. Number, elapsed time in minutes.

DOI: 10.7287/peerj.preprints.500v1/supp-8

Extravasation of Sunitinib-treated Cancer Cells

Extravasation of RFP-HeLa cells in the presence of sunitinib. Number, elapsed time in minutes.

DOI: 10.7287/peerj.preprints.500v1/supp-9

Additional Information

Competing Interests

The authors have declared that no competing interests exist.

Author Contributions

Masamitsu Kanada conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Jinyan Zhang performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper.

Libo Yan performed the experiments, analyzed the data, contributed reagents/materials/analysis tools.

Takashi Sakurai contributed reagents/materials/analysis tools, in vivo microscopy.

Susumu Terakawa conceived and designed the experiments, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

Maintenance of the transgenic zebrafish and the experimental design for this study were approved by the Hamamatsu University School of Medicine animal welfare.

Funding

This work was supported by the Japan Society for the Promotion of Science (JSPS) fellowship to M.K., and partly supported by a grant from the Japan Science and Technology Agency (JST) to S.T. and also by an intramural project, “Cancer research by optical means.” The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


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