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Automated whole animal bio-imaging assay for human cancer dissemination.

Ghotra VP, He S, de Bont H, van der Ent W, Spaink HP, van de Water B, Snaar-Jagalska BE, Danen EH - PLoS ONE (2012)

Bottom Line: Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer.Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model.This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.

View Article: PubMed Central - PubMed

Affiliation: Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, the Netherlands.

ABSTRACT
A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.

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Automated multiparametric quantification of PC3 tumor foci.(A) Extended depth image of 6 dpi fixed embryo after realignment. (B) Embryo outline from segmented GFP channel and Y-axis intersecting X-axis at 75% from extreme left. (C) Calculated injection point at 75% distance from the extreme left and 75% from the top Y position. (D) Segmented red channel showing tumor foci burden in the embryo. (E) Identified tumor foci. (F) Multiple parameters of tumor foci burden calculated per embryo. Each number in the image corresponds to one tumor focus. (G) Tumor foci dissemination in a single embryo represented as scatter plot (coordinates 0,0 represents calculated injection site). (H) Combined scatter plot showing tumor foci dissemination from 39 injected embryos. (I) Quantification of cumulative distance (CD). Each filled square represents cumulative distance from injection point of all identified tumor foci in a single embryo. Mean cumulative distance (MCD) in the 39 injected embryos in this experiment is 15024 µm. Scale bar = 200 µm in A.
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pone-0031281-g003: Automated multiparametric quantification of PC3 tumor foci.(A) Extended depth image of 6 dpi fixed embryo after realignment. (B) Embryo outline from segmented GFP channel and Y-axis intersecting X-axis at 75% from extreme left. (C) Calculated injection point at 75% distance from the extreme left and 75% from the top Y position. (D) Segmented red channel showing tumor foci burden in the embryo. (E) Identified tumor foci. (F) Multiple parameters of tumor foci burden calculated per embryo. Each number in the image corresponds to one tumor focus. (G) Tumor foci dissemination in a single embryo represented as scatter plot (coordinates 0,0 represents calculated injection site). (H) Combined scatter plot showing tumor foci dissemination from 39 injected embryos. (I) Quantification of cumulative distance (CD). Each filled square represents cumulative distance from injection point of all identified tumor foci in a single embryo. Mean cumulative distance (MCD) in the 39 injected embryos in this experiment is 15024 µm. Scale bar = 200 µm in A.

Mentions: Having established conditions for automated imaging and image pre-processing of tumor cell implanted ZF embryos; we subsequently developed an algorithm for automated analysis of tumor foci burden in the post-processed ZF images. For this, Image-Pro based software was developed, which performed essentially three major functions (see materials and methods section for detailed information on the macro's). 1) Reorientation of the images (Figure 2): all embryos were automatically reoriented to a horizontal orientation, with the head towards the right and the yolk sac towards the bottom. 2) Determination of the injection position of labeled tumor cells (Figure 3A–C): the injection position was calculated from the images based on the segmented GFP channel (and confirmed by visual inspection using the red channel). 3) Detection of tumor foci (Figure 3D and E): The red channel was segmented using an intensity threshold and minimum and maximum area filters.


Automated whole animal bio-imaging assay for human cancer dissemination.

Ghotra VP, He S, de Bont H, van der Ent W, Spaink HP, van de Water B, Snaar-Jagalska BE, Danen EH - PLoS ONE (2012)

Automated multiparametric quantification of PC3 tumor foci.(A) Extended depth image of 6 dpi fixed embryo after realignment. (B) Embryo outline from segmented GFP channel and Y-axis intersecting X-axis at 75% from extreme left. (C) Calculated injection point at 75% distance from the extreme left and 75% from the top Y position. (D) Segmented red channel showing tumor foci burden in the embryo. (E) Identified tumor foci. (F) Multiple parameters of tumor foci burden calculated per embryo. Each number in the image corresponds to one tumor focus. (G) Tumor foci dissemination in a single embryo represented as scatter plot (coordinates 0,0 represents calculated injection site). (H) Combined scatter plot showing tumor foci dissemination from 39 injected embryos. (I) Quantification of cumulative distance (CD). Each filled square represents cumulative distance from injection point of all identified tumor foci in a single embryo. Mean cumulative distance (MCD) in the 39 injected embryos in this experiment is 15024 µm. Scale bar = 200 µm in A.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275564&req=5

pone-0031281-g003: Automated multiparametric quantification of PC3 tumor foci.(A) Extended depth image of 6 dpi fixed embryo after realignment. (B) Embryo outline from segmented GFP channel and Y-axis intersecting X-axis at 75% from extreme left. (C) Calculated injection point at 75% distance from the extreme left and 75% from the top Y position. (D) Segmented red channel showing tumor foci burden in the embryo. (E) Identified tumor foci. (F) Multiple parameters of tumor foci burden calculated per embryo. Each number in the image corresponds to one tumor focus. (G) Tumor foci dissemination in a single embryo represented as scatter plot (coordinates 0,0 represents calculated injection site). (H) Combined scatter plot showing tumor foci dissemination from 39 injected embryos. (I) Quantification of cumulative distance (CD). Each filled square represents cumulative distance from injection point of all identified tumor foci in a single embryo. Mean cumulative distance (MCD) in the 39 injected embryos in this experiment is 15024 µm. Scale bar = 200 µm in A.
Mentions: Having established conditions for automated imaging and image pre-processing of tumor cell implanted ZF embryos; we subsequently developed an algorithm for automated analysis of tumor foci burden in the post-processed ZF images. For this, Image-Pro based software was developed, which performed essentially three major functions (see materials and methods section for detailed information on the macro's). 1) Reorientation of the images (Figure 2): all embryos were automatically reoriented to a horizontal orientation, with the head towards the right and the yolk sac towards the bottom. 2) Determination of the injection position of labeled tumor cells (Figure 3A–C): the injection position was calculated from the images based on the segmented GFP channel (and confirmed by visual inspection using the red channel). 3) Detection of tumor foci (Figure 3D and E): The red channel was segmented using an intensity threshold and minimum and maximum area filters.

Bottom Line: Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer.Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model.This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.

View Article: PubMed Central - PubMed

Affiliation: Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, the Netherlands.

ABSTRACT
A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.

Show MeSH
Related in: MedlinePlus