In this study we have utilized a novel bone biopsy technique to develop bone marrow stromal cell cultures from CLL patients that permit functional in vitro assays of leukemic cell–stromal interactions. This has provided us with an opportunity to study the ability of these cell cultures to modulate spontaneous apoptosis and rescue CLL B-cells from drugs that exert their cytotoxic effects through a variety of mechanisms. The stromal cells cultures were found to be robust, long-lived, and able to sustain apoptosis resistance in primary CLL B-cells. In addition, we found that protection from drug-induced cell death by CLL derived bone marrow stromal cells or MSE was observed for both alkylating agents and drugs that bring about cell death by inducing oxidative stress.
Importantly, these experiments suggest that stromal protection is mediated, in part, by soluble factors produced by marrow stromal elements. The nature of the soluble factor(s) mediating this protection is currently not known, but preliminary work by us indicates that the molecular weight is >10 kDa. The candidates for this soluble factor are multiple and include shed receptors (CD25, CD23) or certain cytokines including IL-4, IL-2, BLyS, APRIL, bFGF and VEGF. The latter cytokines have all been shown to enhance CLL B-cell survival or induce drug resistance. Further work is ongoing to identify the factor(s) since maneuvers to reduce the activity or levels of the protective factor may augment the efficacy of some therapeutic approaches. Similar to other investigators [36
], we have not seen the same kind of protection with MSE soluble factors for CLL B-cells exposed to fludarabine where physical contact between stroma and CLL B-cells appears to be necessary for protection to occur (data not shown). Therefore, the mechanisms by which MSE protect CLL B-cells from drug induced death appears to depend to a certain extent on the drugs mechanism of action.
We have also demonstrated that interactions between CLL B-cells and primary marrow stromal elements derived from CLL bone biopsy specimens affect levels of anti-apoptotic proteins and engage in functional interactions that induce dramatic alterations in the levels of secreted pro- and anti-angiogenic cytokines. We examined the angiogenic cytokine profile for MSE and CLL B-cells when cultured together for several reasons. First, there is extensive neovascularization in both marrow and nodes of CLL patients. Second, serum/plasma levels of angiogenic cytokines have been shown to correlate with clinical outcomes in patients with CLL [16
]. Third, signaling through angiogenic receptors appears to relate to a number of biologically important events in the survival of CLL B-cells [17
]. Specifically, we have defined the presence of a VEGF-based autocrine pathway in CLL B-cells [17
]. Neovascularization and angiogenic signaling depends on the balance of pro- to anti-angiogenic cytokines, the so-called angiogenic switch. Thus, our finding that interactions between CLL B-cells and MSE lead to an increase in some pro-angiogenic cytokines (bFGF) along with a diminution of at least one anti-angiogenic cytokine (TSP-1) is notable and identifies one mechanism by which stroma may enhance CLL B-cell survival. Since previous work has also shown that bFGF can increase CLL B-cell resistance to fludarabine [35
], these findings also have implications for drug resistance. Accordingly, therapeutic strategies aimed at neutralizing angiogenic cytokines may enhance the benefits of purine nucleoside therapy [17
Finally, we believe, the use of bone biopsy derived stromal cells is an advance over the use of bone marrow aspirate cells or stromal cell lines for studying interactions between CLL B-cells and marrow stromal cells. This approach recapitulates the cellular heterogeneity of the marrow stromal and provides a long-lived culture system that can be regenerated by secondary culture or even after long-term tissue storage. Because of the ability to sustain these stromal elements, this type of stromal cell culture system allows the study of CLL B-cells isolated from the same patient over time with autologous bone marrow without requiring repeat marrow biopsies. This latter approach will help determine if marrow stromal elements have a greater or reduced ability to alter CLL B-cell survival as the clonal B-cells evolve in a given patient. While a direct comparison of normal versus CLL derived stromal cells (i.e., MSE) was beyond the scope of this study, we did not find any dramatic differences in the ability of these two sources of stroma to promote CLL B-cell survival (data not shown). However, in future studies, we intend to do additional studies to more intensively compare and contrast the features of the normal vs. leukemic derived stromal cells for their ability to modulate CLL B cell apoptosis.
In total, these studies demonstrate the utility of a bone biopsy derived stromal cell system to study the intimate cross-talk that occurs between CLL B-cells and the marrow microenvironment. We find that this interaction influences CLL B-cell resistance to both spontaneous and drug induced apoptosis. While the mechanisms for these effects are shown to be complex, it is apparent that this leukemic cell–stromal cell model will yield more insights that reflect in vivo mechanisms related to CLL disease progression. In turn these insights may ultimately provide a valuable framework for rational approaches to test and evaluate drugs that interrupt leukemic cell–stromal interactions to achieve more effective removal of CLL B-cells.