Leukemia cells induce changes in human bone marrow stromal cells

Journal of Translational Medicine Leukemia cells induce changes in human bone marrow stromal cells Sara Civini 0 Ping Jin 0 Jiaqiang Ren 0 Marianna Sabatino 0 Luciano Castiello 0 Jianjian Jin 0 Huan Wang 0 Yuanlong Zhao 0 Francesco Marincola 1 2 David Stroncek 0 0 Cell Processing Section, Department of Transfusion Medicine, Clinical Center, National Institutes of Health (NIH) , Building 10, Room 3C720, 9000 Rockville Pike, Bethesda, MD 20892-1184 , USA 1 Sidra Medical and Research Centre , Doha , Qatar 2 Infectious Disease and Immunogenetics Section (IDIS), Department of Transfusion Medicine, Clinical Center, National Institutes of Health (NIH) , Bethesda, MD 20892 , USA Background: Bone marrow stromal cells (BMSCs) are multipotent cells that support angiogenesis, wound healing, and immunomodulation. In the hematopoietic niche, they nurture hematopoietic cells, leukemia, tumors and metastasis. BMSCs secrete of a wide range of cytokines, growth factors and matrix proteins which contribute to the pro-tumorigenic marrow microenvironment. The inflammatory cytokines IFN- and TNF- change the BMSC secretome and we hypothesized that factors produced by tumors or leukemia would also affect the BMSC secretome and investigated the interaction of leukemia cells with BMSCs. Methods: BMSCs from healthy subjects were co-cultured with three myeloid leukemia cell lines (TF-1, TF-1 and K562) using a trans-well system. Following co-culture, the BMSCs and leukemia cells were analyzed by global gene expression analysis and culture supernatants were analyzed for protein expression. As a control, CD34+ cells were also cocultured with BMSCs. Results: Co-culture induced leukemia cell gene expression changes in stem cell pluripotency, TGF- signaling and carcinoma signaling pathways. BMSCs co-cultured with leukemia cells up-regulated a number of proinflammatory genes including IL-17 signaling-related genes and IL-8 and CCL2 levels were increased in co-culture supernatants. In contrast, purine metabolism, mTOR signaling and EIF2 signaling pathways genes were up-regulated in BMSCs co-cultured with CD34+ cells. Conclusions: BMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profiles. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable for leukemia stem cells. Bone marrow stromal cells (BMSCs); Leukemia; Tumor microenvironment; Hematopoietic niche - Background Acute myeloid leukemia (AML) is a clonal, malignant disorder. Treatment of AML is often complicated by disease propagation and relapse due to a small subset of cells called leukemia stem cells (LSC). LSC show a less mature phenotype compared with leukemia cells and they display a constitutive activation of factors such as NF-B, Akt, and Wnt/-Catenin which are involved in survival and self-renewal [1-3]. Leukemia stem cells are a heterogeneous population, which were first found among CD34+CD38- populations, but they are also present among CD34+CD38+ and CD34- cells [4]. Normal hematopoietic stem cells and LSCs reveal a high degree of similarity and although LSCs show increased expression of CD44, CD96, CD47 and the loss of CD90 expression, no unique LSC marker has yet been found [5-9]. In the hematopoietic niche, LSCs interact with bone marrow stromal cells (BMSCs) to create a microenvironment that is favorable for LSC survival [10,11]. The interactions between leukemia cells and the niche encompass membrane receptors and soluble factors. These factors include CXCR4/CXCL12 (SDF-1) signaling, which is involved in the homing, survival, and proliferation of leukemia cells in AML [12,13] and chronic myeloid leukemia (CML) [14]. It is also important to note that CD44 and VLA-4 receptors expressed by leukemia cells play a role in their adhesion to stromal cells in the niche and the consequent induction of anti-apoptotic effects that support leukemia cell survival [15,16]. BMSCs, which are also known as mesenchymal stromal cells or mesenchymal stem cells, are a multipotent population that plays an active role in the hematopoietic niche. They maintain hematopoietic stem cells (HSCs) dormant within the niche and they play a role in the release of activated HSCs [17-24]. These cells secrete a wide range of cytokines, growth factors and matrix proteins involved in the hematopoiesis and hematopoietic stem cells maintenance [25-30]. It has been shown that in chronic lymphocytic leukemia (CLL), BMSCs through cysteine-cysteine metabolism provide leukemia cells with the antioxidant species (GSH) and promote cell survival in oxidative stress conditions [31,32]. In multiple myeloma, BMSCs up-regulate the secretion of several factors (IL-6, IGF-1, VEGF, FGF, SDF-1 and TNF) as a result of their direct interaction with myeloma cells through integrins and soluble factors produced by myeloma cells. This interaction of myeloma cells and BMSCs in turn promotes a pro-tumorigenic environment in which the survival, growth and drug resistance of multiple myeloma cells is guaranteed [33-35]. To further understand the interaction between BMSCs and leukemia stem cells in the bone marrow microenvironment, we selected three myeloid leukemia cell lines with different degrees of stemness and co-cultured them with BMSCs from healthy donors. We found that BMSCs responded to leukemia cells by up-regulating many proinflammatory and IL17-signaling related genes. Methods Study design BMSCs from healthy donors were co-cultured with three different myeloid leukemia cell lines. AML cell lines TF-1 and TF-1 were selected because of their phenotype: CD34+/CD38+ and CD34+/CD38-, respectively; the TF-1 phenotype being less mature than the TF-1 phenotype. We also selected K562, a CD34- chronic myeloid leukemia cell line, as a third cell line of bone marrow origin. A 1-m Transwell system (BD Biosciences, San Jose, CA USA) was used to maintain the cultured BMSC and leukemia cell populations separate from each other. BMSCs were also co-cultured under the same conditions with CD34+ cells isolated from G-CSF-mobilized peripheral blood stem cells from healthy donors BMSCs, leukemia and CD34+ cells cultured alone (mono-cultures) were used as controls. Cells from both mono- and co-culture conditions were harvested at 4 h, 10 h, and 24 h. Supernatants were harvested at 48 h. Cells were analyzed for global gene expression profiles, culture media for selected cytokines and chemokines. These studies were approved by a NIH Institution Review Board. Bone marrow stromal cells, leukemia cell lines and hematopoietic stem cells Passage 2 BMSCs from 4 healthy donor bone marrow aspirates were provided by the Bone Marrow Stromal Cell Transplant Center, NIH, Bethesda, Maryland. BMSCs were expanded and characterized as described in our previous work [29,36]. Briefly, cells from bone marrow aspirates were seeded in complete media ( minimal essential medium (-MEM), 2 mM glutamine, 10 g/ml gentamicin a (...truncated)