Molecular Determinants and Functional Characteristics of Leukemic Stem Cells and Their Interaction with the Niche.

We have recently described a subset of leukemic stem cells (LSC) with a high activity of aldehyde dehydrogenase (ALDH^br) that was prospectively isolated from the bone marrow of patients with newly diagnosed AML. These cells, among other features, were characterized by their increased affinity to the stem cell niche. We therefore hypothesized that the niche interaction may be a regulatory and protective factor for LSC. Hence, in the current study, we further characterized the LSC-niche interaction on single cell level, identified molecules that propagated LSC adhesion to the niche, and have correlated those features to functional characteristics like cell proliferation, chemotherapy resistance, and clinical outcome.

Mononuclear cells from the bone marrow or peripheral blood of patients with primary AML were obtained and LSC candidates were freshly isolated by Aldeflour staining followed by flow cytometry sorting based on their side scatter characteristics (SSC) and high ALDH activity (ALDH^br); cells from the same SSC-region with low ALDH activity (ALDH^dim) served as a control population. Subpopulations were further narrowed down by additional staining for CD34, CD133, or CD38. Expression of adhesion molecules like CXCR4, Cdh2, or CD44 was comparatively measured by quantitative real-time PCR and flow cytometry. The influence of the niche interaction on adhesion molecule expression was evaluated by co-cultures with mesenchymal stromal cells (MSCs) which served as a surrogate niche model. Cell fate, divisional kinetics, and clonal evolution of LSC candidates - both with and without contact to the stromal niche - were evaluated on a single cell level. Additionally, leukemic subsets were treated with chemotherapeutic agents, with and without MSC co-cultures, and vital cells were quantified by PI-/AxV-staining followed by flow cytometry. Co-incubation with novel CXCR4-antagonists and functional in vivo assays are currently underway.

Adhesion molecules, such as CD44s, Cdh2, and CXCR4, were consistently increased in the LSC subpopulations - both on mRNA and protein level (n=30; p<0.05). Their expression was further upregulated upon niche interaction in a time-dependent manner. For instance, the CXCR4 expression was elevated 3- to 6-fold within 24 hours cultivation with MSCs in our in vitro culture system, then gradually decreased, and was even downregulated at later timepoints. After chemotherapy treatment, significantly more vital cells were found in the ALDH^br subset as compared to ALDH^dim cells; even more leukemic cells survived upon co-culture with MSC. The descendants of single LSC clones expressed the original aberrations that were present at diagnosis.

Our current data confirmed that a LSC subset can be isolated based on a high ALDH activity. The increased expression of adhesion molecules in this subset mediates a close affinity to the marrow niche, which, in turn, protects LSC against proapoptotic stimuli and helps them maintain their stem cell features. Our present study and our ongoing research will help in developing novel therapeutic methods, i.e. interfering with the niche interaction and, thus, render LSC susceptible to conventional chemotherapy treatment.

No relevant conflicts of interest to declare.