Microenvironmental Changes In An In Vivo Model of Myeloid Leukemia Negatively Regulate Osteoblastic Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1219-1219
Author(s):  
Benjamin J. Frisch ◽  
John M. Ashton ◽  
Craig T. Jordan ◽  
Laura M. Calvi
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
Osteoblastic Cells ◽  
Long Bones ◽  
Endosteal Surface ◽  
Osteoblastic Activity ◽  
Severe Reduction

Abstract Abstract 1219 Patients with myelogenous leukemias can present with symptoms of bone pain and pathologic fractures, however little is known about the interactions between malignant cells and the bone marrow microenvironment. Additionally leukemia is known to severely interfere with normal hematopoiesis. To further characterize interactions between leukemic cells and the microenvironment, we used a model of blast crisis CML (bcCML) in which immature murine hematopoietic cells are engineered to express the BCR/ABL and Nup98/HoxA9 translocation products. Injection of these cells into naïve mice results in rapid accumulation of leukemic cells in the bone marrow (Dash et al. PNAS, 2002). We investigated the effect of leukemia on the bone marrow microenvironment by first performing immunohistochemical analyses. Leukemic cells were observed to preferentially localize in close contact with bony trabeculae. In addition, leukemic mice also exhibited a dramatic loss of trabecular bone as measured by micro-CT scanning (22.8 ± 1.5% vs 13.6 ± 1.5%, BV/TV n=5 in each group p=0.0048), prompting us to examine bone resorption and the abundance of osteoclasts. Histologic sections from leukemic mice showed an increase in mature osteoclasts (TRAP+, multinucleated cells) at the endosteal surface of the long bones (51 ± 4 OC/section vs 64 ± 3 OC/section p=0.0229). Additionally leukemic mice had a 50% increase in serum C-telopeptide (CTX), a well-established marker of global bone resorption (15.5 ± 0.3 ng/ml vs 21.8 ± 1.0 ng/ml, p=0.0003). Therefore, the presence of leukemic cells appears to strongly stimulate osteoclastogenesis and bone resorption. In addition to increased osteoclasts, a rapid and severe reduction in bone formation was identified in leukemic mice by decreased serum osteocalcin, a well-established marker of bone formation (70.8 ± 6.9 ng/ml vs 39.9 ± 3.2 ng/ml, p=0.0036). To determine effects of leukemia on bone-forming cells, we analyzed osteoblastic cells from the long bones of leukemic animals. Marrow was flushed from the bone and minced bone fragments were digested in collagenase. Cells isolated in this fashion have strong osteoblastic activity and can support hematopoietic stem cells (HSCs) (Chitteti et al, Blood 2010). Leukemic cells were present in the isolated fraction confirming that leukemic cells were closely associated with the bone and not entirely removed when the marrow was flushed. Total cells isolated by collagenase digestion from the long bones of leukemic mice were cultured with leukemic cells and evaluated for osteoblastic colony-forming ability. These cultures demonstrated a reduced ability to form osteoblastic colonies compared to controls (26 ± 2 colonies vs 13 ± 2 colonies p=0.0014). Freshly isolated cells were CD45 depleted to remove leukemic cells and again evaluated for colony-forming ability. CD45 negative cells from leukemic mice also demonstrated reduced ability to form mineralizing osteoblastic colonies in vitro compared to controls when an identical number of cells were cultured (10 ± 2 colonies vs 0 colonies) suggesting that their previous exposure to leukemic cells in vivo was sufficient to decrease their osteoblastic activity. Further in vivo osteoblastic evaluation in leukemic mice showed reduced immunohistochemical staining for the osteoblastic marker osteopontin at the endosteal surface, supporting a leukemic-induced reduction of the mature osteoblastic population. These data demonstrate a severe reduction in both number and function of osteoblastic cells in a leukemic environment. Together these data show severe effects of leukemia on both osteoblastic and osteoclastic cells, which could contribute to the bone-specific problems associated with leukemic disease. Moreover, we propose that osteoblastic defects observed in this model may contribute to the leukemia-induced inhibitory effects on normal hematopoiesis. Studies are ongoing to assess the microenvironmental support for normal hematopoiesis in the leukemic setting and to identify the leukemic signals that modify the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Human Extramedullary Bone and Bone Marrow in Mice: First In Vivo Model of a Genetically Controlled Hematopoietic Environment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1323-1323
Author(s):  
Ye Chen ◽  
Rodrigo O Jacamo ◽  
Nicole A. Hofmann ◽  
Yue-xi Shi ◽  
Rui-yu Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Microenvironment ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Leukemic Cells ◽  
Artificial Bones ◽  
Hematoxylin And Eosin ◽  
Bone Structures

Abstract Abstract 1323 The importance of the tumor microenvironment for cancer development, progression and resistance to treatment has recently been recognized. Our group was first to report the contribution of bone marrow (BM) derived mesenchymal stromal cells (MSCs) for tumor development and metastasis. BM is also the dynamic microenvironment (niche) for normal and malignant hematopoietic stem cells (HSC) with high local concentrations of growth factors, chemokines and cytokines. The maintenance of HSCs quiescence and normal hematopoiesis require complex bidirectional interactions between the BM niches and HSCs. Accumulating evidence has shown that the BM microenvironment also plays a pivotal role in the pathophysiology and propagation of leukemia. Leukemia cells undergo spontaneous apoptosis once they are removed from the in vivo microenvironment and placed in suspension cultures without supportive stroma. The understanding of the interactions between leukemic cells and their BM niche is also critically important for leukemia therapy. We here describe a novel artificial bone and bone marrow model mimicking the human hematopoietic microenvironment by using human BM derived MSCs and endothelial colony-forming cells (ECFCs). MSCs and ECFCs were isolated from heparinized human bone marrow or peripheral blood through an initial adhesion step, grown in specific media and then subcutaneously injected into the flanks of the NOD/SCID/IL-2r-gammanull mice, where they developed into bone-like tissues with high osteoblast activity after 10 weeks (Figure 1). Histochemical stains confirmed the bone structures and also showed that these artificial bones contained typical bone marrow cavities constituting a robust hematopoietic environment. In vivo imaging with Osteosense confirmed the presence of hydroxylapatite, and luciferase imaging of firefly luciferase labeled human leukemic cells demonstrated the engraftment of MOLM13/Luc/GFP leukemic cells in the extramedullary BM sites. The extramedullary BM was markedly hypoxic, as shown by Pimonidazole staining, another critical feature of the BM microenvironment. Factors critical for MSC to support the normal and leukemic hematopoiesis are largely unknown and cannot be studied since human MSC do not engraft reliably in xenograft models. We therefore investigated the possibility of genetically modifying MSC in this system and found a significant reduction (50 ± 6%, p<0.001) in MOLM13 cell engraftment in extramedullary BM generated with HIF1-alpha knockdown MSCs (1449 ± 194 cells/mm2), compared to vector controls (3037 ± 496 cells/mm2). This finding indicates that the HIF1-alpha expression in stromal cells is a critical component for the engraftment of leukemic cells in the physiologically hypoxic BM microenvironment. These results, for the first time, establish an in vivo bone and bone marrow model with a genetically controlled human microenvironment.Figure 1Establishment a human bone marrow microenvironment in NOD/SCID/IL-2r-gammanullmice. Representative hematoxylin and eosin (H&E) staining (shown at low magnification) shows an overview of the extramedullary bones with the typical bone structures. Scale bar: 1 mm.Figure 1. Establishment a human bone marrow microenvironment in NOD/SCID/IL-2r-gammanull mice. Representative hematoxylin and eosin (H&E) staining (shown at low magnification) shows an overview of the extramedullary bones with the typical bone structures. Scale bar: 1 mm. Disclosures: No relevant conflicts of interest to declare.

Hijacking the Niche: The Role of Stromal Osteopontin in the Induction of Leukemic Dormancy within the Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 754-754
Author(s):  
Benjamin Boyerinas ◽  
Ali Ekrem Yesilkanal ◽  
Andrea Pontier ◽  
Dorothy A. Sipkins
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
In Vivo Imaging ◽  
Tumor Burden ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Bone Marrow Biopsies ◽  
Stromal Microenvironment

Abstract Abstract 754 Introduction: Acute lymphoblastic leukemia (ALL) is a treatable malignancy where initial induction chemotherapy achieves clinical remission in the majority of patients. Relapsed disease, however, occurs in many patients and is significantly more difficult to treat. The majority of relapsed cases are a result of minimal residual disease (MRD) that persists within the bone marrow (BM) after initial chemotherapy. Our evolving knowledge of the importance of the host microenvironment in tumor progression suggests that the stromal microenvironment can protect leukemic cells from chemotherapeutic assault, and that inhibiting the supportive relationship between leukemic blasts and the bone marrow microenvironment (BMM) will provide novel therapeutic opportunities. We therefore aimed to identify and characterize novel stromal signaling mechanisms that retain and support blasts within the malignant BMM. Our preliminary data suggest that osteopontin (OPN), normally secreted by osteoblasts within the marrow, is one such signaling chemokine that is highly upregulated in the leukemic niche. OPN has well-defined roles in both solid tumor metastasis and normal hematopoietic stem cell function within the BMM. Specifically, OPN expression at the endosteal bone surface functions to recruit hematopoietic progenitors to bone where they are induced to become quiescent and maintain long term repopulating potential. We hypothesized that a similar relationship exists between leukemia and OPN resulting in a quiescent population of chemoresistant leukemic blasts at the BM endosteum. Here, we demonstrate that stromal OPN negatively regulates leukemia cell proliferation in the BMM. Methods: A GFP expressing clone of the pre-B ALL cell line Nalm-6 (10 × 106 cells) was engrafted into SCID hosts (6-8 weeks old) via tail vein injection. In vivo imaging was accomplished in live anesthetized mice by reflecting the scalp and imaging the calvarial marrow compartment using real-time multi-photon confocal microscopy. OPN expression in the malignant marrow was imaged by injecting engrafted mice with fluorescently conjugated anti-OPN antibodies 18hrs prior to imaging. For OPN neutralizing experiments, engrafted mice were injected with a cocktail of anti-mouse and anti-human OPN antibodies at a dose of 3 mg/kg. Results: Using PCR, Western blot and ELISA assays, we show that the ALL cell line Nalm-6 expresses OPN and secretes large quantities of OPN into conditioned media. Flow cytometric analysis demonstrates that Nalm-6 also express the cell surface OPN receptors VLA-4 and VLA-5. Furthermore, Nalm-6 cells specifically adhere to OPN in vitro via specific engagement of these integrin receptors. In vivo imaging demonstrates that OPN expression in the BM increases as leukemia progresses and that OPN is highly expressed adjacent to areas of high tumor burden. Specifically, a significant amount of OPN is detected in bony tunnels surrounding the vasculature at the invading tumor front. Using Q-PCR and western analysis, we demonstrate that both host-derived and leukemia-derived OPN are upregulated in malignant BM. In vivo inhibition of the OPN signaling axis in the Nalm-6 xenograft model using neutralizing antibodies directed at both human and murine OPN increased overall tumor burden two-fold as measured by flow cytometry and in vivo imaging (p=0.02, N=7) while simultaneously increasing the Ki-67 positive proliferative tumor population (p=0.029, N=4). Furthermore, IHC analysis of a panel of diagnostic bone marrow biopsies from a diverse cohort of ALL patients demonstrated high OPN expression in the marrow of these patients. Conclusion: Leukemic blasts that have hijacked normal stromal interactions to become quiescent may represent a major source of MRD and patient relapse in ALL. Our data demonstrate that the interaction of leukemic blasts with OPN in the stromal microenvironment reduces the number of cycling blasts and constrains tumor proliferation within the marrow. Current investigations are aimed at combining OPN neutralization with an in vivo model of MRD to determine whether OPN neutralization induces cycling of quiescent blasts, ultimately rendering them sensitive to chemotherapy. The ultimate goal of this work is the development of clinically relevant therapies designed to render leukemic cells more susceptible to chemotherapy by disengaging them from protective interactions with the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

Massive Mobilization of AML Cells into Circulation by Disruption of Leukemia/Stroma Cell Interactions Using CXCR4 Antagonist AMD3100: First Evidence in Patients and Potential for Abolishing Bone Marrow Microenvironment-Mediated Resistance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 568-568 ◽  
Author(s):  
Michael Andreeff ◽  
Sergej Konoplev ◽  
Rui-Yu Wang ◽  
Zhihong Zeng ◽  
Teresa McQueen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemia Cell ◽  
Surface Expression ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
Fish Analysis ◽  
Cxcr4 Antagonist ◽  
Induced Apoptosis

Abstract The chemokine receptor CXCR4 is critically involved in migration of hematopoietic cells to the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. CXCR4 is regulated in part by mutant FLT3 signaling, but in a series of 122 AML samples with diploid karyotype and lack of FLT3 mutation (ITD), high CXCR4 expression negatively correlated with DFS and OS (p=0.03 and p=0.04, respectively), after multivariate analysis (Konoplev, ASH 2006). We hypothesized that inhibition of SDF-1α-/CXCR4 interactions would result in mobilization of leukemic blasts from the bone marrow into circulation. The in vivo effect of the CXCR4 antagonist AMD3100 was studied in three patients with AML, who had insufficient mobilization of CD34+ cells for autologous stem cell transplantation with G-CSF and/or cytoxan. The combination of G-CSF (10 μg/kg QD) and AMD3100 (240 μg/kg QD SC starting on d4 and repeated for 3–4 days) resulted in massive mobilization of leukemic cells into the circulation in a time-dependent fashion, as determined by flow cytometry and interphase FISH analysis of their respective cytogenetic abnormalities. Patient # Cytogenetics % (+) cells % (+) cells Apheresis FCM Day 2 Day 4/5 CD34x106/kg 1 Trisomy 21 22.6 57.0 FCM CD7/33 22.0 2 Trisomy 9 28.6 68.6 Inv 16 29.0 75.8 4.8 FCM CD13/33 74.0 3 Mono 17 40.4 53.4 5q31 37.5 49.6 8.7 FCM CD13/33 50.0 We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva et al, Leukemia2002:1713). We then tested the hypothesis that CXCR4 inhibition would result in increased sensitivity to chemotherapy, using AMD3465, the second generation small-molecule CXCR4 inhibitor with greater potency than AMD3100. Results demonstrate inhibition of surface expression of CXCR4 and of SDF-1α-, and stroma(MS-5)-induced migration of AML cells. In vitro co-culture systems with stromal cells significantly protected leukemic cells (p < 0.01), while AMD3465 decreased stroma-mediated protection from AraC and Busulfan apoptosis and downregulated AKT signaling in AML cells. In a murine model of luciferase labeled Baf-FLT3ITD leukemias, AMD3465 induced massive dissemination of leukemia, which was abrogated by treatment with Sorafenib, a potent FLT3ITD inhibitor (Zhang, ASH 2006). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis. Disruption of these interactions by CXCR4 inhibition results in leukemia dissemination and chemosensitization. Our results in leukemia patients provide first in man proof-of principle for a novel strategy of targeting the leukemia cell/bone marrow microenvironment interactions. A clinical trial testing this concept in patients with AML is under development.

scholarly journals Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML

Blood ◽  
2009 ◽  
Vol 113 (24) ◽  
pp. 6215-6224 ◽  
Author(s):  
Zhihong Zeng ◽  
Yue Xi Shi ◽  
Ismael J. Samudio ◽  
Rui-Yu Wang ◽  
Xiaoyang Ling ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Fetal Liver ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
In Vivo Studies ◽  
Protective Effects ◽  
Cxcr4 Signaling ◽  
Induced Apoptosis

Abstract SDF-1α/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow–derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1α (SDF-1α)–induced and stroma-induced chemotaxis and inhibited SDF-1α–induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor– and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.

scholarly journals Restoration of the Bone Marrow Microenvironment Improves Hematopoietic Function in a Murine Model of Myelodysplastic Syndrome

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 358-358
Author(s):  
Allison J. Li ◽  
Sophia R. Balderman ◽  
Benjamin J. Frisch ◽  
Mark W. LaMere ◽  
Michael W. Becker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Murine Model ◽  
Hematopoietic Cells ◽  
Fold Increase ◽  
Regulatory Elements ◽  
Bone Marrow Microenvironment ◽  
Osteoblastic Cells ◽  
In Vivo Model

Abstract While data suggest that, in the myelodysplastic syndromes (MDS), the bone marrow microenvironment (BMME) is abnormal, the lack of examination of the BMME in a robust in vivo model has limited progress in the understanding of reciprocal MDS-BMME interactions. If microenvironmental defects contribute to disease progression, targeting the BM niche may offer an alternative approach for therapeutic benefit. We sought to define the MDS BMME in a well-established transgenic murine model that recapitulates hallmark features of human MDS. In this model, hematopoietic tissue-specific expression of the NUP98-HOXD13 (NHD13) fusion gene is driven by Vav regulatory elements, resulting in peripheral cytopenias by 16 weeks of age and mortality from transformation to leukemia at a median time of 11 months of age. Mice were analyzed at 15-36 weeks of age, when the MDS phenotype is prominent in the absence of leukemia. Flow cytometric quantification of BM stroma in 23-week old NHD13 mice showed a 6.5-fold increase in frequency of CD51+/Sca1- osteoblastic cells (OBC) compared to WT (p<0.05). CD51+/Sca1+ multipotent stromal cells (MSC) and CD31+/Sca1+ endothelial cells were also significantly increased in NHD13 compared to WT mice. This was not due to loss of hematopoietic cells in the marrow of NHD13 mice. While an expansion of functional MSCs and osteoblastic cells could result in skeletal changes, micro CT imaging of the femora and tibiae of 20-week old NHD13 mice revealed no differences in skeletal parameters compared to WT mice. These data suggest that the expanded osteolineage cells in NHD13 mice are not functional bone-forming cells. While stromal populations were not altered in bone-associated cells of 23-week old NHD13 compared to WT mice, 36-week old NHD13 mice also showed increased bone-associated OBCs, MSCs, and endothelial cells. Therefore, there are significant time-dependent shifts in critical stromal populations in this in vivo model of MDS, which may contribute to an abnormal BMME. To determine if the MDS BMME contributes to hematopoietic failure, NHD13 BM (CD45.2) was transplanted with WT competitor BM (CD45.1) in a 1:1 ratio into lethally irradiated NHD13 or WT (CD45.2) recipients, thus exposing the same MDS hematopoietic cells to either MDS or WT microenvironments. Using this transplantation paradigm, we previously reported improvement of hematopoiesis when NHD13 BM is exposed to a WT BMME. Surprisingly, CD45.1+ WT competitor-derived cells exhibited myeloid skewing when transplanted into NHD13 recipients compared to WT recipients, suggesting that interaction of WT BM with an MDS BMME can induce myeloid skewing, a feature of the NHD13 model. NHD13 BM was next transplanted non-competitively into lethally irradiated NHD13 and WT mice. At 10 weeks post-transplant, WT recipients had a 2.5-fold increase in peripheral leukocytes (p<0.05), significant improvement of anemia, and significant mitigation of BM long term-HSC loss compared to NHD13 recipients, suggesting that WT BMME components can rescue hematopoietic function in MDS. Together, our studies strongly suggest that a murine model recapitulates MDS microenvironmental abnormalities, and that exposure of MDS hematopoietic cells to a non-malignant microenvironment is sufficient to improve hematopoietic function. Thus, improvement of the BM microenvironment represents a novel therapeutic strategy to ameliorate hematopoietic function in MDS. Disclosures Becker: Millenium: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties.

Disruption of Leukemia/Stroma Cell Interactions by CXCR4 Antagonist CTCE-9908 Enhances Chemotherapy-Induced Apoptosis in AML

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2415-2415
Author(s):  
Hongbo Lu ◽  
Zhihong Zeng ◽  
Yuexi Shi ◽  
Sergej Konoplev ◽  
Donald Wong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Leukemia Cell ◽  
Bone Marrow Microenvironment ◽  
Cell Interactions ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Dose Dependent

Abstract The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells towards the stromal derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stroma/leukemia interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis (Konopleva, Leukemia 2002). Using a peptide analog of SDF-1α designated CTCE-9908, we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. Our results showed that CTCE-9908 significantly inhibits SDF-1α-induced migration of U937 (43% inhibition) and OCI-AML3 cells (40% inhibition) in a dose-dependent manner. In three of the four primary AML samples which expressed CXCR4 on cell surface and migrated in response to SDF-1α, 50 μg/ml CTCE-9908 reduced SDF-1α-induced migration of leukemic blasts (60%, 19% and 50% inhibition respectively). In in vitro co-culture systems, stromal cells significantly protected OCI-AML3 cells from chemotherapy induced apoptosis [no MS-5, 75.2±5.2% annexinV(+); with MS-5, 59±1.1% annexinV(+)]. Western blot analysis revealed that CTCE-9908 inhibits Akt and Erk phosphorylation in a dose-dependent manner in the OCI-AML3 cell line stimulated by SDF-1α. Blockade of CXCR4 expression with CTCE-9908 markedly abrogated the protective effects of stromal cells on OCI-AML3 [Ara-C, 59±1.1% annexinV(+); Ara-C + CTCE-9908, 76.9±1.35 annexinV(+)]. Most importantly, it decreased stroma-mediated protection from AraC-induced apoptosis in four out of five primary AML samples with surface expression of functional CXCR4 (mean increase, 25.1±9.3% compared to chemotherapy alone). In vivo, subcutaneous administration of 1.25mg CTCE-9908 induced mobilization of leukemic cells from primary AML patient transplanted into NOD/Scid-IL2Rγ-KO mice (from 15% to 27% circulating leukemic cells 1 hour post CTCE-9908 injection). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis via retention of leukemic cells in the bone marrow microenvironment niches. Disruption of these interactions by the potent CXCR4 inhibitor CTCE-9908 represents a novel strategy for targeting leukemia cell/bone marrow microenvironment interaction. Based on these observations, in vivo experiments are ongoing to characterize the efficacy of chemotherapy combined with CTCE-9908.

scholarly journals Dendritic cells differentiate into osteoclasts in bone marrow microenvironment in vivo

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 264-265 ◽  
Author(s):  
Mawadda Alnaeeli ◽  
Yen-Tung A. Teng
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Bone Marrow Microenvironment

scholarly journals Polymeric siRNA Delivery Targeting Integrin-β1 Could Reduce Interactions of Leukemic Cells with Bone Marrow Microenvironment

2021 ◽  
pp. 100021
Author(s):  
Daniel Nisakar Meenakshi Sundaram ◽  
Cezary Kucharski ◽  
Remant Bahadur KC ◽  
Ibrahim Oğuzhan Tarman ◽  
Hasan Uludağ
Keyword(s):  
Bone Marrow ◽  
Sirna Delivery ◽  
Bone Marrow Microenvironment ◽  
Leukemic Cells ◽  
Integrin Β1

scholarly journals Pivotal Role of OCL-Derived IGF1 in Drug Resistance and Bone Destruction in MM

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4336-4336
Author(s):  
Jumpei Teramachi ◽  
Kazuaki Miyagawa ◽  
Delgado-Calle Jesus ◽  
Jolene Windle ◽  
Noriyoshi Kurihara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Bone Resorption ◽  
Bone Marrow Cells ◽  
Critical Role ◽  
Bone Destruction ◽  
Bone Marrow Microenvironment ◽  
Rank Ligand ◽  
Marrow Cells

Multiple myeloma (MM) is largely incurable, and is characterized by devastating bone destruction caused by increased osteoclast (OCL) differentiation and bone resorption in more than 85% of MM patients. OCLs in MM not only promote bone resorption but also increase MM cell growth and drug resistance. Despite recent advances in anti-myeloma treatment, development of anti-MM drug resistance is a major limitation of MM therapy. Therefore, new treatment modalities are urgently needed to overcome drug resistance and decrease bone resorption. IGF1 is a crucial factor for tumor cell growth and survival of malignant cells, especially in MM. IGFI also contributes to development of drug resistance of MM cells to anti-MM agents, including proteasome inhibitors and immunomodulatory agents, but how OCLs contribute to drug resistance is still not clearly delineated. We found that IGF1 was highly expressed in OCLs attached to bone and bone marrow myeloid cells in vivo, and the expression levels of IGF1 in OCLs from MM bearing mice is higher than in normal OCLs. Intriguingly, OCLs produced more IGF1 (0.8 ng/ml/protein) than MM cells (not detected) and bone marrow stromal cells (BMSCs) (0.4 ng/ml/protein) in vitro. In addition, IGF1 protein expression in OCLs was upregulated (1.8 fold) by treatment with conditioned media (CM) from 5TGM1 murine MM cells, TNF-α or IL-6, major paracrine factors that are increased in the bone marrow microenvironment in MM. These results suggest that OCLs are a major source of local IGF1 in the MM bone marrow microenvironment. To further characterize the role of OCL-derived IGF1, we generated a novel mouse with targeted deletion of Igf1 in OCLs (IGF1-/--OCL), and assessed the role of OCL-derived IGF1 in drug resistance of MM cells and bone destruction. Treatment of 5TGM1 cells with bortezomib (BTZ) (3 nM, 48 hours) decreased the viability of 5TGM1 cells by 50%. Importantly, the cytotoxic effects of BTZ on MM cells were decreased (by 5%) when MM cells were cocultured with OCLs from wild type (WT) mice. In contrast, coculture of MM cells with IGF1-/--OCLs or WT-OCLs treated with IGF1 neutralizing antibody (IGF1-ab) did not block BTZ's effects on MM cell death. Consistent with these results, coculture of MM cells with IGF1-/--OCLs or WT-OCLs treated with IGF1-ab resulted in BTZ-induced caspase-dependent apoptosis in MM cells. We next examined the effects of OCLs on the signaling pathways responsible for MM cell survival. WT-OCL-CM promptly induced the phosphorylation of Akt and activation of p38, ERK and NF-κB in MM cells. However, these pathways were not activated by MM cells treated with IGF1-/--OCL-CM or IGF1-ab-treated WT-OCL-CM. Since adhesion of MM cells to BMSCs via interaction of VLA-4 and VCAM-1 plays a critical role in cell adhesion-mediated drug resistance (CAMDR) in MM, we tested if treatment of human BMSCs with human OCL-CM upregulated VCAM-1 expression. We found that OCL-CM upregulated VCAM-1 expression on BMSCs (x fold). In contrast, treatment of BMSCs with OCLs treated with IGF1-ab blocked VCAM-1 induction. These data suggest that OCL-derived IGF1 can contribute to MM cell drug resistance in the bone marrow microenvironment. We then examined the role of IGF1 inhibition on osteoclastogenesis and the bone resorption capacity of OCLs. RANK ligand induced the expression of cathepsin K and NFATc1 in CD11b+ bone marrow cells from WT mice, differentiation markers of OCLs, and the formation of TRAP-positive multinucleated OCLs. However, OCLs formed by RANK ligand treatment of CD11b+ bone marrow cells from IGF1-/- mice had markedly decreased cathepsin K and NFATc1 expression and OCL formation. Next, we tested the bone resorption capacity of OCLs formed by CD11b+ bone marrow cells from IGF1-/- mice vs. WT mice. Similar numbers of OCLs were cultured with RANK ligand on bone slices for 72 hours. The bone resorption activity of Igf1-/--OCLs was significantly decreased (70%) compared with WT-OCLs. These results suggest that OCL-derived IGF1 plays a critical role in MM drug resistance and bone destruction, and that inhibition of the effect of IGF1 in OCLs should decrease MM drug resistance and bone destruction. Disclosures Roodman: Amgen trial of Denosumab versus Zoledronate: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Bone marrow from children in relapse with pre-B acute lymphoblastic leukemia proliferates and disseminates rapidly in scid mice

Blood ◽  
1991 ◽  
Vol 78 (11) ◽  
pp. 2973-2981 ◽  
Author(s):  
S Kamel-Reid ◽  
M Letarte ◽  
M Doedens ◽  
A Greaves ◽  
B Murdoch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Poor Prognosis ◽  
Lymphoblastic Leukemia ◽  
Growth Characteristics ◽  
Scid Mice ◽  
Leukemic Cells ◽  
Murine Bone Marrow ◽  
In Vivo Growth

Bone marrow samples from patients with pre-B acute lymphoblastic leukemia (pre-B ALL), either at diagnosis or at relapse, were transplanted into scid mice to determine whether these freshly obtained leukemic cells could proliferate in vivo and whether there were any differences in their in vivo growth characteristics. Cells from three patients who relapsed within 13 months of diagnosis proliferated rapidly in the murine bone marrow, spleen, and thymus, invaded peripheral organs, and resulted in morbidity and mortality of the animals within 4 to 16 weeks. Cells from two patients who relapsed 3.5 years after diagnosis grew much slower than the early relapse samples, taking up to 30 weeks to infiltrate the bone marrow of recipient mice. In contrast, leukemic cells were absent or were detected at low numbers in scid mice transplanted with cells obtained at diagnosis from three patients who have not yet relapsed. These results show an increased ability of leukemic cells from patients with aggressive lymphoblastic leukemia of poor prognosis to proliferate in scid mice.

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