Molecular Mechanisms in Murine Syngeneic Leukemia Stem Cells

Abstract

Acute Myeloid Leukemia (AML) is a severe disease with a very high relapse rate. AML relapse may be attributable to leukemic stem cells (LSC). Notably, the "cancer stem cell" theory, which relates to LSCs, is controversial and criticized due to the technical peculiarities of the xenotransplant of human cells into mice. In this study, we searched for possible LSCs in an immunocompetent synergetic mice model. First, we found phenotypic heterogeneity in the ML23 leukemia line. We prospectively isolated a sub-population using the surface markers cKit⁺CD9^-CD48⁺Mac1^-/low, which have the potency to relapse the disease. Importantly, this sub-population can pass in syngeneic hosts and retrieve the heterogeneity of the parental ML23 leukemia line. The LSC sub-population resides in various organs. We present a unique gene expression signature of the LSC in the ML23 model compared to the other sub-populations. Interestingly, the ML23 LSC sub-population expresses therapeutic targeted genes such as CD47 and CD93. Taken together, we present the identification and molecular characterization of LSCs in a syngeneic murine model.

Keywords: AML; leukemic stem cell; syngeneic model.

Figure 1

ML23 leukemia line passage serially with heterogeneous phenotype: (a) Mice were injected with leukemic ML23 cells from frozen stocks. After leukemia detection in PB, cells were harvested from the BM and injected into other recipients. FACS plot of the primary recipient is of one month after transplantation, and, for the secondary recipient, is from 2 to 3 wk after passage. (b) mFACS plots showing the expression of the indicated markers (X axis), along with the ZsGreen reporter (Y axis).

Figure 2

Prospective isolation of LSCs in ML23 (a) schematic illustration of experimental design. Fresh Leukemic cells were sorted into four sub-populations, and each sub-population was transferred into a new host and its effects were examined. Gating for ML23 sub-populations using cKit/CD9 and CD48/Mac1. (b) FACS plots of leukemic cells, pre-gated ZsGreen+, as sorted into defined sub-populations. (c) ZsGreen+ expression levels from each transplanted sub-population. P11 sub-population transplanted mice presented high ZsGreen+ expression; others did not. (d) Organs from animals that received the indicated sub-population of ML23. Enlarged spleen, white bones, and enlarged LN are shown from P11. Representative data are shown from one out of three independent experiments.

Figure 3

P11 LSC can reconstitute the heterogeneity of ML23: (a) Sorted P11 sub-population (CD9-cKit+Mac1-CD48+), which were transplanted into the recipient mice. (b) P11 sub-population reconstituted the heterogeneity of ML23 in the recipient mice (the primary recipient of P11-sorted cells). Representative data are shown from one out of three independent experiments.

Figure 4

The sub-populations of ML23 distribute broadly in various organs. The average percentage expression (from ZsGreen+ cells) of each of the four sub-populations (P9—12) as measured using FACS analysis for cells extracted from six different tissues of ML23 leukemic mice. Cells were PRE-GATED to ZsGreen+, at least 90% in each sample (not shown). Data are shown from three independent experiments.

Figure 5

LSCs presenting a unique gene expression signature: (a) Some 470 genes (out of 20,020) are differentially expressed between the LSC sub-population (P11) and the other populations (FDR <0.1, fold change >2 or <−2). (b) PCA showing the 4 sub-populations (P9—P12). (c) Heatmap showing the 470 differentially expressed genes. (d) RNA expression signature of the four surface markers was found close to its expression at the protein level and used for validation (compare with Figure 3).

Figure 6

CD34 and CD84 are differentially expressed on ML23 sub-populations, in agreement with RNAseq data: (a) Heatmap of mRNA levels of Cd34 and Cd84 in the ML23 sub-populations. (b,c) Fresh ML23 cells were stained for the primary four surface markers in addition to CD34 and CD84 proteins. The expression level of each of the markers was examined in each of the sub-populations. Representative data are shown from one out of three independent experiments.

Figure 7

Specific drug-targeted genes expressed in ML23 cells. Differential expression levels of current treatment targets that are under study or clinical application were examined in the RNA-seq results of ML23 cells. Two of them (CD47 and CD93) presented overexpression on ML23 LIC (P11).