Motility, Proliferation and Egress of Human AML Cells in Transplanted NOD/SCID Mice Are Regulated by Membrane Bound and Secreted Elastase.

Neutrophil -or leukocyte- elastase is a serine protease stored in azurophilic granules of myeloid cells and is released upon activation and degranulation. Elastase degrades a number of extracellular matrix proteins as well as various cytokines and their receptors and may facilitate neutrophil motility. Lately, others and we have shown that elastase is also involved in G-CSF induced mobilization of stem cells. In the present study we examined the role of elastase in motility and proliferation of leukemic AML cells. We demonstrate a correlation between abnormal high levels of secreted elastase in the peripheral blood plasma of AML patients and the number of leukemic blast cells in the circulation. Interestingly, we found that AML cells not only secrete elastase, but also express constitutively membrane-bound elastase on their cell surface. Confocal microscopy revealed uniform localization of elastase on the cell surface. Of note, incubation of some AML cells with the chemokine SDF-1 increased elastase cell surface expression whereas treatment with neutralizing anti CXCR4 Abs decreased it. Pretreatment of primary human AML cells and cell lines with various elastase inhibitors and neutralizing anti elastase Abs led to reduced spontaneous and SDF-1-induced transwell migration of the leukemic cells. In vivo homing of AML mononuclear cells and CD34-enriched progenitors cells to the BM and spleen of transplanted NOD/SCID/B2mnull mice was significantly impaired by elastase inhibition. Moreover, when leukemic AML cells were pretreated with elastase inhibitors, the formation of spontaneous and SDF-1 induced protrusions was prevented suggesting that elastase participates in leukemic cell motility through direct regulation of cytoskeletal rearrangements and cell polarization. In addition, we found that the proliferation rate of AML cells was likewise elastase-dependent. In contrast, treatment of enriched human cord blood CD34+ progeniotrs with elastase inhibitors enhanced their proliferation rate, the percentage of undifferentiated CD34+/38− cells and their in vivo homing to the BM and spleen of immune deficient mice. Finally, we examined whether elastase may play a role in the abnormal AML cell egress from the BM to the circulation. Primary AML cells from different patients with different FAB subtypes were injected into sublethally irradiated NOD/SCID mice to establish human AML chimeras and 3–4 weeks later elastase inhibitor (1mg) was administrated daily for 4 consecutive days. Egress of AML cells to the blood circulation including blast cells was significantly decreased as compared to untreated chimeric mice. Taken together, these results demonstrate that elastase participates in the regulation of human AML progenitor cell proliferation and cell emigration from the BM to the circulation and that elastase inhibition can efficiently prevent AML cell egress in NOD/SCID chimeric mice.