Causes of refractory acute myeloid leukemia (AML) are unknown, but may be related to the bone marrow (BM) vascular network given the close relationship between hematopoiesis and the vasculature. We hypothesized that endothelial cells (ECs) provide a protective advantage to AML cells. To test this hypothesis, we first cultured human AML cells with and without primary bone marrow endothelial cells (BMECs). Co-cultures of AML cells and BMECs were then exposed to increasing doses of cytarabine chemotherapy. There was a 2-fold decrease in leukemia cell death of AML cells when adhered to BMECs compared to non-adhered (30% vs. 60%, P < 0.0001). Even irradiated BMECs protected AML cells from cytarabine chemotherapy. To identify adhesion molecules mediating this protective effect, we analyzed cell membranes and supernatants of the cytarabine-treated co-cultures using protein microarrays. After cytarabine exposure, the Lutheran blood group glycoprotein basal cell adhesion molecule (BCAM) was upregulated in both human AML cells and BMECs. Prior work has shown BCAM as a receptor for Laminin and VLA5. As AML cells are known to express VLA5, we hypothesized that blocking BCAM may represent a novel therapeutic strategy. Blocking BCAM with neutralizing antibodies resulted in a 75% increase in non-adherent AML cells. Together, these in vitro results support the concept that ECs may be a protective reservoir for AML cells, at a minimum by means of adhesion molecules. BCAM represents a viable target. Because of the complex nature of the leukemia microenvironment, we sought to test this concept in vivo. Prior intravital efforts have focused on calvaria bone, which may over-represent the endosteal niche and under-represent the vascular niche due to the very close approximation of bone surfaces. Therefore, we created an intravital animal model of human AML to track single AML cells in the bone marrow of mouse long bones. In brief, we irradiated NOD/scid/IL2Rγnull (NSG) mice, drilled a window on the tibia surface, xenotransplanted fluorescently tagged human AML cells via IV injection, and then analyzed the tibias by fluorescent microscopy for the presence of AML cells at various time points after transplant. Initially the AML cells homed to endosteal surfaces of the bone marrow as early as one day after transplant. Over time, the AML cells lining the endosteum remained as single cells or very small clusters of a few cells. However, in the central marrow region the AML cells proliferated into massive clusters around blood vessels. Ongoing experiments are being performed to determine the disruption and resurgence of AML cells after chemotherapy and blockade of adhesion molecules. In sum, ECs protect human AML cells from chemotherapy by direct contact and the BCAM/Laminin/VLA5 axis may be a therapeutic target. Using our unique intravital imaging model of bone marrow in long bones, the endosteal niche appears to be the first site of homing and engraftment while the vascular niche appears to be the site for leukemia proliferation/progression.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.