Human acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rγc-deficient mice

Abstract

Human leukemic stem cells, like other cancer stem cells, are hypothesized to be rare, capable of incomplete differentiation, and restricted to a phenotype associated with early hematopoietic progenitors or stem cells. However, recent work in other types of tumors has challenged the cancer stem cell model. Using a robust model of xenotransplantation based on NOD/SCID/IL2Rγc-deficient mice, we confirmed that human leukemic stem cells, functionally defined by us as SCID leukemia-initiating cells (SL-ICs), are rare in acute myelogenous leukemia (AML). In contrast to previous results, SL-ICs were found among cells expressing lineage markers (i.e., among Lin+ cells), CD38, or CD45RA, all markers associated with normal committed progenitors. Remarkably, each engrafting fraction consistently recapitulated the original phenotypic diversity of the primary AML specimen and contained self-renewing leukemic stem cells, as demonstrated by secondary transplants. While SL-ICs were enriched in the Lin-CD38- fraction compared with the other fractions analyzed, SL-ICs in this fraction represented only one-third of all SL-ICs present in the unfractionated specimen. These results indicate that human AML stem cells are rare and enriched but not restricted to the phenotype associated with normal primitive hematopoietic cells. These results suggest a plasticity of the cancer stem cell phenotype that we believe has not been previously described.

Figure 1. Heterogeneity of the disease immunophenotype compared among patients diagnosed with AML.

A representative of CD34^positive specimen (specimen 348) (top row), a representative of CD34^dim specimen (specimen 53) (second row), a representative of CD34^negative primary specimen (specimen 325) (third row), and an example of CD34⁺-enriched samples from normal BM (bottom row) are shown. Boxes represent all the different fractions sorted in this study. SSC, side scatter. Numbers in flow plots represent the percent of gated cells within each indicated region.

Figure 2. Engraftment level of human AML cells in BM of NSG mice after 12 weeks after transplantation of Lin^negative and Lin^dim subsets sorted from 6 primary AML patient samples.

Black diamonds show the engraftment level in each individual mouse, injected with at least 0.3 million sorted human cells. Numbers above each graph indicate the specimen identification number. Numbers by each horizontal bar indicate the mean of human blast engraftment in NSG mice for each sorted and injected fractions.

Figure 3. SL-IC do not necessarily express CD34.

Engraftment level of human AML cells in BM of NSG mice 12 weeks after transplantation of CD34^– and CD34⁺ subsets sorted from 6 primary AML patient samples expressing (A) CD34 or (B) not. Black diamonds show the engraftment level in each individual mouse, injected with 0.1 to 1.0 million sorted human cells.

Figure 4. SL-ICs are not restricted to CD38^– populations.

(A) Engraftment level of human AML cells in BM of NSG mice after 12 weeks after transplantation of CD38^– and CD38⁺ subsets sorted from AML primary samples. (B) Sixteen-hour incubation of AML mononuclear cells from primary specimen 53, with anti-CD38 antibody affects the human cell engraftment in NSG mice. (C) Comparison of the engraftment level after 12 weeks after transplantation of unsorted, unstained (–) samples versus unsorted, stained (+) samples for 4 primary AML specimens. Black diamonds show the engraftment level in individual mice, injected with 0.1 to 1.0 million sorted human cells. *P < 0.05, Mann-Whitney U test.

Figure 5. Engraftment level of human AML cells in BM of NSG mice 12 weeks after transplantation of CD38⁺CD45RA^– and CD38⁺CD45RA⁺ subsets sorted from 5 AML primary samples.

Black diamonds show the engraftment level in each individual mouse, injected with 0.1 to 1.0 million sorted human cells.

Figure 6. Reconstitution of the original AML phenotype from all sorted fractions from CD34^negative specimen 325.

(A, B, and F) Original immunophenotype of the patient cells. (C–E) Phenotype of BM cells from NSG mice injected with Lin^–CD38^– cells and harvested 12 weeks after transplantation. (G–I) Phenotype of BM cells from NSG mice injected with Lin^dimCD38⁺ cells and harvested 12 weeks after transplantation. Numbers in flow plots represent the percent of gated population within each indicated region.

Figure 7. The absolute distribution of SL-ICs does not correlate with their immunophenotypic distribution.

We assessed the percentage of the total cells found in each phenotype by extensive flow cytometry (top bar). Subsequently, we calculated the absolute numbers of SL-ICs associated with each fraction (bottom bar) using the SL-ICs frequency and the percentage of the total cells found in each phenotype. For instance, Lin^–CD38^– cells contain the highest frequency of SL-ICs (1 in 38,030 cells); however, Lin^–CD38^– cells represent only 3% of all mononuclear cells in patient 53 (top bar). Normalizing to 100 SL-ICs (i.e., 100 × [1/430,240] = 43 × 10⁶ mononuclear cells), we determined that, in spite of having the highest frequency of SL-ICs, the Lin^–CD38^– fraction contains only 34% (43 × 10⁶ × 0.03/38,030 = 34) of all SL-ICs (bottom bar).

Figure 8. Proposed models for leukemic stem hypothesis.

The original model described a binary model of tumor heterogeneity, with most differentiated cells losing all LSC activity (top row). LSCs exist but are not discriminated by current markers (missing marker model; second row). LSCs are enriched but not entirely restricted to immature cell phenotype, with limited capacity to dedifferentiate (modified differentiation model; third row). LSCs are not enriched in the immature cell fraction; probability of retaining stem cell property is independent of maturation markers (tumor plasticity model; bottom).