Functional Niche Competition Between Normal Hematopoietic Stem and Progenitor Cells and Myeloid Leukemia Cells

Abstract

Hematopoietic stem and progenitor cells (HSPCs) reside in a specialized niche that regulates their proliferative capacity and their fate. There is increasing evidence for similar roles of marrow niches on controlling the behavior of leukemic cells; however, whether normal hematopoietic stem cell (HSC) and leukemic cells reside in or functionally compete for the same marrow niche is unclear. We used the mixed lineage leukemia-AF9 (MLL-AF9) murine acute myeloid leukemia (AML) in a competitive repopulation model to investigate whether normal HSPC and leukemic cells functionally compete for the same marrow niches. Irradiated recipient mice were transplanted with fixed numbers of MLL-AF9 cells mixed with increasing doses of normal syngeneic whole bone marrow (WBM) or with purified HSPC (LSK). Survival was significantly increased and leukemic progression was delayed proportional to increasing doses of normal WBM or normal LSK cells in multiple independent experiments, with all doses of WBM or LSK cells studied above the threshold for rapid and complete hematopoietic reconstitution in the absence of leukemia. Confocal microscopy demonstrated nests of either leukemic cells or normal hematopoietic cells but not both in the marrow adjacent to endosteum. Early following transplantation, leukemic cells from animals receiving lower LSK doses were cycling more actively than in those receiving higher doses. These results suggest that normal HSPC and AML cells compete for the same functional niche. Manipulation of the niche could impact on response to antileukemic therapies, and the numbers of normal HSPC could impact on leukemia outcome, informing approaches to cell dose in the context of stem cell transplantation.

Keywords: Acute myeloid leukemia; Bone marrow; Competition; Hematopoietic stem cells; Murine; Niche.

Figure 1. Impact of normal bone marrow cell dose on survival following MLL-AF9 leukemia initiation

Kaplan-Meier survival curves for recipient C57L/6J mice co-transplanted with fixed-numbers of MLL-AF9 cells (4 × 10⁵) mixed with increasing numbers of normal marrow hematopoietic cells, either whole bone marrow (WBM, panel A), or purified hematopietic stem and progenitor cells (LSK, panel B). Controls in each experiment received MLL-AF9 alone or normal hematopoietic cells alone. Animals were euthanized when premorbid based on predefined objective criteria. (A) Whole bone marrow. (B) Purified LSK cells. P values are shown comparing survivals between groups via logrank testing. Comparisons with p <0.05 are considered significant and are designated “*” or “**” for p <0.025.

Figure 2. Impact of normal hematopoietic stem and progenitor cell dose on MLL-AF9 leukemic burden

Groups of recipients were co-transplanted with 4 × 10⁵ MLL-AF9 cells alone or 4 × 10⁵ MLL-AF9 cells and increasing numbers of purified LSK cells, and then assessed for leukemia burden (via % GFP + MLL-AF9 cells) in the femoral bone marrow (A) and spleen (B) at 7 and at 12 days following transplantation. At 7 days there was a significant reduction in leukemic infiltration comparing 50,000 versus 10,000 LSK cells for both femurs (A) p=2.71×10⁻⁶ and spleens (B) p= 1.152×10⁻⁵. By day 12, all groups were grossly leukemic. As expected, MLL-AF9 cells administered alone rapidly constituted the entirety of the marrow, due to the ablation of endogenous hematopoiesis via the total body irradiation.

Figure 3. Impact of normal hematopoietic cell dose on organ leukemic infiltration

Representative hematoxylin and eosin stained sections of liver from mice transplanted with normal WBM alone (A), fixed numbers of MLL-AF9 cells (4 ×10⁵) mixed with 1 or 50 million WBM cells (panels B and C), purified LSK cells alone (D), or fixed numbers of MLL-AF9 mixed with 10,000 (E) or 50,000 (F) LSK cells. Magnification 100X. Animals were euthanized on day 8 for WBM and day 7 for LSK.

Figure 4. Impact of normal hematopoietic cell dose on cycling of leukemia cells at time of disease progression

At the time of euthanasia due to a pre-morbid state, the cycling status of marrow leukemic cells was measured. The % of GFP+ leukemic cells that expressed Ki67 is shown. (A) Representative Ki67 staining of the GFP+ leukemic cells. (B) Comparison of % Ki67+ tumor cells at different LSK doses at the time of euthanasia. A dose of 50,000 purified LSKs resulted in a significantly increased fraction of Ki67+ leukemic cells, compared to groups transplanted with lower doses of purified LSKs (p=0.0075).

Figure 5. Impact of normal hematopoietic cell dose on cycling of leukemia cells early post-transplantation

When animals were sacrificed at set times post-transplantation, the proportion of Ki67+ leukemic cells was reduced at higher (50,000 LSK) versus lower (10,000 LSK) normal cell doses at day 7 (p=0.0007, marked “***”), but by day 12 had increased compared to the lower cell dose (p=0.0232, marked “**”).

Figure 6. Sternal bone marrow confocal combined with two-photon imaging demonstration of leukemic and normal HSCs foci

Representative micrographs of sternal marrow sections from mice transplanted with normal WBM (marked by dsRED) only compared with fixed amount of MLL-AF9 cells (marked by GFP) along with different LSKs doses. Scale bars indicate 300 μm. Two mice were imaged for each time point in each group, in two independent experiments.