HUMAN BONE-MARROW MESENCHYMAL STEM CELLS PROTECT B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS FROM APOPTOSIS IN VITRO BY UPREGULATING NOTCH-3 AND -4 ON BOTH CELL TYPES
AH Nwabo Kamdje, F Mosna, F Bifari, V Lisi, E Mimiola,
O Perbellini, G Pizzolo, M Krampera
Background. Notch signaling pathway plays a crucial role in promoting self-renewal in either normal hematopoietic and leukemic stem cells. In fact, deregulation of Notch signaling is involved in the pathogenesis of several forms of acute lymphoblastic leukemia (ALL), most importantly in the T-ALL with the translocation t(7;9)(q34;q34.39). However, little is known about the role of Notch signaling in B-lineage ALL. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are non-hematopoietic precursors that may support hematopoietic progenitors using Jagged-1/Notch-1 signaling. Aims. to evaluate the role of the Notch signaling pathway in the maintenance of B-ALL cells in coculture with BM-MSCs. Methods. MSCs were obtained from normal BM samples collected after informed consent, and expanded up to passage 3-4, displaying a homogeneous mesenchymal immunophenotype and multipotent differentiation capability. B-ALL cells were obtained by density gradient centrifugation from BM samples of 5 newly-diagnosed patients with high blast count (mean purity: 91.2%, range: 82-97), and were cocultured with BM-MSCs at 10/1 ratio for 3, 7 and 28 days. Apoptosis of B-ALL cells was evaluated by Annexin-V/7-AAD staining, while proliferation was assessed with the Carboxyfluorescein-diacetate Succinimyl-ester (CFSE) method and flow cytometry. Expression of Jagged-1 and Notch-1, -2, -3 and -4 was assessed on both cell types with flow cytometry. Results. At each time point the number of surviving B-ALL cells was increased by the coculture with BM-MSCs (day 3: 56.5±1.6% vs 73.5±0.2%, P<0.001; day 7: 15.8±2.7% vs 58.0±1.4%, P<0.001; day 28: 4.6±1.5% vs 30.6±2.1%, P<0.001). This finding was due to a dramatic reduction in apoptosis rather than to the proliferation of B-ALL cells, which was negligible. At the same time, the expression of Notch-1, Notch-3, Notch-4 and Jagged-1 by B-ALL was markedly upregulated by the coculture with BM-MSCs (Notch-1: 1.7±0.6% vs 21.0±1.3%, P<0.001; Notch-3: 5.3±0.7% vs 16.3±0.7%, P<0.001; Notch-4: 1.8±0.5% vs 3.8±0.8%, P<0.001; Jagged-1: 8.4±0.7% vs 37.4±1.5%, P<0.001; all at day +3). On the other hand, Notch-3 and -4 were upregulated on BM-MSCs (Notch-3: 8.8±1.1% vs 54.8±1.1%, P<0.001 at day +3; 7.9±0.7% vs 88.5±1.7%, P<0.001 at day +7; Notch-4: 1.0±0.2% vs 5.8±0.5%, P<0.001 at day +3; 2.2±0.3% vs 48.5±1.4%, P<0.001 at day +7). Notch-2, expressed at low levels at basal conditions on both B-ALL and MSCs, became undetectable from day +3 onwards. We then blocked all the Notch signaling with the γ-secretase inhibitor XII: consequently, B-ALL cells were all dead at day +3 when cultured alone, but still 42.5±0.5% of the total population was alive by coculturing B-ALL with BM-MSCs. We next blocked single Notch receptors through specific neutralizing antibodies: 61.2±0.4%, 51.4±0.5% and 30.8±0.8% of B-ALL cells were still alive using anti-Notch-1, -3 and -4 antibodies, respectively (Figure 1). Conclusions. considered together, these data show how BM-MSCs contribute to the survival of B-ALL blasts by activating Notch signaling. Notch-3 and -4 appear mainly responsible for these phenomena and might become future additional targets in the treatment of B-ALL.
Figure 1. Viable B-ALL cells at day +3 of culture.