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Quantification of cancer cell extravasation in vivo

Nature Protocols volume 11, pages 937–948 (2016)Cite this article

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Abstract

Cancer cell 'invasiveness' is one of the main driving forces in cancer metastasis, and assays that quantify this key attribute of cancer cells are crucial in cancer metastasis research. The research goal of many laboratories is to elucidate the signaling pathways and effectors that are responsible for cancer cell invasion, but many of these experiments rely on in vitro methods that do not specifically simulate individual steps of the metastatic cascade. Cancer cell extravasation is arguably the most important example of invasion in the metastatic cascade, whereby a single cancer cell undergoes transendothelial migration, forming invasive processes known as invadopodia to mediate translocation of the tumor cell from the vessel lumen into tissue in vivo. We have developed a rapid, reproducible and economical technique to evaluate cancer cell invasiveness by quantifying in vivo rates of cancer cell extravasation in the chorioallantoic membrane (CAM) of chicken embryos. This technique enables the investigator to perform well-powered loss-of-function studies of cancer cell extravasation within 24 h, and it can be used to identify and validate drugs with potential antimetastatic effects that specifically target cancer cell extravasation. A key advantage of this technique over similar assays is that intravascular cancer cells within the capillary bed of the CAM are clearly distinct from extravasated cells, which makes cancer cell extravasation easy to detect. An intermediate level of experience in injections of the chorioallantoic membrane of avian embryos and cell culture techniques is required to carry out the protocol.

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Figure 1: Comparison of Transwell invasion assay with the chorioallantoic membrane (CAM) to quantify cancer cell extravasation and imaging intravascular and extravascular cancer cells in the CAM.
Figure 2: Preparation of ex ovo chicken embryos and microinjector assembly for i.v. injection of cancer cells or labeling agents.
Figure 3: I.v. injections of cells or lectin and setting up the extravasation efficiency assay.
Figure 4: Distinguishing intravascular and extravascular cancer cells in the CAM.
Figure 5: Representative schematic of how an extravasation efficiency assay is performed.

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Acknowledgements

This study was supported by a Translational Breast Cancer Research Unit Fellowship and a Canadian Institutes for Health Research Post-doctoral Fellowship Award to K.C.W., a Tier I Canada Research Chair in Oncology to A.F.C. and a Prostate Cancer Canada/Movember Rising Stars Grant to H.S.L. (grant 2013-56). This work was also supported by a Prostate Cancer Canada New Investigator Pilot Grant to H.S.L. (2012-888).

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Author notes

  1. Yohan Kim and Karla C Williams: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Surgery, Schulich School of Medicine, Western University, London, Ontario, Canada

    Yohan Kim, Karla C Williams, Carson T Gavin, Emily Jardine & Hon S Leong

  2. Translational Prostate Cancer Research Laboratory, Lawson Health Research Institute, London, Ontario, Canada

    Yohan Kim, Carson T Gavin, Emily Jardine & Hon S Leong

  3. Translational Breast Cancer Research Unit, London Health Sciences Centre, London, Ontario, Canada

    Karla C Williams & Ann F Chambers

  4. Department of Oncology, Schulich School of Medicine, Western University, London, Ontario, Canada

    Ann F Chambers

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Contributions

Y.K., K.C.W., C.T.G., E.J. and H.S.L. developed the animal models and performed example extravasation experiments. Y.K., K.C.W., A.F.C. and H.S.L. wrote the protocol.

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Correspondence to Hon S Leong.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

Underlying data behind Figure 5 showing how Extravasation Efficiency Assay calculations are performed. (XLSX 11 kb)

Three-Dimensional Rendering of Image Set Revealing Intravascular Cancer Cells in Relationship to Blood Lumen and CAM Capillary Bed.

Red signal represents Dextran-Alexa555, purple signal represents Lectin-DyLight649, blue signal represents nuclei stained by Hoechst 33456, and the green signal represents two PC-3M-LN4-zsGreen cells present in the intravascular space of the CAM (AVI 20520 kb)

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Kim, Y., Williams, K., Gavin, C. et al. Quantification of cancer cell extravasation in vivo. Nat Protoc 11, 937–948 (2016). https://doi.org/10.1038/nprot.2016.050

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