Metastasis to distant organs is the major cause of cancer-related death. The Division of Toxicology, LACDR, Leiden University has developed advanced 3D in vitro imaging and microscopy-based genetic screens. These approaches have recently led to the unraveling of some of the mechanisms controlling metastasis and provided clues as to how to interfere with this process.
Cancer cells use various strategies to escape from the primary tumor, enter the bloodstream to disseminate to distant organs, and form metastases. Key to these strategies is the ability of cancer cells to migrate. Cell migration involves remodeling of the cytoskeleton and of the “cell adhesion complexes” that mediate attachment of cells to each other and to the extracellular matrix (ECM). Cellular signal transduction cascades that are typically rewired in cancer cells control such remodeling. We have developed technology to create arrays of 3D in vitro small tumor-like structures embedded in ECM using a microinjection-based approach (see images). This allowed automated 3D microscopy for analysis of breast cancer cell migration.
The images on the left show an automated injection-derived array of ECM-embedded tumor like 3D cultures. Different columns were treated with different drugs. The top image shows a low zoom overview; the bottom image shows a higher zoom of some structures.
Using this approach we discovered that some types of breast cancer lose their epithelial shape and become much more motile when receptors for the ECM are lost. For this switch, the cancer cells rewired an intracellular signaling network very much like normal epithelial cells do when they have to migrate to other locations during embryonic development. As a result, these breast cancer cells were no longer glued together but migrated as individual cells (see images to the left). In vivo models subsequently showed that such cells disseminated more effectively and were more metastatic (
Science Signaling 7(312):Ra15, 2014).
To identify novel drug targets relevant to breast cancer cell migration; quantitative, multiparametric, high-content microscopy has been developed for genetic screens (see below Figure 1 and 2). This approach was used to screen a library of ~1500 small interfering RNAs (siRNAs) for their effect on cancer cell migration speed, directionality, and persistence. After validation steps ~30 candidate drug targets were identified for interfering with cancer cell migration and a subset was selected that showed strong, significant association with metastasis-free survival in breast cancer patients. One such gene encoded a kinase that was highly expressed in aggressive breast cancer subtypes and was found to control the dynamics of cell-ECM adhesions (see Figure 3 below). Subsequent in vivo studies confirmed that this kinase is required for breast cancer metastasis and hence, represents a new candidate drug target (J Clin Invest, 2015 in press).
Figure 1: Phagokinetic track assay – an automated brightfield microscopy method for high throughput multiparametric analysis of cell migration. Different migration features are indicated on the left for cells transfected with control siRNAs and siRNAs targeting several genes.
Figure 2: Random 2D migration assay – a real time automated epifluorescence imaging microscopy method for analysis of cell migration coupled to a cell-tracking feature. Original images (top row), time overlay showing cell movement (bottom row), and trajectories of individual cells (middle row) are shown for cells transfected with control siRNAs and siRNAs several genes.
Figure 3: Real time confocal microscopy to analyze dynamics of cell-ECM adhesions. Original images (left column) and time overlay showing displacement of adhesions are shown for cells transfected with control siRNAs and siRNAs several genes.