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ActivinA enhances BCP-ALL cell migration
have high engraftment levels into the BM.24 NALM-6 cells, either pretreated or not with ActivinA, were injected (i.v.) at day 0 in NSG mice (1×106/mouse) to evaluate their engraftment in different leukemia-targeted organs (Figure 8A). Mice were subsequently monitored for weight loss over two weeks after transplantation, and leukemia bur- den was evaluated 11 and 14 days after injection by flow cytometry, determining the percentage of human CD19 and CD10 positive cells in several organs.
First, we observed a significant difference in terms of change in body weight between the two experimental groups starting from day 4 after injection (P<0.05) with an even greater difference on days 11 and 14 after injection (P<0.01), suggesting a different disease progression (Figure 8B).
Moreover, we found that, on injection, both untreated and pretreated cells disseminated through the peripheral blood to different organs, such as BM, spleen, liver, meninges, and brain. Notably, we found that ActivinA- pretreated cells were able to engraft more rapidly in the BM of recipient mice (median leukemic percentage at day +11: 17.7%, range: 6.1-42.3%) compared to their untreat- ed counterparts (median leukemic percentage at day +11: 10.3%, range: 0-21.4%, n=12). We also observed an increased leukemic percentage in the central nervous sys- tem (meninges and brain) of NSG mice following in vitro exposure to ActivinA (P<0.05) (Figure 8C), indicating that ActivinA stimulation was able to enhance the metastatic potential of leukemic cells in vivo.
Therefore, in these two mouse models, we were able to demonstrate that ActivinA stimulation makes leukemic cells more aggressive.
Discussion
There is ample evidence correlating aberrant TGF-b family growth factor activity to carcinogenesis. Despite its prominent role in solid cancer progression,7 the involve- ment of ActivinA, a member of the TGF-b family, in the pathogenesis of hematologic malignancies has never been explored. To the best of our knowledge, here we show for the first time that ActivinA is highly expressed in the BM of BCP-ALL patients at diagnosis compared to HDs. Interestingly, we demonstrated that BM-MSCs are an important source of ActivinA, the production of which is strongly up-regulated following direct contact with leukemic cells or through leukemia-released soluble fac- tors. This finding is in accordance with the recently revised “seed and soil” theory, showing that leukemic cells are able to alter the BM stroma, creating a fertile ground which fuels tumor cell survival and progression.1,25,26 Of note, we observed that MSCs isolated from the BM of BCP-ALL patients were able to produce higher levels of ActivinA compared to HD-MSCs, even after several in vitro passages. This means that they 'remember' the pro- found alterations that had occurred within the leukemic BM niche.
Nowadays, there is general agreement that inflamma- tion could play a pivotal role in the transformation, sur- vival and proliferation of leukemic cells. In particular, sev- eral studies have demonstrated that BM cells in ALL are able to create a pro-inflammatory microenvironment that impairs frequency and function of normal HSCs within the BM.18,20 In line with this evidence, we demonstrate that
the BM of BCP-ALL patients is characterized by increased levels of the pro-inflammatory cytokines IL-1b, IL-6 and TNF-α, which can synergize with BCP-ALL cells in stimu- lating ActivinA production and release by BM-MSCs. Accordingly, it has been demonstrated that ActivinA expression was increased in several inflammato- ry diseases, such as septicemia, inflammatory bowel dis- ease, and rheumatoid arthritis.27 The abundance of ActivinA within the leukemic BM niche, and its identifica- tion as a new MSC-secreted leukemia-driven molecule, prompted us to investigate its possible effects on BCP-ALL cells.
In accordance with recent literature reporting that ActivinA increases the migration and invasive properties of several solid tumors,12-14,28-33 our GEP analysis of ActivinA-treated leukemic cells showed a crucial effect on different biological processes linked to cell motility. In detail, we used time-lapse microscopy to demonstrate that this molecule was able to increase the spontaneous cell motility of both immortalized and primary BCP-ALL cells.
In addition to increased random motility, ActivinA- treated leukemic cells were more responsive to the CXCL12 chemokine, which plays an essential role in maintaining the quiescent BM HSC pool, thus regulating physiological hematopoiesis.34 The increase in BCP-ALL migration towards CXCL12 was selectively inhibited by ActivinA/Activin receptor blocking through SB431542,16 ensuring the specific contribution of ActivinA in mediat- ing this advantage. Importantly, the effect of ActivinA on cell chemotaxis was highly cell-specific. While increasing leukemic cell migration in response to even suboptimal concentrations of CXCL12, ActivinA markedly impaired the ability of healthy CD34+ cells to migrate toward a CXCL12 gradient. This opposite effect could be particular- ly relevant in the context of the altered BCP-ALL BM niche, where we observed a significantly decreased CXCL12 concentration, in agreement with recent litera- ture.19 Concerning the molecular mechanisms underlying ActivinA action, a protein-mediated regulation of the CXCL12 receptors CXCR4 and CXCR7 was ruled out. Moreover, in contrast to what Sozzani et al. described in dendritic cells,16 ActivinA did not stimulate either leukemic cell chemotaxis itself (Online Supplementary Figure S10) or their secretion of CXCL12 (data not shown). On the contrary, our GEP analysis demonstrated that this molecule mainly induces an overall positive regulation of pathways associated with cell motility, such as RAS, PI3K/AKT, and calcium homeostasis.
It has been demonstrated that Ca2+ co-ordinates struc- tural components of the cell migration machinery and sig- naling molecules crucial for proper cell motility. Through the activation of actin-interacting molecules such as pro- tein kinase C35 and calmodulin-dependent kinases,36 and the regulation of Rho GTPases, Ca2+ signaling finely tunes actin cytoskeleton dynamics.37 Interestingly, we demon- strated that ActivinA is able to increase the motility of BCP-ALL cells through an increase in the pool of free cytosolic calcium, resulting in an increased rate of F-actin polymerization. Strikingly, the increase in intracellular Ca2+ was not observed in ActivinA-stimulated CD34+ cells, thus explaining the possible molecular mechanism medi- ating the differential activity of this molecule on the migration of leukemic versus healthy hematopoietic cells. Recent literature28 describing the ability of ActivinA to
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