High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia

Abstract

Self-renewal is a feature of cancer and can be assessed by cell transplantation into immune-compromised or immune-matched animals. However, studies in zebrafish have been severely limited by lack of these reagents. Here, Myc-induced T-cell acute lymphoblastic leukemias (T-ALLs) have been made in syngeneic, clonal zebrafish and can be transplanted into sibling animals without the need for immune suppression. These studies show that self-renewing cells are abundant in T-ALL and comprise 0.1% to 15.9% of the T-ALL mass. Large-scale single-cell transplantation experiments established that T-ALLs can be initiated from a single cell and that leukemias exhibit wide differences in tumor-initiating potential. T-ALLs also can be introduced into clonal-outcrossed animals, and T-ALLs arising in mixed genetic backgrounds can be transplanted into clonal recipients without the need for major histocompatibility complex matching. Finally, high-throughput imaging methods are described that allow large numbers of fluorescent transgenic animals to be imaged simultaneously, facilitating the rapid screening of engrafted animals. Our experiments highlight the large numbers of zebrafish that can be experimentally assessed by cell transplantation and establish new high-throughput methods to functionally interrogate gene pathways involved in cancer self-renewal.

Figure 1

T-ALLs from CG1-strain zebrafish engraft into non-IR CG1-strain recipients. GFP-labeled T-ALLs were isolated from primary leukemic fish, and 10³ FACS-sorted GFP-labeled leukemia cells were transplanted into non-IR CG1- and AB-strain animals (A-C and G-I, respectively) or IR AB-strain fish (D-F). Transplant fish were scored for engraftment at 10, 20, and 30 days after transplantation. Panels are merged images of fluorescent and brightfield photographs. The engraftment kinetics differ greatly when T-ALLs are introduced into CG1 (J) or IR AB-strain zebrafish (AB + IR; K). Percent engraftment is the percentage of recipient animals that have visibly engrafted T-ALL at each time point. Panels J and K are combined data from 3 independent experiments (n = 238 transplants total). The raw data for this experiment are available in supplemental Table 2.

Figure 2

Single T-ALL cells can be efficiently transplanted into non-IR CG1 recipient. Transplant recipient fish receiving 1 FACS-sorted dsRED⁺ T-ALL cell at 20 (A,D), 30 (B,E), and 45 days after transplantation (C,F). One representative animal engrafted T-ALL by 20 days (A-C), whereas one never developed leukemia (D-F). Images photographed at 7.0×. All panels are merged images of fluorescent and brightfield photographs.

Figure 3

T-ALLs from CG1 fish are capable of engraftment into CG1 outcrossed animals. A GFP-labeled T-ALL engrafted into the progeny of CG1 by albino fish (CG1/Alb; A-C). Engraftment of T-ALLs into AB-strain zebrafish required 2 rounds of outcrossing to CG1 fish (D-I). A dsRED-labeled T-ALL engrafted into the progeny of a CG1 by CG1/AB rag2-GFP transgenic animal (D-F) or an Amcyan-labeled T-ALL engrafted into the progeny of a CG1 by CG1/AB mylz2-mCherry (G-I). Images photographed at 7.0×. All panels are merged images of fluorescent and brightfield photographs. GFP-labeled thymus is marked by T.

Figure 4

The LED fluorescence macroscope can image 5 fluorescent fluorophores in normal muscle and T-cell acute lymphoblastic leukemia (T-ALL). Whole animal imaging of wild-type (Negative), mylz2-Amcyan, mylz2-GFP, creatine kinase-zsYellow, mylz2-mCherry transgenic zebrafish (A-D). Amcyan was imaged by the use of a blacklight and 480/30 filter (A,J,N), GFP (blacklight, 520/30 filter; B,K,O), zsYellow (blue light, 575/52 filter; C), dsRED express (green light, 610/40 filter; L,P), and mCherry (green light, 610/40 filter; D). Transgenic zebrafish imaged using an epi-fluorescence stereomicroscope at the lowest magnification (7.0×; E-H). The LED fluorescence macroscope is also capable of imaging engraftment of T-cell acute lymphoblastic leukemia in recipient animals. Whole-animal imaging of recipient fish transplanted with 1.5 × 10⁴ Amcyan-, GFP-, or dsRED express–labeled leukemia cells at 30 days after transplantation (I-L). Animals from panels I through L were mixed to demonstrate that fluorescent-labeled animals can be easily delineated (M-P). Transplant recipients imaged by the use of an epi-fluorescence stereomicroscope at low magnification (7.0×, Q-T). Scale bars are 2 cm in panels A through D and I through L, and 5-mm in panels E through H and Q through T.

Figure 5

The LED fluorescence macroscope can detect tumor engraftment of single T-ALL cells and is capable of multispectral, real-time imaging. Animals engrafted with a single dsRED-labeled T-ALL cell can be easily distinguished from nonengrafted animals at 45 days after transplantation (n = 1 of 10 fish, A; n = 2 of 20 fish, B; n = 3 of 29 fish panel C) by LED fluorescence macroscopy. Multispectral imaging with the LED fluorescence macroscope (D-I). Still image capture with dual-spectrum imaging of unanesthetized mylz2-Amcyan and mylz2-mCherry transgenic animals (D, blue and black light with 520/40 filter and 640/35 filter), mylz2-GFP and mylz2-mCherry (E, blue light with 520/40 filter and 640/35 filter), and creatine kinase-zsYellow and mylz2-mCherry (F, blue light with 575/52 filter and 640/35 filter). Animals were imaged from below at 30 frames per second, and a single frame is shown. Multispectral imaging also can be used to visualize fluorescent-labeled T-ALLs engrafted into fluorescent recipient animals. A mylz2-mCherry animal engrafted with an Amcyan-labeled T-ALL at 30 days after transplantation imaged by epi-fluorescence stereomicroscopy (G, 7.0× magnification) or by use of the LED fluorescence macroscope (blue light with 480/30 and 640/35 filters; H-I). Panel H shows a control animal (top) along with a dual transgenic leukemic fish (bottom). Three leukemic fish imaged from below at 30 frames per second and a single frame is shown (I). Scale bars are 1 cm in panels A through F and H through I; 2 mm in panel G.