RESUMO
Acute lymphoblastic leukemia (ALL) is the most common pediatric, and ninth most common adult, cancer. ALL can develop in either B or T lymphocytes, but B-lineage ALL (B-ALL) exceeds T-ALL clinically. As for other cancers, animal models allow study of the molecular mechanisms driving ALL. Several zebrafish (Danio rerio) T-ALL models have been reported, but until recently, robust D. rerio B-ALL models were not described. Then, D. rerio B-ALL was discovered in two related zebrafish transgenic lines; both were already known to develop T-ALL. Here, we report new B-ALL findings in one of these models, fish expressing transgenic human MYC (hMYC). We describe B-ALL incidence in a large cohort of hMYC fish, and show B-ALL in two new lines where T-ALL does not interfere with B-ALL detection. We also demonstrate B-ALL responses to steroid and radiation treatments, which effect ALL remissions, but are usually followed by prompt relapses. Finally, we report gene expression in zebrafish B lymphocytes and B-ALL, in both bulk samples and single B- and T-ALL cells. Using these gene expression profiles, we compare differences between the two new D. rerio B-ALL models, which are both driven by transgenic mammalian MYC oncoproteins. Collectively, these new data expand the utility of this new vertebrate B-ALL model.
RESUMO
Zebrafish (Danio rerio) are a powerful model to study lymphocyte development. Like mammals, D. rerio possess an adaptive immune system that includes B and T lymphocytes. Studies of zebrafish lymphopoiesis are difficult because antibodies recognizing D. rerio cell surface markers are generally not available, complicating isolation and characterization of different lymphocyte populations, including B-lineage cells. Transgenic lines with lineage-specific fluorophore expression are often used to circumvent this challenge. The transgenic lck:eGFP line has been used to study D. rerio T cell development, and has also been utilized to model T cell development and acute lymphoblastic leukemia (T-ALL). Although lck:eGFP fish have been widely used to analyze the T-lineage, they have not been used to study B cells. Recently, we discovered that many zebrafish B cells also express lck, albeit at lower levels. Consequently, lck:eGFP B cells likewise express low levels of GFP. Based on this finding, we developed a protocol to purify B-lineage cells from lck:eGFP zebrafish, which we report here. Our method describes how to utilize a fluorescent-activated cell sorter (FACS) to purify B cells from lck:eGFP fish or related lines, such as double-transgenic rag2:hMYC; lck:eGFP fish. In these lines, B cells, particularly immature B cells, express GFP at low but detectable levels, allowing them to be distinguished from T cells, which express GFP highly. B cells can be isolated from marrow, thymus, spleen, blood, or other tissues. This protocol provides a new method to purify D. rerio B cells, enabling studies focused on topics like B cell development and B lymphocyte malignancies.
Assuntos
Linfócitos B/citologia , Linfócitos B/patologia , Separação Celular/métodos , Proteínas de Fluorescência Verde/genética , Peixe-Zebra/imunologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Linhagem da Célula , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Linfócitos T/citologia , Peixe-Zebra/genéticaRESUMO
Precursor-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric cancer, but there are no useful zebrafish pre-B ALL models. We describe the first highly- penetrant zebrafish pre-B ALL, driven by human MYC. Leukemias express B lymphoblast-specific genes and are distinct from T cell ALL (T-ALL)-which these fish also develop. Zebrafish pre-B ALL shares in vivo features and expression profiles with human pre-B ALL, and these profiles differ from zebrafish T-ALL or normal B and T cells. These animals also exhibit aberrant lymphocyte development. As the only robust zebrafish pre-B ALL model and only example where T-ALL also develops, this model can reveal differences between MYC-driven pre-B vs. T-ALL and be exploited to discover novel pre-B ALL therapies.