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

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.

SpCas9- and LbCas12a-Mediated DNA Editing Produce Different Gene Knockout Outcomes in Zebrafish Embryos.

CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) genome editing is a powerful technology widely used in current genetic research. In the most simple and straightforward way it can be applied for a gene knockout resulting from repair errors, induced by dsDNA cleavage by Cas nuclease. For decades, zebrafish (Danio rerio) has been known as a convenient model object of developmental biology. Both commonly used nucleases SpCas9 (Streptococcus pyogenes Cas9) and LbCas12a (Lachnospiraceae bacterium Cas12a) are extensively used in this model. Among them, LbCas12a is featured with higher specificity and efficiency of homology-directed editing in human cells and mouse. But the editing outcomes for these two nucleases in zebrafish are still not compared quantitatively. Therefore, to reveal possible advantages of one nuclease in comparison to the other in the context of gene knockout generation, we compare here the outcomes of repair of the DNA breaks introduced by these two commonly used nucleases in zebrafish embryos. To address this question, we microinjected the ribonucleoprotein complexes of the both nucleases with the corresponding guide RNAs in zebrafish zygotes and sequenced the target gene regions after three days of development. We found that LbCas12a editing resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies (percentage of mutated copies of the target gene to all gene copies in the embryo) of both nucleases were the same. On the other hand, overlapping of protospacers resulted in similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.

A new zebrafish model for experimental leukemia therapy.

The efficacy of cyclophosphamide (CY), vincristine (VCR) and prednisolone (PRE) were studied in leukemia-bearing zebrafish larvae. A transplantable T-cell acute lymphoblastic leukemia (T-ALL) line ZL1 was induced by mosaic expression of zRag2-EGFP-mMyc transgene and underwent more than 20 consecutive transplantations in adult syngeneic fish prior to the experiments. Drug efficiency was assessed by an increase of lifespan (ILS) of treated leukemia-bearing animals as compared with untreated leukemia-bearing animals. Different doses of the drugs and length of the treatment were tested. CY and VCR demonstrated therapeutic effect which was dose- and time course-dependent. The maximal increase of ILS reached 61.1% after CY (400 mg/L, 72 h) treatment and 44.4%-in VCR (4 mg/L, 72 h) treated animals. None of the tumor-bearing larvae showed complete recovery from leukemia as a result of any VCR and CY monotherapy schedule. PRE was inefficient for treatment of leukemia in zebrafish in a dose range between 1 and 50 mg/L and a treatment length between 24 and 72 h due to it toxicity exclusively towards leukemia-bearing larvae. These data demonstrate that, in addition to morphological and genetic similarities with mammalian leukemia, zebrafish T-ALL is also sensitive to the same chemotherapeutic drugs in vivo as mammals. Therefore, this model can be utilized as a cost effective system for experimental tumor therapy and large-scale screening of anticancer compounds.