CXCR4-targeted PET imaging using 64Cu-AMD3100 for detection of Waldenstrom Macroglobulinemia.
ABSTRACT: Objective: Waldenström Macroglobulinemia (WM) is a rare B-cell malignancy characterized by secretion of immunoglobulin M and cancer infiltration in the bone marrow. Chemokine receptor such as CXCR4 and hypoxic condition in the bone marrow play crucial roles in cancer cell trafficking, homing, adhesion, proliferation, survival, and drug resistance. Herein, we aimed to use CXCR4 as a potential biomarker to detect hypoxic-metastatic WM cells in the bone marrow and in the circulation by using CXCR4-detecting radiopharmaceutical.Methods: We radiolabeled a CXCR4-inhibitor (AMD3100) with 64Cu and tested its binding to WM cells with different levels of CXCR4 expression using gamma counter in vitro. The accumulation of this radiopharmaceutical tracer was tested in vivo in subcutaneous and intratibial models using PET/CT scan. In addition, PBMCs spiked with different amounts of WM cells ex vivo were detected using gamma counting.Results: In vitro, 64Cu-AMD3100 binding to WM cell lines demonstrated a direct correlation with the level of CXCR4 expression, which was increased in cells cultured in hypoxia with elevated levels of CXCR4, and decreased in cells with CXCR4 and HIF-1? knockout. Moreover, 64Cu-AMD3100 detected localized and circulating CXCR4high WM cells with high metastatic potential.Conclusions: In conclusion, we developed a molecularly targeted system, 64Cu-AMD3100, which binds to CXCR4 and specifically detects WM cells with hypoxic phenotype and metastatic potential in the subcutaneous and intratibial models. These preliminary findings using CXCR4-detecting PET radiopharmaceutical tracer indicate a potential technology to predict high-risk patients for the progression to WM due to metastatic potential.
Project description:UNLABELLED: BACKGROUND: Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources. METHODS: To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1? protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions. RESULTS: Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression level-dependent) negative impact of CXCR4 expression (p?<?0.04). A role for the CXCR4-CXCL12 axis in Ewing sarcoma growth was suggested by our observations that i) CXCR4 expression correlated positively with tumor volume at diagnosis (p?=?0.013), ii) CXCL12 was present within the microenvironment of virtually all cases, iii) CXCL12 induced proliferation of CXCR4-positive Ewing sarcoma cell lines, which could be abrogated by AMD3100. CXCR4 expression was not correlated with occurrence of metastatic disease. Also, therapy-naïve tumors demonstrated higher CXCR4 expression as compared to metastases (p?=?0.027). Evaluation of in vivo hypoxia-inducible factor-1? expression and culture of cells under hypoxic conditions revealed no role for hypoxia in CXCR4 expression. CONCLUSIONS: Together, our results imply a crucial role for the CXCR4-CXCL12 axis in auto- and/or paracrine growth stimulation. Integration of CXCR4-targeting strategies into first- and/or second-line treatment regimens may represent a promising treatment option for Ewing sarcoma.
Project description:We have demonstrated that the ?-chemokine stromal-derived factor (SDF)-1-CXCR4 axis plays an important role in rhabdomyosarcoma (RMS) metastasis. With the recent description of CXCR7, a new receptor for SDF-1 that also binds the interferon-inducible T-cell ? chemoattractant (ITAC) chemokine, we became interested in the role of the CXCR7-SDF-1/ITAC axis in RMS progression. To address this issue, we evaluated 6 highly metastatic alveolar (A)RMS and 3 less metastatic embryonal (E)RMS cell lines and found that all these cell lines express CXCR7. Although CXCR4 was expressed at a much higher level by highly metastatic ARMS lines, CXCR7 was present at a high level on ERMS lines. We also noticed that CXCR7 expression on RMS cells was downregulated in hypoxic conditions. More importantly, the CXCR7 receptor on RMS cell lines was functional after stimulation with ITAC and SDF-1 as evidenced by mitogen-activated protein kinase (MAPK)p42/44 and AKT phosphorylation as well as CXCR7 internalization, chemotaxis, cell motility and adhesion assays. Similarly to CXCR4, signaling from activated CXCR7 was not associated with increased RMS proliferation or cell survival. Moreover, CXCR7(+) RMS cells responded to SDF-1 and I-TAC in the presence of CXCR4 antagonists (T140, AMD3100). Furthermore, while intravenous injection of RMS cells with overexpressed CXCR7 resulted in increased seeding efficiency of tumor cells to bone marrow, CXCR7 downregulation showed the opposite effect. In conclusion, the CXCR7-SDF-1/ITAC axis is involved in the progression of RMS; targeting of the CXCR4-SDF-1 axis alone without simultaneous blockage of CXCR7 will be an inefficient strategy for inhibiting SDF-1-mediated prometastatic responses of RMS cells.
Project description:Glioblastoma is the most aggressive and malignant primary brain tumor in adults and has a poor patient survival of only 20 months after diagnosis. This poor patient survival is at least partly caused by glioblastoma stem cells (GSCs), which are slowly-dividing and therefore therapy-resistant. GSCs are localized in protective hypoxic peri-arteriolar niches where these aforementioned stemness properties are maintained. We previously showed that hypoxic peri-arteriolar GSC niches in human glioblastoma are functionally similar to hypoxic peri-arteriolar hematopoietic stem cell (HSC) niches in human bone marrow. GSCs and HSCs express the receptor C-X-C receptor type 4 (CXCR4), which binds to the chemoattractant stromal-derived factor-1? (SDF-1?), which is highly expressed in GSC niches in glioblastoma and HSC niches in bone marrow. This receptor-ligand interaction retains the GSCs/HSCs in their niches and thereby maintains their slowly-dividing state. In acute myeloid leukemia (AML), leukemic cells use the SDF-1?-CXCR4 interaction to migrate to HSC niches and become slowly-dividing and therapy-resistant leukemic stem cells (LSCs). In this communication, we aim to elucidate how disruption of the SDF-1?-CXCR4 interaction using the FDA-approved CXCR4 inhibitor plerixafor (AMD3100) may be used to force slowly-dividing cancer stem cells out of their niches in glioblastoma and AML. Ultimately, this strategy aims to induce GSC and LSC differentiation and their sensitization to therapy.
Project description:Pharmacological mobilization of hematopoietic progenitor cells (HPCs) is used clinically to harvest HPCs for bone marrow transplants. It is now widely accepted that the CXCR4:CXCL12 chemokine axis plays a critical role in the retention of HPCs in the bone marrow, and CXCR4 antagonists have been developed for their mobilization. The first of this class of drugs to be US Food and Drug Administration-approved was the bicyclam AMD3100. In addition to mobilizing HPCs and leukocytes in naïve mice, AMD3100 has been shown to mobilize mesenchymal progenitor cells (MPCs) in vascular endothelial growth factor (VEGF-A) pretreated mice. AMD3100 binds to the transmembrane region of CXCR4 and is thought to mobilize HPCs by reversing the gradient of CXCL12 across the bone marrow endothelium. Consistent with this hypothesis, our data show that selective neutralization of CXCL12, with chalcone 4-phosphate (C4P), inhibited AMD3100-stimulated mobilization of HPCs and leukocytes in naïve mice and MPCs in VEGF-A pretreated mice. In contrast it is shown here that the CXCR4 antagonist KRH3955 that binds to the extracellular loop of CXCR4 does not reverse the CXCL12 chemokine gradient. However, this drug efficiently mobilizes HPCs, a response that is not inhibited by C4P. In contrast, KRH3955 does not mobilize MPCs in VEGF-A pretreated mice. These data suggest that CXCR4 antagonists that bind to distinct regions of the receptor mobilize progenitor cells by distinct molecular mechanisms.
Project description:Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4(+) human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas ?2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.
Project description:The antagonism of CXC-chemokine receptor 4 (CXCR4) with AMD3100 improves cardiac performance after myocardial infarction by augmenting the recruitment of endothelial progenitor cells (EPCs) from the bone marrow to the regenerating vasculature. We investigated whether AMD3100 may accelerate diabetes-impaired wound healing through a similar mechanism. Skin wounds were made on the backs of leptin receptor-deficient mice and treated with AMD3100 or saline. Fourteen days after treatment, wound closure was significantly more complete in AMD3100-treated mice (AMD3100: 87.0 ± 2.6%, saline: 33.1 ± 1.8%; P<0.0001) and was accompanied by greater collagen fiber formation, capillary density, smooth muscle-containing vessel density, and monocyte/macrophage infiltration. On day 7 after treatment, AMD3100 was associated with higher circulating EPC and macrophage counts, and with significantly upregulated mRNA levels of stromal cell-derived factor 1 and platelet-derived growth factor B in the wound bed. AMD3100 also promoted macrophage proliferation and phagocytosis and the migration and proliferation of diabetic mouse primary dermal fibroblasts and 3T3 fibroblasts, which express very little CXCR4. In conclusion, a single topical application of AMD3100 promoted wound healing in diabetic mice by increasing cytokine production, mobilizing bone marrow EPCs, and enhancing the activity of fibroblasts and monocytes/macrophages, thereby increasing both angiogenesis and vasculogenesis. Not all of the AMD3100-mediated effects evolved through CXCR4 antagonism.
Project description:Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst2-ANT was determined to have a high affinity for SSTR2. Additionally, 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst2-ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64Cu-CB-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst2-ANT (64Cu-CB-TE2A-sst2-ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors.Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64Cu-CB-TE2A-sst2-ANT was compared with that of the 64Cu-labeled agonist 64Cu-CB-TE2A-tyrosine3-octreotate (64Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and small-animal PET/CT of 64Cu-CB-TE2A-sst2-ANT was performed.The dissociation constant value for the radiopharmaceutical was determined to be 26 +/- 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64Cu-CB-TE2A-sst2-ANT showed significantly less internalization than did 64Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64Cu-CB-TE2A-sst2-ANT from the blood was rapid, whereas the clearance of 64Cu-CB-TE2A-sst2-ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64Cu-CB-TE2A-sst2-ANT than those for 64Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64Cu-CB-TE2A-sst2-ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h.The PET radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptor-positive tumors.
Project description:AMD3100, also known as plerixafor, was originally developed as an anti-human immunodeficiency virus (HIV) drug, and later characterized as a C-X-C chemokine receptor type 4 (CXCR4) antagonist. Previous reviews have focused on the application of AMD3100 in the treatment of HIV, but a comprehensive evaluation of AMD3100 in the treatment of leukemia, solid tumor, and diagnosis is lacking. In this review, we broadly describe AMD3100, including the background, functional mechanism and clinical applications. Until the late 1990s, CXCR4 was known as a crucial factor for hematopoietic stem and progenitor cell (HSPC) retention in bone marrow. Subsequently, the action and synergy of plerixafor with Granulocyte-colony stimulating factor (G-CSF) led to the clinical approval of plerixafor as the first compound for mobilization of HSPCs. The amount of HSPC mobilization and the rapid kinetics promoted additional clinical uses. Recently, CXCR4/CXCL12 (C-X-C motif chemokine 12) axis was found to be involved in a variety of roles in tumors, including leukemic stem cell (LSC) homing and signaling transduction. Thus, CXCR4 targeting has been a treatment strategy against leukemia and solid tumors. Understanding this mechanism will help shed light on therapeutic potential for HIV infection, inflammatory diseases, stem-cell mobilization, leukemia, and solid tumors. Clarifying the CXCR4/CXCL12 axis and role of AMD3100 will help remove malignant cells from the bone marrow niche, rendering them more accessible to targeted therapeutic agents.
Project description:CXC-chemokine receptor 4 (CXCR4) regulates the retention of stem/progenitor cells in the bone marrow (BM), and the CXCR4 antagonist AMD3100 improves recovery from coronary ligation injury by mobilizing stem/progenitor cells from the BM to the peripheral blood. Thus, we investigated whether AMD3100 also improves recovery from ischemia/reperfusion injury, which more closely mimics myocardial infarction in patients, because blood flow is only temporarily obstructed.Mice were treated with single subcutaneous injections of AMD3100 (5 mg/kg) or saline after ischemia/reperfusion injury. Three days later, histological measurements of the ratio of infarct area to area at risk were smaller in AMD3100-treated mice than in mice administered saline, and echocardiographic measurements of left ventricular function were greater in the AMD3100-treated mice at week 4. CXCR4(+) cells were mobilized for just 1 day in both groups, but the mobilization of sca1(+)/flk1(+) cells endured for 7 days in AMD3100-treated mice compared with just 1 day in the saline-treated mice. AMD3100 upregulated BM levels of endothelial nitric oxide synthase (eNOS) and 2 targets of eNOS signaling, matrix metalloproteinase-9 and soluble Kit ligand. Furthermore, the loss of BM eNOS expression abolished the benefit of AMD3100 on sca1(+)/flk1(+) cell mobilization without altering the mobilization of CXCR4(+) cells, and the cardioprotective effects of AMD3100 were retained in eNOS-knockout mice that had been transplanted with BM from wild-type mice but not in wild-type mice with eNOS-knockout BM.AMD3100 prolongs BM progenitor mobilization and improves recovery from ischemia/reperfusion injury, and these benefits appear to occur through a previously unidentified link between AMD3100 and BM eNOS expression.
Project description:Patients with metastatic ovarian cancer continue to have a dismal prognosis, emphasizing the need for new strategies to identify and develop new molecular targets for therapy. Chemokine CXCL12 and its receptor CXCR4 are upregulated in metastatic ovarian cancer cells and the intraperitoneal tumor microenvironment. CXCL12-CXCR4 signaling promotes multiple steps in proliferation and dissemination of ovarian cancer cells, suggesting that targeted inhibition of this pathway will limit tumor progression. To investigate CXCL12-CXCR4 signaling in ovarian cancer and establish effects of inhibiting this pathway on tumor progression and survival, we designed a Gaussia luciferase complementation imaging reporter system to detect CXCL12 binding to CXCR4 in ovarian cancer cells. In cell-based assays, we established that the complementation imaging reporter could detect CXCL12 binding to CXCR4 and quantify specific inhibition of ligand-receptor interaction. We monitored CXCL12-CXCR4 binding and inhibition in a mouse xenograft model of metastatic human ovarian cancer by imaging Gaussia luciferase complementation and assessed tumor progression with firefly luciferase. Bioluminescence imaging studies in living mice showed that treatment with AMD3100, a clinically approved inhibitor of CXCL12-CXCR4, blocked ligand-receptor binding and reduced growth of ovarian cancer cells. Treatment with AMD3100 also modestly improved overall survival of mice with metastatic ovarian cancer. The Gaussia luciferase complementation imaging reporter system will facilitate further preclinical development and optimization of CXCL12-CXCR4 targeted compounds for treatment of ovarian cancer. Our research supports clinical translation of existing CXCR4 inhibitors for molecular therapy for ovarian cancer.