CLL B Cell Interaction with Bone Biopsy Generated Marrow Stromal Elements Enhances Their Apoptosis Resistance in Association with an Angiogenic Switch.

CLL B cells have significant opportunity to interact with multiple tissue sites that are critical to the leukemic cell’s ability to survive. The marrow stromal elements (MSE) in B-chronic lymphocytic leukemia (B-CLL) are very likely one of the most frequently involved in this important cell-cell interaction. We have utilized a novel bone marrow biopsy technique (Alvi S, Leuk Res, 2001) that consistently generates robust and long-lived marrow stromal elements from B-CLL patients in order to study CLL B cell apoptosis status in relation to angiogenic factors. The latter angiogenic parameters were studied as we have previously detected both a VEGF based autocrine pathway for CLL B cells and found an extensive neovascularization in CLL marrows. Bone biopsy material from B-CLL patients (N=25) yielded active cell growth in vitro which expresses the features of marrow stromal constituents. Importantly, these marrow constituents were able to be sustained for several months (range, 3 – 52 weeks) and also could be transferred repetitively to new culture dishes. CLL B cells were able to spontaneously release VEGF and TSP-1 as we have previously published (Kay N, Leukemia, 2002). MSE from CLL marrow (N=5) were found to spontaneously secrete basic fibroblast growth factor (bFGF) 5.2 ± 0.9 pg/ml (mean ± one SEM), vascular endothelial growth factor (VEGF) 162 ± 48 pg/ml and thrombospondin-1 (TSP-1) 90 ± 26 ng/ml into the culture medium. Significant reduction in CLL B cell spontaneous apoptosis,measured by Annexin/PI incorporation (mean ± one SEM), was seen after exposure to the CLL MSE (N=6) both after 24 hours (i.e., Non exposed CLL B cells vs. MSE co-cultured CLL B cells was 70% ± 9 vs. 39% ± 10 respectively for 24 hours, p < 0.01) and 72 hrs (data not shown). Exposure to MSE also enhanced the in vitro drug resistance of CLL B cells to chlorambucil (C). Thus, for CLL B cells (n=5) cultured with CLL MSE and C (1μM) for 24 hours, apoptosis levels were reduced from 58.8% ± 7.8 for B cells without MSE compared to 39.8% ± 3.8 for B cells exposed to MSE. Apoptosis resistance enhancement for CLL B cells was found to be similar with normal marrow stromal elements and a stromal cell line (HS-5). Anti-apoptotic proteins detected by immunoblot were found to increase for CLL B cells exposed to stromal elements from bone marrow biopsies and included Mcl-1, Bcl-2, Survivin and XIAP. While Mcl-1 was found to increase, there was a decrease in Bcl-2 and Survivin detected by immunoblot when CLL B cells were exposed to the HS-5 cell line implying a different mechanism for apoptosis enhancement. When CLL B cells were added to CLL MSE for 24 hours, there was an increase in VEGF released into the culture medium (283 pg ± 114 for the co-culture vs. 14 pg ± 1.7 and 162 pg ± 48.8 for CLL B cells and MSE cultured alone respectively). In contrast co-culture of CLL B cells with MSE (24 hours) resulted in less TSP-1 in the culture medium than expected (106 ng ± 28.8 for co-cultured CLL and MSE cells vs. 89.5 ng ± 27.5 and 90.9 ng ± 26.4 for CLL B cells and MSE cultured alone respectively). This change in the ratio of pro- to anti-angiogenic factors favoring VEGF strongly suggests that CLL B cell interaction with stroma can facilitate an angiogenic switch and may be linked to disease progression. In addition, we have found that bone biopsies from B-CLL patients can generate long-term marrow stromal elements that are able to reliably enhance CLL B cell apoptosis resistance.