Publications

Publications

Quantitative Analysis of human Cancer Cell Extravasation Using Intravital Imaging

Methods Mol Biol. 2016;1458:27-37

Willetts L, Bond D, Stoletov 1, Lewis JD

Abstract

Metastasis, or the spread of cancer cells from a primary tumor to distant sites, is the leading cause of cancer-associated death. Metastasis is a complex multi-step process comprised of invasion, intravasation, survival in circulation, extravasation, and formation of metastatic colonies. Currently, in vitro assays are limited in their ability to investigate these intricate processes and do not faithfully reflect metastasis as it occurs in vivo. Traditional in vivo models of metastasis are limited by their ability to visualize the seemingly sporadic behavior of where and when cancer cells spread (Reymond et al., Nat Rev Cancer 13:858-870, 2013). The avian embryo model of metastasis is a powerful platform to study many of the critical steps in the metastatic cascade including the migration, extravasation, and invasion of human cancer cells in vivo (Sung et al., Nat Commun 6:7164, 2015; Leong et al., Cell Rep 8, 1558-1570, 2014; Kain et al., Dev Dyn 243:216-28, 2014; Leong et al., Nat Protoc 5:1406-17, 2010; Zijlstra et al., Cancer Cell 13:221-234, 2008; Palmer et al., J Vis Exp 51:2815, 2011). The chicken chorioallantoic membrane (CAM) is a readily accessible and well-vascularized tissue that surrounds the developing embryo. When the chicken embryo is grown in a shell-less, ex ovo environment, the nearly transparent CAM provides an ideal environment for high-resolution fluorescent microcopy approaches. In this model, the embryonic chicken vasculature and labeled cancer cells can be visualized simultaneously to investigate specific steps in the metastatic cascade including extravasation. When combined with the proper image analysis tools, the ex ovo chicken embryo model offers a cost-effective and high-throughput platform for the quantitative analysis of tumor cell metastasis in a physiologically relevant in vivo setting. Here we discuss detailed procedures to quantify cancer cell extravasation in the shell-less chicken embryo model with advanced fluorescence microscopy techniques.

PubMed

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Why did the chicken cross the road…?

Konstantin Stoletov and Lian Willetts co-first-authored an article published recently in Nature Communications titled “Quantitative in vivo whole genome motility screen reveals novel therapeutic targets to block cancer metastasis“. These two researchers, along with fellow Lewis lab members and collaborators from the University of Calgary and Vanderbilt University set out to determine what genes and signaling networks are involved in the rate-limiting steps of solid tumour cell motility, in vivo. But the team was hampered by the lack of an effective, quantitative, in vivo imaging platform. They wanted to visualize the movement of tumour cells, or lack of, in real-time AND use this intravital imaging platform to screen a large bank of tumour cells harboring single gene mutations for cells that show a loss of motility.

https://www.nature.com/articles/s41467-018-04743-2

When tumour cells metastasize they get into (intravasate) the hosts’ bloodstream and use the vascular system like roadways to travel throughout the body. This lets the tumour cells colonize new microenvironments where they will proliferate and form new tumours. So metastatis is really dependent on tumour cell motility. Although there are many different types of solid tumours known, previous research suggests that if the tumour cells can mobilize and metastasize then the expressed motility-related genes share homology across tumour types. This is great news because it would mean that therapeutic targets aimed at stopping motility could also stop metastasis for many tumour types!

Dr. Konstantin Stoletov

 

Dr. Lian Willetts
Dr. Lian Willetts

The Lewis lab researchers and their collaborators developed an in vivo, fluorescent, time-lapse screening platform that uses shell-less avian embryos for tumour growth and formation. The avian embryo is an excellent tumour model because the tumour cells will grow on the chorioallantoic membrane in a single cell layer, making in vivo cell motility imaging actually doable.

Using this platform the team screened over 30 000 human genes for the ones needed for cell motility and ultimately found 17 genes that looked to be effective metastasis-blocking gene targets. Stoletov, along with other Lewis lab members, are continuing this research by studying these 17 attractive candidates further to determine which one (s) would make therapeutic metastasis-blocking targets.

This article has generated a lot of interest in the scientific community and in the general public! Check out the links below to mentions and articles in the media.

https://www.biocentury.com/bc-innovations/translation-brief/2018-07-18/how-chicken-embryo-screen-identified-entos%E2%80%99-

https://www.ualberta.ca/medicine/news/2018/june/putting-the-brakes-on-metastatic-cancer

Stay tuned for a podcast that will be posted soon from “Parsing Science” where the hosts interview Dr. John Lewis about this work!

https://www.parsingscience.org/coming_soon/
UPDATE Oct 12, 2018: The podcast with John Lewis on Parsing Science called “Halting Cancers’ Spread“, is now available!

- Perrin Beatty