Neutrophil-Mediated Experimental Metastasis Is Enhanced by VEGFR Inhibition in a Zebrafish Xenograft Model

Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time-resolved imaging of both processes at single-cell resolution within 1 week. The tumour vasculature was initiated de novo by remodelling of primitive endothelial cells into a functional network. Roles of myeloid cells in critical tumourigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumour invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two-photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumour vascularization and a localized tumour growth but enhanced migration of neutrophils, which in turn promoted tumour invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signalling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumour invasiveness

Automated Whole Animal Bio-Imaging Assay for Human Cancer Dissemination

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

Systems microscopy approaches to understand cancer cell migration and metastasis

Cell migration is essential in a number of processes, including wound healing, angiogenesis and cancer metastasis. Especially, invasion of cancer cells in the surrounding tissue is a crucial step that requires increased cell motility. Cell migration is a well-orchestrated process that involves the continuous formation and disassembly of matrix adhesions. Those structural anchor points interact with the extra-cellular matrix and also participate in adhesion-dependent signalling. Although these processes are essential for cancer metastasis, little is known about the molecular mechanisms that regulate adhesion dynamics during tumour cell migration. In this review, we provide an overview of recent advanced imaging strategies together with quantitative image analysis that can be implemented to understand the dynamics of matrix adhesions and its molecular components in relation to tumour cell migration. This dynamic cell imaging together with multiparametric image analysis will help in understanding the molecular mechanisms that define cancer cell migration

Neutrophil‐mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model

Inhibition of VEGF signalling effectively suppresses localized tumour growth but accelerates tumour invasiveness and micrometastasis by unknown mechanisms. To study the dynamic and reciprocal interactions between tumour cells and their microenvironment during these processes, we established a xenograft model by injecting tumour cells into the blood circulation of transparent zebrafish embryos. This reproducibly results in rapid simultaneous formation of a localized tumour and experimental micrometastasis, allowing time-resolved imaging of both processes at single-cell resolution within 1 week. The tumour vasculature was initiated de novo by remodelling of primitive endothelial cells into a functional network. Roles of myeloid cells in critical tumourigenesis steps such as vascularization and invasion were revealed by genetic and pharmaceutical approaches. We discovered that the physiological migration of neutrophils controlled tumour invasion by conditioning the collagen matrix and forming the metastatic niche, as detected by two-photon confocal microscopy and second harmonic generation. Administration of VEGFR inhibitors blocked tumour vascularization and a localized tumour growth but enhanced migration of neutrophils, which in turn promoted tumour invasion and formation of micrometastasis. This demonstrates the in vivo cooperation between VEGF signalling and myeloid cells in metastasis and provides a new mechanism underlying the recent findings that VEGFR targeting can promote tumour invasiveness. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Differentiation between poorly and highly aggressive human cancer cell lines using automated bioimaging assay.

<p>(<b>A</b>) Scatter plot representation of tumor cell dissemination for indicated prostate (<i>upper</i> graphs), breast (<i>middle</i>), and colorectal cancer cell lines (<i>lower</i> graphs). Number of injected embryos from 2 biological replicates is indicated. (<b>B</b>) MCD determined from data represented in <b>A</b>. Data are presented as mean ± s.e.m. *p<0.05, ***p<0.001. (<b>C</b>) 6 dpi embryo injected with PC3 showing tumor foci burden determined from segmented red channel (<i>left</i>), and represented as scatter plot (<i>right</i>). (<b>D</b>) Automated determination of region for exclusion of tumor foci around implantation site and in area of intestinal development (<i>left</i>), and remaining tumor foci represented as scatter plot (<i>right</i>). (<b>E</b>) MCD before (<i>black</i>) and after exclusion (<i>white bars</i>) for the indicated prostate (<i>left</i>), breast (<i>middle</i>), and colorectal cancer lines (<i>right graph</i>). Fold difference between poorly and highly aggressive cell lines is indicated. Data are presented as mean ± s.e.m. *p<0.05, ***p<0.001. (<b>F</b>) MCD after exclusion for PC3 and MCF7 in multiple independent experiments demonstrates reproducibility. Data are presented as mean ± s.e.m.</p

Outline of steps involved in embryo orientation.

<p>(<b>A</b>) Extended depth image of 6 dpi ZF embryo. (<b>B</b>) Grey value image from combination of green and red channels. (<b>C</b>) Blurred grey image after applying closing filter to optimize determination of outline. (<b>D</b>) Embryo segmented after applying intensity threshold and area filter. Arrowhead indicates a red object outside the outline that is excluded from segmentation. (<b>E</b>) Cropped image with only selected object. (<b>F</b>) Embryo rotated by x° for horizontal reorientation. (<b>G</b> and <b>H</b>) Determination of the x position value of the center of mass (<i>cm</i>) and center of centroid (<i>cc</i>). (<b>I</b>) Horizontal flip of the image only if <i>cm</i> is on the left side of <i>cc</i>, resulting in images with the head of the embryo always to the right side. (<b>J</b>) Image after applying closed filter to the combined green and red channel to get the outline of the embryo. Point lying at 75% distance from the extreme left of the embryo outline is calculated. Y-axis is drawn at this X-position from upper to lower outline. Upper rectangle 1 is drawn. (<b>K</b>) Lower rectangle 2 is drawn. (<b>L</b>) Vertical flip of the image only if red intensity in rectangle 1 is higher than in rectangle 2. (<b>M</b>) Schematic representation of calculations for steps <b>E–I</b>. Altogether, this procedure results in images where the head is on the right and the yolk sac is on the bottom of the image. Scale bar = 200 µm in <b>E</b> and <b>I</b>.</p

Differentiation between epithelial and mesenchymal cell types using the automated bioimaging assay.

<p>(<b>A</b>) MCD in a panel of human cancer cell lines from different origins. Number of injected embryos is indicated. <i>White bars</i> indicate cell lines showing a scattered phenotype in 2D cell culture. <i>Black bars</i> indicate cell lines growing as epithelial islands in 2D culture. <i>Grey bars</i> indicate cell lines with intermediate/mixed epithelial/mesenchymal characteristics. (<b>B</b>) 4T1 breast cancer cells growing as islands of loosely attached spindle-shaped cells (<i>left</i>) and completely scattered growth of 4T1 cells following E-cadherin silencing (<i>right</i>). (<b>C</b>) E-cadherin surface expression by FACS. (<b>D</b>) Scatter plot representation of indicated 4T1 variants. Number of injected embryos from 2 independent experiments is shown. (<b>E</b>) Representative images of embryos injected with indicated 4T1 variants. (<b>F</b>) MCD determined from data represented in <b>D</b>. Data are presented relative to wild type 4T1 as mean ± s.e.m. **p<0.01. Scale bar is 200 µm in <b>E</b>.</p

Determination of cancer cell dissemination kinetics.

<p>(<b>A</b> and <b>B</b>) LnCAP (<b>A</b>) or PC3 cells (<b>B</b>) were implanted and embryos were fixed at 2, 4, or 6 dpi for imaging (immunofluorescence images and automated image analysis (scatter plots)). Bottom row images (scale bar = 50 µm) show zoom-in of area marked by dotted line in top row images (scale bar = 100 µm). (<b>C</b>) CD at 2, 4 and 6 dpi for LnCAP (grey) and PC3-injected embryos (black) calculated from scatterplots in A and B, respectively. Statistical testing for difference between LnCAP and PC3 at different dpi is indicated. *p<0.05, ***p<0.001.</p

Schematic overview of the procedure.

<p>(<b>A</b>) Yolk sac implantation of CM-DiI labeled tumor cells into Tg (Fli:EGFP) ZF embryos 2 days post-fertilization. (<b>B</b>) Formaldehyde fixed 6 dpi embryos arrayed in 96 well plates. (<b>C</b>) Automated image acquisition using CLSM platform equipped with movable stage captures multiple Z stacks per embryo using 488 and 561 nm laser lines. (<b>D</b> and <b>E</b>) Automated creation of extended depth composite images. (<b>F</b>) Multiple extended depth images depicting embryos lying in different lateral orientations. (<b>G</b>) Automated uniform reorientation of images. (<b>H</b>) Scatter plot representing tumor foci burden in multiple embryos belonging to one experimental condition.</p

Automated multiparametric quantification of PC3 tumor foci.

<p>(<b>A</b>) Extended depth image of 6 dpi fixed embryo after realignment. (<b>B</b>) Embryo outline from segmented GFP channel and Y-axis intersecting X-axis at 75% from extreme left. (<b>C</b>) Calculated injection point at 75% distance from the extreme left and 75% from the top Y position. (<b>D</b>) Segmented red channel showing tumor foci burden in the embryo. (<b>E</b>) Identified tumor foci. (<b>F</b>) Multiple parameters of tumor foci burden calculated per embryo. Each number in the image corresponds to one tumor focus. (<b>G</b>) Tumor foci dissemination in a single embryo represented as scatter plot (coordinates 0,0 represents calculated injection site). (<b>H</b>) Combined scatter plot showing tumor foci dissemination from 39 injected embryos. (<b>I</b>) 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 <b>A</b>.</p