Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies

Abstract

Zebrafish are a major model for chemical genetics, and most studies use embryos to find small molecules that cause interesting phenotypes or that can rescue disease models. Limited studies have dosed adults with small molecules, and relied on water-born exposure or injection techniques. Challenges in drug delivery-related trauma and anesthesia-related toxicity have prevented the adult zebrafish from long-term drug efficacy studies. Here we introduce a novel anesthetic combination of MS-222 and isoflurane to an oral gavage technique for a non-toxic, non-invasive and long-term drug administration platform. As a proof of principle, we established drug efficacy of the FDA-approved BRAFV600E inhibitor, Vemurafenib, in the adult zebrafish harboring BRAFV600E melanoma tumors. In the model, adult casper zebrafish intraperitoneally transplanted with a zebrafish melanoma cell line (ZMEL1) are exposed to daily sublethal dosing at 100 mg/kg of Vemurafenib for 2 weeks via oral gavage and resulted in an average 65% decrease in tumor burden and a 15% mortality rate. In contrast, Vemurafenib-resistant ZMEL1 cell lines, generated in culture from low-dose drug exposure for 4 months, did not respond to the oral gavage treatment regimen when transplanted into irradiated casper zebrafish. Similarly, caspers transplanted with primary BRAFV600E-mutant melanoma tumors resulted in a 65% decrease in tumor burden when gavaged daily with 100 mg/kg Vemurafenib for 2 weeks. This drug treatment regimen can be applied to adult transgenic zebrafish harboring primary melanoma tumors and resulted in a 70% decrease in tumor burden. Taken together, we developed an effective long-term drug treatment system in both a transplantation model using adult casper zebrafish and a primary melanoma model using adult transgenic zebrafish. This drug administration technique of oral gavage will allow adult zebrafish to be used to identify more effective anti-melanoma combination therapies and opens up possibilities for treating adult models of other diseases.