Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker - PubMed (original) (raw)

Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker

Max Jan et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Hematopoietic tissues in acute myeloid leukemia (AML) patients contain both leukemia stem cells (LSC) and residual normal hematopoietic stem cells (HSC). The ability to prospectively separate residual HSC from LSC would enable important scientific and clinical investigation including the possibility of purged autologous hematopoietic cell transplants. We report here the identification of TIM3 as an AML stem cell surface marker more highly expressed on multiple specimens of AML LSC than on normal bone marrow HSC. TIM3 expression was detected in all cytogenetic subgroups of AML, but was significantly higher in AML-associated with core binding factor translocations or mutations in CEBPA. By assessing engraftment in NOD/SCID/IL2Rγ-null mice, we determined that HSC function resides predominantly in the TIM3-negative fraction of normal bone marrow, whereas LSC function from multiple AML specimens resides predominantly in the TIM3-positive compartment. Significantly, differential TIM3 expression enabled the prospective separation of HSC from LSC in the majority of AML specimens with detectable residual HSC function.

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Conflict of interest statement

Conflict of interest statement: I.L.W. is a Director of Stem Cells, Inc., and is a cofounder of Cellerant, Inc. and Stem Cells, Inc. I.L.W. and R.M. have filed International Patent Application No. PCT/US2009/000224 entitled “Markers of Acute Myeloid Leukemia Stem Cells.”

Figures

Fig. 1.

Fig. 1.

TIM3 is more highly expressed on AML LSC than on NBM HSC. (A) Representative flow cytometry histograms indicating TIM3 expression on NBM HSC (Lin−CD34+CD38−CD90+) and AML LSC (Lin−CD34+CD38−CD90−). (B) TIM3 protein expression was assessed by flow cytometry for multiple specimens of NBM HSC, primary AML LSC, and bulk AML. Mean fluorescence intensity was normalized for cell size and against lineage-positive cells for comparison between measurements conducted on different days. Normalized mean expression (and range) for each population were as follows: NBM HSC 8.8 (2.3–18.9), AML LSC 130.0 (3.0–488.5), bulk AML 148.1 (3.5–443.7). Using a two-sided Student's t test, differences in mean expression between NBM HSC and AML LSC (P = 0.01) and between NBM HSC and bulk AML (P = 0.01) were statistically significant, whereas the difference in mean expression between AML LSC and bulk AML (P = 0.67) was not statistically significant. (C) The percentage of cells positive for TIM3 expression by flow cytometry within the Lin−CD34+CD38− compartment of AML and normal bone marrow samples was determined by comparison with isotype control. Mean (and median) percentage of cells positive for TIM3 expression were as follows: NBM 30% (30%), AML 86% (98%). The difference in mean percentage of cells positive for TIM3 expression between NBM and AML (P < 0.0001) was statistically significant using a two-tailed Student's t test. (D) Clinical features of AML patients whose samples were studied here.

Fig. 2.

Fig. 2.

Functional NBM HSC and LSC differ in TIM3 expression. (A) TIM3+ and TIM3− fractions of the Lin−CD34+CD38− compartment from normal human bone marrow were identified by flow cytometry. (B) These cells were purified by two rounds of FACS and transplanted into NSG pups. Twelve weeks later, bone marrow cells were harvested and analyzed by flow cytometry for the presence of human CD45+ leukocyte engraftment (Left) whose lineage was further defined by expression of CD19 on lymphoid cells and CD33 on myeloid cells (Right). (C) Summary of long-term engraftment in NSG mice from normal bone marrow populations. (D) TIM3+ and TIM3− subpopulations were observed in Lin−CD34+ (SU018) or both Lin−CD34+CD38+ and Lin−CD34+CD38− (SU046) AML cells by flow cytometry. (E) Summary of bone marrow engraftment 12 wk after transplantation in NSG mice from multiple AML cases.

Fig. 3.

Fig. 3.

Prospective separation of residual normal HSC from leukemic cells in three AML cases. (A) TIM3 expression was determined on the Lin−CD34+ or Lin−CD34+CD38− fraction of each AML sample by flow cytometry. (B) TIM3− and TIM3+ cells were double-sorted to >99% purity by FACS. For AML SU043, these sorted cells were plated in duplicate into complete methylcellulose media and, 14 d later, myeloid colony formation was determined by microscopy. No leukemic blast colonies were observed. For AML SU014 and SU031, sorted cells were transplanted into NSG pups and, 12 wk later, human engraftment in mouse bone marrow was analyzed by flow cytometry as in Fig. 2_B_. (C) Sorted cell populations, methylcellulose colonies from TIM3− cells, and/or bone marrow cells from mice engrafted with TIM3− cells were assessed by PCR for the presence of the _FLT3_-ITD mutation for AML SU043 and SU014. For AML SU031, sorted cells or human CD45+ cells purified from engrafted mice were assessed by RT-PCR for the presence of the CBFB-MYH11 fusion transcript produced by inv(16) and human β-actin. (D) Summary of long-term engraftment in NSG mice of subpopulations from multiple AML cases.

Fig. 4.

Fig. 4.

Prospective separation of functional HSC and functional LSC from three AML cases. (A) TIM3 expression was determined on the Lin−CD34+ or Lin−CD34+CD38− fractions of each AML sample by flow cytometry. (B) TIM3+ and TIM3− cells were double-sorted to >99% purity by FACS. These sorted cells were transplanted into newborn NSG mice. Twelve weeks after transplantation, bone marrow cells were harvested and analyzed by flow cytometry for the presence of human CD45+ leukocyte engraftment (Left) whose lineage was further defined by expression of CD19 on lymphoid cells and CD33 on myeloid cells (Right). (C) Sorted cell populations and/or bone marrow cells from engrafted mice were assessed by PCR for the presence of the _FLT3_-ITD mutation for AML SU030 and SU047. For AML SU046, FISH analysis was used to assess the presence of the MLL translocation produced by t(6;11) in FACS-sorted cell populations and human CD45+ cells purified from engrafted mice. Representative normal and abnormal MLL FISH results using a break-apart probe are shown.

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