A Humanized Zebrafish Transplant Model Expressing CXCL12 Provides an Enhanced In Vivo Therapeutic Screening Platform for T-ALL
Authors: Rajan Vinothkumar , Melong Nicole , Campbell Clinton JV , Dellaire Graham , Berman Jason N. 
Published: October 2019

Abstract
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia and accounts for approximately 15% of all pediatric ALL cases. Outcomes have improved with contemporary chemotherapy regimens, however, they remain inferior to the 90% survival rates now observed for B-immunophenotype ALL. Perturbations in several signaling pathways, such as the NOTCH and PI3-kinase pathways, have been found to contribute to the pathogenesis of T-ALL, providing opportunities for targeted therapies. We previously developed a zebrafish-based xenotransplantation model of T-ALL, whereby we transplanted primary human T-ALL samples into transparent zebrafish embryos and identified a novel NOTCH mutation based on the differential response of a particular patient sample to the addition of a γ-secretase inhibitor added to the embryo water (Bentley et al, Haematologica 2015). While these results convincingly showed that the zebrafish xenograft model could serve as a preclinical chemical genomics screening platform, the absence of human microenvironmental cues in zebrafish may limit the direct translation of findings to the clinical context.
CXCL12 (SDF1α) is a cytokine recently shown to have tremendous influence on T-ALL survival, proliferation and migration (Passaro D et al, Cancer Cell 2015; Pitt LA et al, Cancer Cell 2015), including potentially an impact on the survival of the leukemia initiating cell population (LICs). SDF1α is duplicated in the zebrafish genome, but both paralogs lack binding capacity to human CXCR4 and hence are not biologically active in the presence of human leukemia cells. In order to humanize the zebrafish microenvironment to more accurately reflect the behavior and response of xenografted human leukemia cells, we generated a transgenic zebrafish that expresses human CXCL12 under the zebrafish sdf1a promoter in the casper double pigment mutant. Fluorescently labeled T-ALL cell lines were injected into the zebrafish yolk sac at 48 hours post-fertilization (hpf) in both casper and CXCL12 transgenic casper embryos. CXCL12 transgenic embryos exhibited T-ALL cell migration beyond the site of injection at 5 days post-injection (dpi), whereas transplanted leukemia cells failed to migrate in the unaltered casper embryos. Importantly, at 7dpi in the CXCL12 transgenic embryo, some of the T-ALL cells homed to the kidney marrow and caudal hematopoietic tissue (CHT), which are the sites of zebrafish hematopoiesis.
This study is the first to demonstrate the ability to enhance the zebrafish xenograft platform through modifications to the microenvironment, analogous to what has been observed in mouse models. These studies set the stage for employing this approach to evaluate novel therapies in T-ALL that are both tumor-directed, as well as those targeted to the microenvironment, such as the CXCR4 antagonist, AMD3100. Given the ability to easily engraft patient-derived samples and test individual or combination therapies in a one-week timeframe, this approach has tremendous potential to provide rapid real time preclinical response data to help personalize therapy for patients with T-ALL and other hematologic malignancies.

Disclosures
No relevant conflicts of interest to declare.