While prolonged survival of malignant cells in peripheral blood and bone marrow is a hallmark of B-CLL, B-ALL is characterized by high proliferation rates and aggressive invasion of lung, liver and lymphoid tissues by lymphoblasts. In a co- culture based experiment we addressed the following questions: (1) whether CSCs can be maintained in vitro, (2) if CSCs depend on the same specialized bone marrow niche cells like non-malignant immature hematopoietic cells do and (3)
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how their frequency changes on Ebf2-expressing and non-expressing feeder layers. Therefore we used a well-characterized human B-ALL patient sample with a known CSC frequency (originally determined by directly transplanting the leukemic cells from a mouse into another without in vitro culture) and plated these cells on confluent IEO (express Ebf2) and non-IEO (whole bone marrow stromal cells depleted for Ebf2-expressing niche cells) feeder layers isolated from Ebf2- Gfp+/- mice. These co-cultures were then run for one month.
As the CD phenotype of CSCs in B-ALL is unknown, a FACS analysis would not provide all information needed and we decided to use an in vivo readout system. Therefore the human B-ALL cells were transplanted into NOD/LtSz-scid IL2Rγnull
mice (NSG) mice (Shultz et al., 2005). In this animal model the polygenic NOD/ShiLtJ strain (Anderson and Bluestone, 2005) was combined with the severe combined immune deficiency (Prkdcscid) mutation (Blunt et al., 1995) and the targeted IL2 receptor gamma chain (Il2rgtm1Wjl) deficiency (Cao et al., 1995). As a consequence, NSG mice lack mature B and T cells and have almost no NK cells. Furthermore they have defects in innate immunity and cytokine signaling pathways. As human cells develop normally upon transplantation, this animal model is widely used in the studies of hematopoietic stem cells and human disease like HIV, EBV and various types of cancer.
Co-cultures were examined under the microscope every week and no signs of cell death or proliferation were observed. Therefore, B-ALL cells were most likely resting in G0 phase during co-culture. After four weeks the total B-ALL cell number was determined. We detected a mild reduction in total cell numbers on the IEO feeder layer compared to the numbers initially seeded while the number of B-ALL cells cultured on non-IEO stromal cells were almost unchanged. This indicates that IEO cells do not support the main B-ALL cell population as well as non-IEO cells do. However, no conclusion regarding the CSC frequency can be drawn from this result. To determine the CSC frequency, the harvested B-ALL cells were transplanted at limiting dilutions into NSG mice. Disease progression is driven by CSCs and their frequency is indirectly determined at the threshold limit, where the transplanted cell number does not cause leukemia in NSG mice.
11 days post injection, animals that had been injected the highest dose of leukemic cells were detectably engrafted in the bone marrow (fluorescent signal from the knee). Although the B-ALL cells were originally isolated from the spleen of leukemic mice, they home to the bone marrow first. This is the regular pattern of engraftment detected in FACS analysis on prepared organs from leukemic mice (personal communication, Catarina Alves, HMGU). This result suggests, that the
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B-ALL cells are very potent and survive in the spleen, but preferably home to the bone marrow where they originate from. Interestingly, in those mice injected with higher cell numbers, B-ALL cells cultured on non-IEO cells engrafted slightly better, leading to faster disease progression. This argues, that either the CSC frequency was maintained better or their number even increased on non-IEO cells compared to IEO cells. However, at lower cell numbers non-significant differences were observed.
At the lowest cell number not all mice were engrafted one-month post injection. Based on these results the CSC frequency was calculated (about one in 3x103 B- ALL cells) and was found to be unchanged compared to the initial frequency of the sample without in vitro culture. These results implicate that B-ALL cells do not specifically depend on the support of IEO niche cells but can be maintained on whole bone marrow stromal cells. This argues that they do not depend on the same niches like normal HSCs but they can be maintained in these niches.
The detection of an unaltered CSC frequency after one month in vitro is a very interesting finding and has not previously been reported in the literature. In future experiments, we want to use this experimental setup to study different types of feeder layers to elucidate the minimal needs of B-ALL cells. This will provide valuable information why B-ALL can aggressively infiltrate other organs.
6
Outlook
Within the course of this study, we applied gene targeting in murine embryonic stem cells to generate mice with a conditional allele of Ebf2. Functional testing of this mouse model proved its functionality and confirmed an important functional role for the transcription factor Ebf2 in hematopoietic stem cell homeostasis. Furthermore, our results obtained from stromal cell type-specific deletions implicate, that specifically Ebf2-expressing osteoblastic cells are required for HSC homeostasis. However, our data suggest that this support is required only at certain stages of development and at the present point in time the contribution of other cell types cannot be excluded.
Therefore, we will breed Ebf2fl/fl mice with additional stromal Cre lines and analyze these animals at the age of three to four weeks. As we speculate, based upon our DNA microarray data, that Ebf2 is required specifically for the homing and
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proliferation of hematopoietic cells, it may be worth to investigate the hematopoietic recovery in adult Ebf2 conditional knockout mice after bone marrow suppression with 5-fluorouracil (5-FU). 5-FU is a chemotherapeutic drug that kills actively cycling bone marrow cells resulting in a partial bone marrow ablation. The recovery from this challenge requires intensive proliferation of hematopoietic stem- and progenitor cells and this process may require Ebf2.
Our results obtained from the B-CLL co-cultures show, that the transcription factor Ebf2 can induce or repress genes that affect the survival of malignant B-CLL cells. To identify molecules that mediate this increased survival and reveal pathways of communication between feeder and B-CLL cells, we will analyze the gene expression profiles of EL28 mock- and EL28 Ebf2-transduced cells by DNA microarray. This data shall then be combined with the results from the DNA microarray experiment performed on IEO-Ebf2+/- and IEO-Ebf2-/- cells. In-depth bioinformatic analysis will allow for the identification of the signals provided by Ebf2-expressing stromal cells and may help to identify new Ebf2 target genes.
To extend our knowledge on the minimal needs of B-ALL cells, we plan to perform additional co-culture experiments to test more B-ALL patient samples and different feeder layers. Finally, we want to find out whether this co-culture system could be used in drug development. Therefore, we will add well-established B-ALL drugs into co-cultures and study their effect on B-ALL cell proliferation and apoptosis by FACS. Influences on the CSC frequency will be determined by transplanting B-ALL cells at limiting dilutions into immunocompromised mice.