ABSTRACT
Leukemia-initiating cells (LICs) in acute myeloid leukemia (AML) are believed to be restricted to the CD34(+) fraction. However, one of the most frequently mutated genes in AML is nucleophosmin (NPM), and this is associated with low CD34 expression. We, therefore, investigated whether NPM-mutated AMLs have LICs restricted to the CD34(+) fraction. We transplanted sorted fractions of primary NPM-mutated AML into immunodeficient mice to establish which fractions initiate leukemia. Approximately one-half of cases had LICs exclusively within the CD34(-) fraction, whereas the CD34(+) fraction contained normal multilineage hematopoietic repopulating cells. Most of the remaining cases had LICs in both CD34(+) and CD34(-) fractions. When samples were sorted based on CD34 and CD38 expression, multiple fractions initiated leukemia in primary and secondary recipients. The data indicate that the phenotype of LICs is more heterogeneous than previously realized and can vary even within a single sample. This feature of LICs may make them particularly difficult to eradicate using therapies targeted against surface antigens.
Subject(s)
Antigens, CD34/metabolism , Leukemia, Myeloid, Acute/pathology , Neoplastic Stem Cells/pathology , Nuclear Proteins/genetics , ADP-ribosyl Cyclase 1/metabolism , Animals , Cell Separation/methods , Erythroid Precursor Cells/metabolism , Erythroid Precursor Cells/pathology , Erythroid Precursor Cells/transplantation , Humans , Immunotherapy, Adoptive/methods , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/therapy , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mutant Proteins/metabolism , Neoplastic Stem Cells/metabolism , Nuclear Proteins/metabolism , Nucleophosmin , Phenotype , Xenograft Model Antitumor AssaysABSTRACT
How melanoma acquire a metastatic phenotype is a key issue. One possible mechanism is that metastasis is driven by microenvironment-induced switching between noninvasive and invasive states. However, whether switching is a reversible or hierarchical process is not known and is difficult to assess by comparison of primary and metastatic tumors. We address this issue in a model of melanoma metastasis using a novel intravital imaging method for melanosomes combined with a reporter construct in which the Brn-2 promoter drives green fluorescent protein (GFP) expression. A subpopulation of cells containing little or no pigment and high levels of Brn2::GFP expression are motile in the primary tumor and enter the vasculature. Significantly, the less differentiated state of motile and intravasated cells is not maintained at secondary sites, implying switching between states as melanoma cells metastasize. We show that melanoma cells can switch in both directions between high- and low-pigment states. However, switching from Brn2::GFP high to low was greatly favored over the reverse direction. Microarray analysis of high- and low-pigment populations revealed that transforming growth factor (TGF)beta2 was up-regulated in the poorly pigmented cells. Furthermore, TGFbeta signaling induced hypopigmentation and increased cell motility. Thus, a subset of less differentiated cells exits the primary tumor but subsequently give rise to metastases that include a range of more differentiated and pigment-producing cells. These data show reversible phenotype switching during melanoma metastasis.
