Establishment of <em>HRAS</em> Transgenic Medaka as a Stable Tumor Model for <em>In Vivo</em> Screening of Anticancer Drugs

<div><p>Most targeted anticancer drugs have been identified by screening at the molecular or cellular level <em>in vitro</em>. However, many compounds selected by such costly and time-consuming screening do not prove effective against tumors <em>in vivo</em>. The development of anticancer drugs would thus be facilitated by the availability of an <em>in vivo</em> screening system based on a multicellular organism. We have now established a transgenic line of the freshwater fish medaka in which melanophores (melanocytes) proliferate in a manner dependent on heat shock–induced signaling by a human RAS oncoprotein. The human <em>HRAS^G12V</em> oncogene was expressed under the control of a melanophore-specific gene promoter in order to allow visualization of tumor growth in live fish maintained in a water tank. The expression of <em>HRAS^G12V</em> was induced as a result of Cre-mediated recombination by exposure of the fish to a temperature of 37°C for 30 min, given that the Cre gene was placed under the control of a medaka heat shock promoter. One of the stable transgenic lines developed abnormal pigment cell proliferation in the eyes and epidermis with 100% penetrance by 6 months postfertilization. Sorafenib, an inhibitor of RAS signaling, was administered to the transgenic fish and was found both to reduce the extent of melanophore proliferation and to improve survival. The transgenic medaka established here thus represents a promising <em>in vivo</em> system with which to screen potential anticancer drugs that target RAS signaling, and this system can readily be adapted for the screening of agents that target other oncogenes.</p> </div

Hematoxylin-eosin staining of <i>Tg</i><i>(</i><i>tyr:HRAS^G12V</i><i>)</i>/<i>Tg</i><i>(</i><i>hsp:cre</i><i>)</i> medaka.

<p>(<b>A</b>) Overall image of fish #7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054424#pone-0054424-g003" target="_blank">Figure 3</a>. (<b>B</b>)– (<b>E</b>), Melanophore layers (arrows) of a wild-type adult fish (<b>B</b>) as well as of fish #7 (<b>C</b>), #6 (<b>D</b>), and #4 (<b>E</b>) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054424#pone-0054424-g003" target="_blank">Figure 3</a>. (<b>F</b>)– (<b>J</b>), Eye, bone and spinal cord, heart, muscle, and gill, respectively, of fish #7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054424#pone-0054424-g003" target="_blank">Figure 3</a>. (<b>K</b>) Enlarged image of the boxed region in (<b>J</b>). (<b>L</b>) Kidney and digestive duct of fish #7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054424#pone-0054424-g003" target="_blank">Figure 3</a>. (<b>M</b>) Enlarged image of the boxed region in (<b>L</b>). Scale bars: 1 mm (<b>A</b>) and 0.1 mm (<b>B</b>–<b>M</b>).</p

Effect of sorafenib (0.1 μM) treatment on melanophore hyperplasia and overall survival in <i>Tg</i><i>(</i><i>tyr:HRAS^G12V</i><i>)</i><i>/Tg</i><i>(</i><i>hsp:cre</i><i>)</i> medaka.

<p>(<b>A</b>) Schedule of drug administration. (<b>B</b>) Photos taken from the dorsal side of all fish at 7 days before (day –7) and 29 days after (day +29) the first drug administration. The area of MPLs in the fish body was measured based on the set of captured images on the right, and the average fold change from day –7 to day +29 was calculated for each group. (<b>C</b>) Kaplan-Meier survival curves for the sorafenib-treated and control (DMSO-treated) groups were generated from the experiment shown in (<b>B</b>). Fish with obvious melanophore hyperplasia were divided into two groups, one of which was treated with 0.1 μM sorafenib (<i>n</i> = 14) and the other with DMSO as a control (<i>n</i> = 13). *<i>P</i> = 0.0267 (log-rank test).</p

Characteristics of <i>Tg</i><i>(</i><i>tyr:HRAS^G12V</i><i>)</i>/<i>Tg</i><i>(</i><i>hsp:cre</i><i>)</i> medaka.

<p>Age-dependent changes in eight double-transgenic fish were monitored after heat treatment at 1 wpf. Five of the eight fish manifested hyperproliferation of pigment cells around the eyes by 8 wpf. All of the fish had developed MPLs with characteristics of infiltrative tumors by 20 wpf, and all had died by 43 wpf. Arrows indicate the first appearance of metastasis-like lesions.</p

Age dependence of MPL incidence and survival in <i>Tg</i><i>(</i><i>tyr:HRAS^G12V</i><i>)</i>/<i>Tg</i><i>(</i><i>hsp:cre</i><i>)</i> medaka.

<p>The transgenic fish were subjected to heat treatment at 1 wpf and then monitored for the development of MPLs and death. A single monitoring was performed with 39 <i>Tg(tyr:HRAS^G12V)</i>/<i>Tg(hsp:cre)</i> medaka at 4 wpf.</p

Generation of transgenic medaka expressing human <i>HRAS^G12V</i>.

<p>A plasmid containing the human <i>HRAS^G12V</i> cDNA downstream of the medaka tyrosinase gene (<i>tyr</i>) promoter as well as the <i>EGFP</i> gene positioned between <i>loxP</i> sequences was constructed for the generation of transgenic medaka. These transgenic fish were crossed with another transgenic line harboring the gene for Cre recombinase (<i>cre</i>) under the control of the medaka <i>hsp70</i> promoter. The resulting double-transgenic animals express Cre recombinase after exposure to heat shock, resulting in excision of the <i>EGFP</i> gene and expression of <i>HRAS^G12V</i>.</p

Penetrance of melanophore proliferative lesions (MPLs) among <i>Tg(tyr:HRAS^G12V)</i>/<i>Tg(hsp:cre)</i> double-transgenic medaka derived from four different <i>Tg(tyr:HRAS^G12V)</i> lines (Tg lines 1 to 4).

<p>The fish were subjected to heat treatment by incubation at 37°C for 30 minutes at 4 wpf and examined at 6 mpf.</p