Protocol for rapid assessment of the efficacy of novel Wnt inhibitors using zebrafish models

Abstract

Summary Dysregulation of Wnt signaling is a hallmark of many cancers, and the development of effective, non-toxic small-molecule Wnt inhibitors is desirable. Off-target toxicities of new compounds are typically tested in mouse models, which is both costly and time consuming. Here, we present a rapid and inexpensive protocol to determine the in vivo toxicity and efficacy of novel Wnt inhibitors in zebrafish using a combination of a fluorescence reporter assay as well as eye rescue and fin regeneration assays. These experiments are completed within 1 week to rapidly narrow drug candidates before moving to more expensive pre-clinical testing. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2020) .

Bioluminescent Zebrafish Transplantation Model for Drug Discovery

“…Usually, a trade-off average concentration has to be selected within the activity range of all small-molecule compounds used. This value is generally within the range of 1–15 µM in most zebrafish in vivo pharmaceutical screens ( Bowman and Zon, 2010 ; Colanesi et al, 2012 ; Precazzini et al, 2020 ; Haney et al, 2021 ; Oprisoreanu et al, 2021 ). Therefore, we decided to use 10 µM as our selected in vivo screening concentration.…”

Bioluminescent zebrafish transplantation model for drug discovery

“…Usually, a trade-off average concentration has to be selected within the activity range of all small-molecule compounds used. This value is generally within the range of 1 – 15 μM in most zebrafish in vivo pharmaceutical screens (Bowman and Zon, 2010; Colanesi et al, 2012; Haney et al, 2021; Oprisoreanu et al, 2021; Precazzini et al, 2020). Therefore, we decided to use 10 μM as our selected in vivo screening concentration.…”

“…Cell proliferation is most often measured until 3-4 dpi by the quantification of fluorescently labeled cancer cells, in vivo (Corkery et al, 2011;Ghotra et al, 2012;Hason and Bartunek, 2019;Patton et al, 2021b;Tulotta et al, 2016;Tulotta et al, 2019) or ex vivo (Somasagara et al, 2021). Our screening workflow enables rapid characterization of tumor-inhibitor responsiveness within a time frame of seven days.…”

“…Zebrafish embryos are naturally transparent and can engraft transplanted cancer cells until around the seventh-day post fertilization when the adaptive immune system starts to mature. This allows for simple in vivo tracking and imaging of fluorescently labeled cancer cells and the study of early tumor growth and dissemination with the involvement of the tumor microenvironment (Hason and Bartunek, 2019;Povoa et al, 2021;Tang et al, 2016;White et al, 2013). For longer-term studies, it is possible to use genetically immunocompromised animals, such as the rag2 E450fs or the prkdc D3612fs mutant lines (Tang et al, 2014;Tang et al, 2016;Yan et al, 2019;Yan et al, 2020).…”

Zebrafish drug screening identifies Erlotinib as an inhibitor of Wnt/β-catenin signaling and self-renewal in T-cell acute lymphoblastic leukemia

“…06051910 A) for modulators of the Wnt/β-catenin/TCF-LEF signaling axis modulators. Screening was carried out as previously described [ 39 , 40 ]. Briefly, at 24 h post-fertilization (hpf), zebrafish larvae showing similar GFP fluorescence in the tail fin were selected for use.…”