Diversity of cancer stem cells in acute lymphoblastic leukaemia

Abstract

For many cancers research led to controversial results regarding frequency and identity of cancer stem cells, cells that self-renew and are able to reconstitute the full phenotype of the original malignancy. In some malignancies such as acute myeloid leukaemia the hierarchical stem cell model that suggests that only rare and immunophenotypically immature blasts exhibit stem cell characteristics, resembling the normal physiological haematopoietic hierarchy, has been well established. In contrast to that, the stochastic model states that all or at least a substantial proportion of malignant cells has stem cell potential whereby this is supported by extrinsic stimuli. Initially, several studies suggested that acute lymphoblastic leukaemia (ALL) also is organised in a hierarchy. However, using more immunocompromised mouse strains and refined transplantation techniques for in vivo xenotransplantation models, previous findings regarding frequency and restriction of stem cells to very early B-precursor cell stages have been challenged in more recent studies. In order to address the questions of identity and frequency of stem cells in ALL, a robust orthotopic mouse model with the most immunocompromised mouse strain currently available (NOD/scid IL2Rγnull; NSG) was established. Primary diagnostic patient ALLs or blasts harvested from engrafted mouse bone marrow were sorted for B-cell lineage differentiation markers CD10, CD19, CD20 or CD34 to purify candidate stem cell populations of different maturity. All transplanted cell populations, from the most immature CD34+CD19low stage to the already more differentiated stage of CD19+CD20high cells were able to reconstitute the original ALL in mouse bone marrow and followed a typical dissemination pattern with infiltration of the spleen. Furthermore, this more mature CD19+CD20high subpopulation proved self-renewal ability in serial transplantation experiments. To investigate, whether stem cells in ALL are a rare entity or more abundant, unsorted bulk leukaemia blasts were transplanted in limiting dilutions from 1 x 104 to 10 cells per mouse. Cell numbers required for engraftment varied between leukaemias but the leukaemia engrafted in mice when only 10 to 1,000 cells were transplanted. The limiting dilution experiments were repeated with sorted blast populations according to the surface antigens CD10, CD20 and CD34. Stem cell frequencies in all sorted populations were comparable and as few as 10 to 100 cells were sufficient to reconstitute the leukaemia in mice. Stem cells do not seem to be restricted to immature blast populations as in the hierarchical model but a broad spectrum of different blast immunophenotypes display stem cell capabilities. Furthermore, the frequency of stemness among unsorted bulk ALL cells as well as subpopulations of different maturity according to the blast immunophenotype is high and similar. The results from this thesis provide strong evidence for the stochastic cancer stem cell model in B precursor ALL.