Background

Metastasis is responsible for approximately 90% of cancer-associated mortality. It proceeds through multiple steps: invasion, intravasation, survival in the circulatory system, extravasation, colonization, and metastatic tumor formation in secondary organs with angiogenesis (Nguyen et al., 2009; Chaffer and Weinberg, 2011; Welch and Hurst, 2019). The dissemination of cancer cells is an initial step of metastasis, and its molecular mechanism involves a local breakdown of basement membrane, loss of cell polarity, and induction of epithelial to mesenchymal transition (EMT). EMT plays a central role in early embryonic morphogenesis; its process enables various types of epithelial cells to convert into mesenchymal cells, through a downregulation of epithelial markers such as E-cadherin and an upregulation of mesenchymal markers such as vimentin. Twist, a basic helix-loop-helix transcription factor, plays a critical role in inducing the EMT program (Tsai and Yang, 2013; Lu and Kang, 2019). Past studies showed that elevated expression of Twist is associated with poor survival rates in patients with cancer; also, ectopic expression of Twist confers metastatic properties on cancer cells through induction of EMT (Yang et al., 2004; Tsai et al., 2012).

Cancer research using zebrafish as a model has attracted attention because this model offers many unique advantages that are not readily provided by other animal models (White et al., 2013; Osmani and Goetz, 2019). Furthermore, the zebrafish system has also been increasingly recognized as a chemical screening platform because it provides the advantage of high-throughput screening in an in vivo vertebrate setting with physiologic relevance to humans (Zon and Peterson, 2005; Letrado et al., 2018; Nakayama and Gong, 2020; Nakayama and Makinoshima, 2020; Nakayama et al., 2021b, 2022a and 2022b). Our study previously established a tamoxifen-controllable Twist1a-ER^T2 transgenic zebrafish line that serves as an in vivo drug screening platform for the identification of anti-metastasis drugs targeting metastatic dissemination of cancer cells. By crossing Twist1a-ER^T2 with xmrk (a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish showed spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of an EMT (Nakayama et al., 2020; Lu et al., 2021). The dissemination patterns are generally divided into three categories: (i) local dissemination, in which disseminated mCherry-positive cells exist in close proximity to the liver; (ii) abdominal dissemination, in which the cells spread throughout the abdomen; and (iii) distant dissemination, in which the cells are observed over a broad region from the trunk to the tail (Figure 1A).

This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the discovery of anti-metastasis drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test chemical through comparing the frequencies of the fish showing the abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group. Previous studies confirmed that ki16425 (a LPA1 inhibitor) or Y27632 (an inhibitor of Rho-associated coiled-coil-containing protein kinase), which have been reported to suppress metastasis in mice models of metastasis (Itoh et al., 1999; Boucharaba et al., 2006), could suppress cell dissemination in the fish model. In vivo drug screen using this model identified adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), as having a potential to suppress metastatic dissemination of cancer cells (Figure 1B and 1C). Furthermore, pharmacologic and genetic inhibition of HSD11β1 were validated to suppress metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model (Nakayama et al., 2020 and 2021a). Taken together, our model offers an in vivo drug screening platform for the identification of anti-metastatic drugs.

Figure 1. Twist1a-ER^T2/xmrk double transgenic zebrafish offers an in vivo drug screening platform for the identification of anti-metastatic drugs. A. Representative images of the dissemination of mCherry-labeled hepatic cells from the liver in Twist1a-ER^T2/xmrk double transgenic zebrafish at 16 days post-fertilization (dpf). The fish were treated with doxycycline and 4-hydroxytamoxifen (4-OHT). Some of the disseminated mCherry-positive cells are indicated by arrowheads. The images are shown as Z-stack images using 100× magnification. Scale bar, 200 μm. B. Representative images of the effect of adrenosterone on the dissemination of mCherry-positive cells in the fish at 16 dpf. Fish were treated with either vehicle (left) or adrenosterone (right). The images are shown as Z-stack images using 100× magnification. Scale bar, 100 μm. C. Mean frequencies of the fish showing the dissemination patterns of mCherry-positive cells in the vehicle- or adrenosterone-treated groups. Each value is presented as mean ± SEM from three independent experiments. Images are reprinted from Nakayama et al. (2020).