Interaction Of CXCL12 Research Articles

New Insights into Cellular Defects during Haematopoiesis of Sickle Cell Disease Using the Townes Mouse Model

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β globin gene leading to the synthesis of an abnormal haemoglobin (HbS), that under low oxygen tension polymerises driving the sickling of red blood cells and reducing their lifespan. SCD is characterized by anaemia, vaso-occlusion and haemolysis, progressive multiorgan damage and increased mortality. For decades, peripheral haemolysis has been considered as the sole driver of anaemia in SCD. Recently, we have demonstrated the occurrence of ineffective erythropoiesis (IE) in SCD suggesting that anaemia can be impacted by defects of central origin and hinting to abnormalities in the haematopoietic compartment. To gain insight into SCD haematopoiesis, we investigated the haematopoietic compartment of the humanized transgenic Townes mouse model by conducting an in-depth flow cytometry-based analysis in the bone marrow (BM) and spleen, comparing SS mice to control littermates (AA and AS) at 8 and 16 weeks of age (termed 8-w and 16-w, respectively). BM and spleen are two key hematopoietic tissues in adult mice: BM is involved in maintaining homeostatic haematopoiesis and spleen responds to stress haematopoiesis. First, we assessed haematopoietic differentiation in the BM of SS, AA and AS mice. Total number of BM cells and frequency of Lin Neg and Lin NegKit Pos (LK) were identical between SS and control littermates whereas a minor increase in the percentages of Lin NegSca PosKit Pos (LSK) was observed in 16-w SS mice. Within the LSK compartment, we found a 1.81- and 2.5-fold reduction of long-term haematopoietic stem cell (lt-HSC) frequency in 8-w and 16-w SS mice, respectively (Fig1 A). HSCs differentiate into multipotent progenitors (MPPs), which have decreased self-renewal capacity and progressively engage in the megakaryocytic (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) lineage. The MPP3 compartment showed a 1.6-fold increased frequency in 8-w and 16-w SS mice indicating a skewing toward myeloid cell production and the onset of extramedullary haematopoiesis as early as 8 weeks after birth (Fig1 A). Next, we analysed the downstream lineages of the committed progenitors (lymphoid, granulo-macrophagic, megakaryocytic, and erythroid) and found a 1.4 and 2.3-fold increase of th megakaryocyte-erythroid progenitor cells (MEPs) and pre-CFU-Es in 8-w and 16-w SS mice, respectively (Fig1 A,B). We assessed the potential of SS hematopoietic progenitors (HSPCs) to differentiate into myelomonocytic and erythroid lineages by performing methylcellulose colony forming unit (CFU) assays with LSK and LK cells sorted from 8-w mice. Although no difference was observed in the number and type of colonies formed in vitro from SS and control HSPCs, SS colonies were much smaller than the AA/AS ones. Furthermore, serial replating showed a phenotype of stemness exhaustion in SS mice, with a dramatic decrease in the number of colonies formed by LSK cells (27.5 SS CFUs vs 51.6 AA/AS CFUs), suggesting lower self-renewal capacities of SS HSPCs. In spleen, in addition to the expected splenomegaly of SS mice, we found a trend toward a decreased frequency of the LSK compartment and an increase of the LK compartment. There was a 2.25- and 1.42-fold increase in the percentage of MPP-2 and MPP-3 cells, respectively, and a 2.1-fold increase in the pre-CFU-Es confirming an intense splenic haematopoiesis in SS mice (Fig 1B). HSPCs are known to be retained by the BM stromal cells through the chemotactic interaction of CXCL12 and CXCR4 and the adhesive interaction of VCAM-1 and VLA-4. Measuring HSC-related cytokines in BM supernatants, we found severely diminished levels of CXCL12 in SS mice as compared to control littermates (18 pg.mL -1 in SS vs 84 pg.mL -1 in AA; N=80), suggesting HSC mobilization from the BM to the spleen with disease onset. In addition to the well-established stress erythropoiesis phenotype reported in SCD mice, our study describes cellular abnormalities in the haematopoietic compartment shedding light on potential new mechanisms that contribute to SCD pathology in relation to anaemia and bone marrow stemness.

Loss of bone morphogenetic protein signaling in fibroblasts results in CXCL12-driven serrated polyp development

Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling–CXCL12 interaction could have a role in the etiology of serrated polyp formation.

Colorectal Cancer: The Contribution of CXCL12 and Its Receptors CXCR4 and CXCR7.

Simple SummaryMany signaling pathways are involved in cancer progression, and among these pathways, the CXCL12 axis and its two receptors CXCR4 and CXCR7 are well described for many cancers. This review presents the current knowledge on the role played by each of the actors of this axis in colorectal cancer and on its consideration in the development of new therapeutic strategies.Colorectal cancer is one of the most common cancers, and diagnosis at late metastatic stages is the main cause of death related to this cancer. This progression to metastasis is complex and involves different molecules such as the chemokine CXCL12 and its two receptors CXCR4 and CXCR7. The high expression of receptors in CRC is often associated with a poor prognosis and aggressiveness of the tumor. The interaction of CXCL12 and its receptors activates signaling pathways that induce chemotaxis, proliferation, migration, and cell invasion. To this end, receptor inhibitors were developed, and their use in preclinical and clinical studies is ongoing. This review provides an overview of studies involving CXCR4 and CXCR7 in CRC with an update on their targeting in anti-cancer therapies.

CXCL12/CXCR4 axis gene variants contribute to an increased vulnerability to HPV infection and cervical oncogenesis.

Every year, more than half a million women are diagnosed with cervical cancer (CC). Individual factors may contribute to the cervical cancer development, such as immunogenetic variation. CXCL12/CXCR4 axis is involved in tumor progression and aggressiveness. In the present study, we aimed to investigate a possible association between two single-nucleotide variants (CXCL12 rs1801157 and CXCR4 rs2228014) with HPV infection and cervical cancer development. PCR technique was used to test HPV positivity in 424 women, in which the allelic frequency of CXCL12 rs1801157 and CXCR4 rs2228014 was also assessed by PCR-restriction fragment length polymorphism. CXCL12 rs1801157 was associated with HPV infection in the allelic distribution as well in the codominant, dominant and recessive genetic models; as well with squamous intraepithelial lesions (SIL) and CC in the codominant and dominant models. CXCR4 rs2228014 was associated to HPV infection in the codominant model and allelic distribution; as well with SIL/CC in the codominant, dominant and allelic models. Independent associations were found for CXCL12 AA genotype and HPV infection, SIL and CC development, as well as, CXCR4 allele T and HPV infection and CC. The variants interaction analysis demonstrated that the presence of both polymorphisms increases the susceptibility of HPV infection in 10.1 times, SIL (2 times) and CC development in 4.2 times. This is the first study demonstrating that the interaction of CXCL12 and CXCR4 variants contributes to the increased susceptibility of HPV infection, squamous intraepithelial lesions and cervical cancer development.

CXCR4 Signaling in AML Is Dampened By the Endogenous CXCR4 Inhibitory Peptide EPI-X4

CXCR4 - CXCL12 interaction belongs to one the most fundamental loops regulating in particular cell anchorage in the BM and trafficking of cells to distant organs. This regulatory principle is highly conserved between species and is also maintained in neoplasms, shaping the behavior of malignant cells. CXCR4 is overexpressed in 25-30% of patients with acute myeloid leukemia (AML) and is associated with poor prognosis. Functional assays have proven that the CXCR4 - CXCL12 crosstalk is critical in regulating the interaction between leukemic stem cells (LSCs) and their niche. We now demonstrate that an endogenous peptide called EPI-X4 is a key candidate to balance CXCR4 signaling in AML. EPI-X4 was identified as a peptide (EPI-X4 endogenous inhibitor of CXCR4) specifically binding to CXCR4. It is generated at low pH chopped from serum albumin by Cathepsin D and E, forming a 16-mer peptide. By screening 283 primary AML patient samples, we could demonstrate that AML bulk as well as functionally validated human AML-LSCs are highly positive for CXCR4. Upon EPI-X4 treatment we could observe a marked deprivation in clonogenic growth and migration of CXCR4 positive AML cells towards a CXCL12 gradient (reduction by 66 and 77 % compared to inactive peptide as control, respectively; p<0.0005 and p<0.03). The growth inhibitory effect of EPI-X4 was accompanied by a significant decrease in CXCL12 stimulated phosphor-ERK levels, suggesting reshuffling of signaling pathways by the peptide. Furthermore, homing and engraftment potential of primary CXCR4 expressing AML patient sample in NSG mice was reduced by EPI-X4 (engraftment by 76%; p<0.004). These antagonizing and intrinsic effects were even increased when treating the cells with optimized derivatives of EPI-X4 with higher anti-CXCR4 activity. We further performed RNA-Seq on the AML cell line OCI-AML3 treated with EPI-X4 or an inactive control for 24 h in serum free conditions. In total, we found 629 genes differentially expressed between EPI-X4 and the control (FDR<0,05; Fold change >0,5). Detailed gene enrichment analysis revealed alteration of genes involved in carbohydrate and lipid metabolism pathways. The same set-up was used to assess the protein profile of EPI-X4 treated OCI-AML3 cells and resulted in quantification of 3500 proteins of which 26 were differentially expressed, among them PYCARD involved in Caspase - mediated apoptosis regulation and ECD, associated with TP53 stability. Taken together, these data demonstrate that a naturally occurring peptide exist, which counteracts CXCR4 signaling in AML. Understanding these endogenous systems which balance CXCR4 activity in malignant diseases will finally help to develop novel therapeutic tools by generating optimized highly specific anti-CXCR4 peptides. Disclosures Buske: Roche, Janssen, Bayer, MSD: Research Funding; Roche, Janssen, AbbVie, Pfizer, Celltrion: Honoraria, Speakers Bureau; Morphosys: Membership on an entity's Board of Directors or advisory committees.

CXCL12γ isoform inhibits adverse left ventricular remodeling after acute myocardial infarction

The chemokine CXCL12 is essential for cardiovascular system development and plays a major role in physio-pathological processes such as inflammation, angiogenesis but also tissue remodeling after an ischemic insult. In addition to the binding to its receptors, CXCR4 and CXCR7, CXCL12 interaction with heparan-sulfates (HS) commands its biological activity. Studies in experimental models of myocardial infarction (MI) revealed a protective role for CXCL12α. However, in mice, three CXCL12 isoforms (α, β and γ) have been identified and the CXCL12γ isoform shows an unchallenged ability to cooperate with HS, suggesting a pivotal role in tissue repair. The aim of the study is to analyze the role of CXCL12γ isoform and the importance of CXCL12/HS interactions in post-ischemic cardiac remodeling in an acute model of MI. MI are induced in mice carrying a Cxcl12 gene mutation that precludes interactions with HS (Cxcl12Gagtm) and in Cxcl12γ knock-in animals (Cxcl12γ-KI), harboring CXCL12γ deficiency. Alternatively, the impact of CXCL12γ overexpression is also evaluated in wild-type (WT) mice receiving transcutaneous echo-guided injections of adenovirus encoding Cxcl12γ in cardiac tissue. Cardiac function and remodeling have been assessed through echocardiography analysis and evaluation of infarct size, interstitial fibrosis, capillary and arteriole densities and as well as inflammatory cell infiltration. After MI, Cxcl12Gagtm and Cxcl12γ-KI animals exhibit reduction in cardiac function and adverse left ventricular remodeling when compared to their respective WT littermates. Interestingly, overexpression of CXCL12γ in WT mice normalizes cardiac function by reducing the size of the infarcted area, interstitial fibrosis and promoting vascular growth. We show that CXCL12γ isoform plays an important role in the regulation of cardiac function after MI and that its interactions with HS seems essential for adequate cardiac repair.

Development and Evaluation of an 18F-Radiolabeled Monocyclam Derivative for Imaging CXCR4 Expression.

In humans, C–X–C chemokine receptor type 4 (CXCR4) is a protein that is encoded by the CXCR4 gene and binds the ligand CXCL12 (also known as SDF-1). The CXCR4–CXCL12 interaction in cancer elicits biological activities that result in tumor progression and has accordingly been the subject of significant investigation for detection and treatment of the disease. Peptidic antagonists have been labeled with a variety of radioisotopes for the detection of CXCR4, but the methodology utilizing small molecules has predominantly used radiometals. We report here the development of a 18F-radiolabeled cyclam-based small molecule radioprobe, [18F]MCFB, for imaging CXCR4 expression. The IC50 value of [19F]MCFB for CXCR4 was similar to that of AMD3465 (111.3 and 89.8 nM, respectively). In vitro binding assays show that the tracer depicted a differential CXCR4 expression, which was blocked in the presence of AMD3465, demonstrating the specificity of [18F]MCFB. Positron emission tomography (PET) imaging studies showed a distinct uptake of the radioprobe in lymphoma and breast cancer xenografts. High liver and kidney uptakes were seen with [18F]MCFB, leading us to further examine the basis of its pharmacokinetics in relation to the tracer’s cationic nature and thus the role of organic cation transporters (OCTs). Substrate competition following the intravenous injection of metformin led to a marked decrease in the urinary excretion of [18F]MCFB, with moderate changes observed in other organs, including the liver. Our results suggest involvement of OCTs in the renal elimination of the tracer. In conclusion, the 18F-radiolabeled monocyclam, [18F]MCFB, has potential to detect tumor CXCR4 in nonhepatic tissues.

CXCR4-CXCL12 interaction is important for plasma cell homing and survival in NZB/W mice.

Antibody-secreting cells (ASCs), including short-lived plasmablasts and long-lived memory plasma cells (LLPCs), contribute to autoimmune pathology. ASCs, particularly LLPCs, refractory to conventional immunosuppressive drugs pose a major therapeutic challenge. Since stromal cells expressing C-X-C motif chemokine-12 (CXCL12) organize survival niches for LLPCs in the bone marrow, we investigated the effects of CXCL12 and its ligand CXCR4 (C-X-C chemokine receptor 4) on ASCs in lupus mice (NZB/W). Fewer adoptively transferred splenic ASCs were retrieved from the bone marrow of recipient immunodeficient Rag1-/- mice when the ASCs were pretreated with the CXCR4 blocker AMD3100. CXCR4 blockade also significantly reduced anti-OVA ASCs in the bone marrow after secondary immunization with OVA. In this study, AMD3100 efficiently depleted ASCs, including LLPCs. After two weeks, it decreased the total number of ASCs in the spleen and bone marrow by more than 60%. Combination with the proteasome inhibitor bortezomib significantly enhanced the depletion effect of AMD3100. Continuous long-term (five-month) CXCR4 blockade with AMD3100 after effective short-term LLPCs depletion kept the number of LLPCs in the bone marrow low, delayed proteinuria development and prolonged the survival of the mice. These findings identify the CXCR4-CXCL12 axis as a potential therapeutic target likely due to its importance for ASC homing and survival.

MEKK1, JNK, and SMAD3 mediate CXCL12-stimulated connective tissue growth factor expression in human lung fibroblasts

BackgroundIn idiopathic pulmonary fibrosis, the interaction of CXCL12 and CXC receptor 4 (CXCR4) plays a critical role in lung fibrosis. Connective tissue growth factor (CTGF) overexpression underlies the development of pulmonary fibrosis. Our previous report showed that the Rac1-dependent extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein (AP)-1 pathways are involved in CXCL12-generated CTGF expression in human lung fibroblasts (WI-38). In present study, we additionally inspected the involvement of mitogen-activated protein kinase kinase kinase 1 (MEKK1)/JNK-dependent SMAD3 in CXCL12-triggered CTGF expression in WI-38 cells.MethodsWI-38 cells were stimulated with CXCL12 in the absence or presence of specific inhibitors or small interfering RNAs (siRNAs). CTGF expression and signaling transduction molecules were assessed by Western blot, luciferase activity assay, or ChIP assay.ResultsCXCL-12-induced CTGF expression was attenuated by SIS3 (a SMAD3 inhibitor) and SMAD3 siRNA, but not by SB431542 (an activin receptor-like kinase 5, ALK5, inhibitor). CXCL12-stimulated CTGF expression was also attenuated by MEKK1 siRNA. Treatment of cells with CXCL12 caused an increase in SMAD3 phosphorylation at Ser208, translocation to nuclei, SMAD3-luciferase activity, and recruitment of SMAD3 to the CTGF promoter. Stimulation of cells with CXCL12 resulted in increase in JNK phosphorylation at Thr183/Tyr185 and MEKK1 phosphorylation at Thr261. Moreover, CXCL12-mediated SMAD3 phosphorylation or SMAD3-luciferase activity was inhibited by MEKK1 siRNA or SP600125. Finally, CXCL12-mediated JNK phosphorylation was attenuated by MEKK1 siRNA.ConclusionIn conclusion, results of this study suggest that CXCL12 activates the MEKK1/JNK signaling pathway, which in turn initiates SMAD3 phosphorylation, its translocation to nuclei, and recruitment of SMAD3 to the CTGF promoter, which ultimately induces CTGF expression in human lung fibroblasts.

The structural investigation of glycosaminoglycan binding to CXCL12 displays distinct interaction sites.

The stromal cell-derived factor 1α (CXCL12) belongs to the CXC chemokine family and plays an important role in tissue regeneration and the recruitment of stem cells. Here, a stable chemotactic gradient is essential that is formed by the interaction of CXCL12 with the extracellular matrix. Binding properties of CXCL12 to naturally occurring glycosaminoglycans (GAGs) as well as to the artificial highly sulfated hyaluronic acid (HA) are investigated by using a combination of NMR spectroscopy, molecular modeling and molecular dynamics simulations. Our results demonstrate a preferred protein binding for the sulfated GAGs heparin (HE) and highly sulfated HA. Furthermore, we could demonstrate that the orientation of the sulfate is crucial for binding. All sulfated GAGs interact with the CXCL12 GAG-binding motif (K24-H25-L26-K27-R41-K43-R47), where K27 and R41 represent the anchor points. Furthermore, differences could be observed in the second interaction interface of CXCL12: both HE and highly sulfated HA interfere with the receptor-binding motif, while chondroitin sulfate binds different amino acids in close proximity to this motif. CXCL12 does not interact with HA, which was directly demonstrated by NMR spectroscopy and molecular modeling and explained by the lack of sulfate groups of the HA molecule.

Role of Chemokine Signalling in the Pathogenesis of Good’s Syndrome-Case Reports, Clinical Characterization from Single-Centre Perspective

Good’s syndrome (GS), is a rare condition defined as a coexistence of thymoma and hipogammaglobulinaemia. Involvement of central lymphoid organ prompts concept about perturbation in lymphocyte migration and differentiation. Cell homing to bone marrow compartment depends on the CXCR4↔CXCL12 interaction. In this paper we describe two patients with GS-mild and severe form, presenting differences in the expression of CXCR4 on cells in peripheral blood and bone marrow. Patient 1 (mild form): (i) Mild hipogammaglobulinaemia (IgG=150 mg/dL), (ii) Low count of peripheral B cells (14%, 60 cells/μL) and NK (18%, 77cells/μL); high level of T cells (93.3%, 4006 cells/ μL), (iii) Moderate thymic enlargement (9 × 15 cm), (iv) CXCR4 expression in BM 82.6% (20139/μL), 34% CXCR4+CD19+ (8289/μL). Patient 2 (severe form): (i) Severe hipogammaglobulinaemia (IgG=20 mg/dL), (ii) Absence of B and NK cells deficiency (in peripheral blood 0.4% i.e., 10/μL, and 6.17%-47.3/μL respectively), (iii) Severe thymic enlargement (20 × 25 cm) (iv) CXCR4 expression in BM 46.3% (552/μL); 6.1%, CXCR4+CD19+ (174/ μL). Interestingly, the bone marrow of patient with severe form of GS contained more CD10-positive B cells than BM of the patient with mild form (80% vs. 5.88% of B cells), but in former as well as letter a significant proportion of the total CXCR4-positive lymphocytes are negative for B cells marker (comparable: 48.1% and 53.1% respectively). In our patients a tenfold decrease in the CD4-positive γδ T cells (CD4+TCRγδ) counts was observed. Both cases went up fatal due to progression of nasopharyngeal cancer (mild form) and breast cancer (severe form). This data confirm that earliest B cell precursors, pre-pro-B cells and end-stage B cells, plasma cells require CXCR4 ↔ CXCL12 interaction. In contrast in GS weak expression of CXCR4 in marrow precursors is source of B cell differentiation arrest on pro-B cell stage. The low level Nk and CD4+γδ T cells in Good syndrome is the new observation.

The chemokine receptors CXCR4/CXCR7 and their primary heterodimeric ligands CXCL12 and CXCL12/high mobility group box 1 in pancreatic cancer growth and development: finding flow.

Novel therapies need to be developed for patients with pancreatic cancer because of the poor outcomes of current regimens. Pancreatic cancer cells respond to the C-X-C chemokine receptor type 4 (CXCR4)/C-X-C chemokine receptor type 7 (CXCR7)/C-X-C motif chemokine 12 (CXCL12)/high-mobility group box 1 signaling axis and this axis presents a novel target for therapy. C-X-C motif chemokine 12 stimulates CXCR4/CXCR7-bearing cells in a paracrine manner. C-X-C chemokine receptor type 4 and CXCR7 are transmembrane G protein-coupled receptors that, upon interaction with ligand CXCL12, activate downstream protein kinases that promote a more aggressive behavior. C-X-C chemokine receptor type 4 is expressed on most pancreatic cancer cells, whereas CXCR7 is primarily expressed on tumor-associated endothelium. High-mobility group box 1 promotes the CXCR4 and CXCL12 interaction, promoting angiogenesis and lymphangiogenesis. Hypoxia-inducible factor 1 is a potent stimulator of CXCR4 and CXCL12 expression, promoting more aggressive behavior. This receptor/ligand interaction can be disrupted by CXCR4 antagonists available and in clinical use to harvest bone marrow stem cells. Novel imaging strategies are now being developed at several centers to evaluate response to therapy and identify early recurrence. Thus, the CXCR4/CXCR7/CXCL12 interaction plays a critical role in cancer cell progression, proliferation, invasion, as well as metastasis and is a suitable target for therapy, imaging, as well as development of novel diagnostics.

Profiling of Cxcl12 receptors, Cxcr4 and Cxcr7 in murine testis development and a spermatogenic depletion model indicates a role for Cxcr7 in controlling Cxcl12 activity.

In mice the chemokine Cxcl12 and its receptor Cxcr4 participate in maintenance of the spermatogonial population during postnatal development. More complexity arises since Cxcl12 also binds to the non-classical/atypical chemokine receptor Cxcr7. We explored the expression pattern of Cxcl12, Cxcr4 and Cxcr7 during postnatal development in mouse testes and investigated the response of Cxcl12, Cxcr4, Cxcr7 and SSC-niche associated factors to busulfan-induced germ cell depletion and subsequent recovery by RNA expression analysis and localization of the proteins. In neonatal testes transcript levels of Cxcl12, Cxcr4 and Cxcr7 were relatively low and protein expression of Cxcr7 was restricted to gonocytes and spermatogonia. During development, RNA expression of Cxcl12 remained stable but that of Cxcr4 and Cxcr7 increased. Cxcr7 was expressed in germ cells located at the basement membrane of the seminiferous tubules. In adult testes, transcript levels of Cxcl12 were highest while the localization of Cxcr7 did not change. Following germ cell depletion, a significantly increased expression of Cxcl12 and a decreased expression of Cxcr7 were observed. Germ cells repopulating the seminiferous tubules were immunopositive for Cxcr7. We conclude that Cxcr7 expression to be restricted to premeiotic germ cells throughout postnatal testicular development and during testicular recovery. Hence, the spermatogonial population may not only be simply controlled by interaction of Cxcl12 with Cxcr4 but may also involve Cxcr7 as an important player.

Abstract 1114: Combination of a novel CXCR4 antagonist with chemotherapy reduces breast cancer bone metastatic tumor burden

Abstract Background: Bone is the most common site of metastasis for patients with breast cancer. Tumor cells migrate to and reside in the protective bone marrow microenvironment niche through adhesive interaction between tumor CXCR4 and stromal CXCL12 (SDF1). CXCL12 is produced by activated osteoblasts, bone marrow and lung stromal cells, and endothelial cells. Nearly 60% of breast cancers express CXCR4 and this is associated with decreased survival. We hypothesized that a Protein Epitope Mimetic (PEM) POL5551, a novel CXCR4 antagonist, will limit the extent of tumor metastasis by disrupting stromal-mediated protection from cytotoxic chemotherapy and in turn may prolong survival. Approach: In vitro, POL5551 had no direct cytotoxic activity and did not reduce proliferation of CXCR4+ MDA-MB-231 or 4T1 osteolytic breast cancer cell lines. However, in an in vitro scratch-wound assay POL5551 inhibited migration of MDA-MB-231 cells. In a Gaussia luciferase (GLuc) complementation model in MDA-MB-231 cells, the interaction of CXCL12 with CXCR4, but not CXCR7, was blocked by low nanomolar concentrations of POL5551. At 20 mg/kg administered from day 10 post inoculation, POL5551 displayed no single agent activity on primary tumor xenografts, and in combination with eribulin there was no synergistic effect on primary tumor xenografts. However, continuation of treatment with POL5551 after surgical tumor removal decreased tumor metastasis and prolonged survival to 58 days compared to control at 45 days and eribulin alone at 51 days. Also, POL5551 showed effects on immune infiltration to the primary tumor. To test the hypothesis that CXCR4 antagonism disrupts the protective bone marrow niche in which tumor cells reside and sensitizes them to cytotoxic chemotherapy, we employed a “framing dosing strategy”. In a second model of metastasis produced by intracardiac injection of MDA-MB-231 cells, POL5551 (20 mg/kg, s.c.) was administered to mice with bone metastases 4 hours before and 4 & 18 hours after eribulin chemotherapy (0.2 mg/kg, i.v.). While bone is the predominant site of metastasis with these cell lines, lung and liver metastasis also occurs. POL5551 in combination with eribulin decreased bone tumor burden more than eribulin alone (reduction in leg bone & liver tumor burden, versus control respectively: eribulin alone 58% & 75%, n.s.; POL5551+eribulin: 89% & 86%, P<0.05; n=4-5 mice per group). Conclusion: These preclinical data support a synergism of CXCR4 antagonism during chemotherapy for the treatment of metastatic breast cancer. These and additional data support initiation of a Phase I clinical trial to evaluate a related PEM CXCR4 antagonist in combination with eribulin chemotherapy in patients with metastatic breast cancer who have failed at least 2 lines of therapy. Note: This abstract was not presented at the meeting. Citation Format: Jingyu Xiang, Michelle A. Hurchla, Kathryn Luker, Garry Douglas, Barbara Romagnoli, Eric Chevalier, Michael Bauer, Johann Zimmermann, Klaus Dembowsky, Gary Luker, Katherine N. Weilbaecher. Combination of a novel CXCR4 antagonist with chemotherapy reduces breast cancer bone metastatic tumor burden. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1114. doi:10.1158/1538-7445.AM2014-1114

Interaction of the chemokines I-TAC (CXCL11) and SDF-1 (CXCL12) in the regulation of tumor angiogenesis of colorectal cancer

The chemokine CXCL12 has a decisive role in tumor progression by mediating pro-angiogenic and pro-metastatic effects through its receptor CXCR4. The CXCL12 pathway is connected with another chemokine, CXCL11, through its second receptor CXCR7. CXCL11 also binds to the CXCR3 receptor. CXCL11 function in tumor angiogenesis is likely receptor dependent because CXCR3 predominantly mediates angiostatic signals whereas CXCR7 mediated signaling is rather angiogenic. We therefore studied the interaction of CXCL12 and CXCL11 in an in vivo model of colorectal cancer metastasis. GFP-transfected CT26.WT colorectal cancer cells were implanted into the dorsal skinfold chamber of syngeneic BALB/c mice. The animals received either peritumoral application of CXCL11 or intraperitoneal injections with neutralizing antibodies against CXCL11, CXCL12 or both. Tumor growth characteristics, angiogenesis, cell migration, invasive tumor growth, tumor cell proliferation and apoptosis were studied by intravital fluorescence microscopy and immunohistochemistry during an observation period of 14days. Local exposure to CXCL11 significantly stimulated tumor growth compared to controls and enhanced invasive growth characteristics without affecting tumor angiogenesis and tumor cell migration. Neither CXCL11 nor CXCL12-blockade had a significant impact on tumor growth and angiogenesis, whereas the combined neutralization of CXCL11 and CXCL12 almost completely abrogated tumor vessel formation. As a consequence, tumor growth and invasive growth characteristics were reduced compared to the other groups. The results of the present study underline the interaction of CXCL12 and CXCL11 during tumor angiogenesis. The combined blockade of both signaling pathways may provide an interesting anti-angiogenic approach for anti-tumor therapy.

Emerging Targets in Pituitary Adenomas: Role of the CXCL12/CXCR4-R7 System.

Chemokines are chemotactic regulators of immune surveillance in physiological and pathological conditions such as inflammation, infection, and cancer. Several chemokines and cognate receptors are constitutively expressed in the central nervous system, not only in glial and endothelial cells but also in neurons, controlling neurogenesis, neurite outgrowth, and axonal guidance during development. In particular, the chemokine CXCL12 and its receptors, CXCR4 and CXCR7, form a functional network that controls plasticity in different brain areas, influencing neurotransmission, neuromodulation, and cell migration, and the dysregulation of this chemokinergic axis is involved in several neurodegenerative, neuroinflammatory, and malignant diseases. CXCR4 primarily mediates the transduction of proliferative signals, while CXCR7 seems to be mainly responsible for scavenging CXCL12. Importantly, the multiple intracellular signalling generated by CXCL12 interaction with its receptors influences hypothalamic modulation of neuroendocrine functions, although a direct modulation of pituitary functioning via autocrine/paracrine mechanisms was also reported. Both CXCL12 and CXCR4 are constitutively overexpressed in pituitary adenomas and their signalling induces cell survival and proliferation, as well as hormonal hypersecretion. In this review we focus on the physiological and pathological functions of immune-related cyto- and chemokines, mainly focusing on the CXCL12/CXCR4-7 axis, and their role in pituitary tumorigenesis. Accordingly, we discuss the potential targeting of CXCR4 as novel pharmacological approach for pituitary adenomas.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Diabetes impairs hematopoietic stem cell mobilization by altering niche function.

Success with transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) in patients depends on adequate collection of these cells after mobilization from the bone marrow niche by the cytokine granulocyte colony-stimulating factor (G-CSF). However, some patients fail to achieve sufficient HSPC mobilization. Retrospective analysis of bone marrow transplant patient records revealed that diabetes correlated with poor mobilization of CD34+ HSPCs. In mouse models of type 1 and type 2 diabetes (streptozotocin-induced and db/db mice, respectively), we found impaired egress of murine HSPCs from the bone marrow after G-CSF treatment. Furthermore, HSPCs were aberrantly localized in the marrow niche of the diabetic mice, and abnormalities in the number and function of sympathetic nerve termini were associated with this mislocalization. Aberrant responses to β-adrenergic stimulation of the bone marrow included an inability of marrow mesenchymal stem cells expressing the marker nestin to down-modulate the chemokine CXCL12 in response to G-CSF treatment (mesenchymal stem cells are reported to be critical for HSPC mobilization). The HSPC mobilization defect was rescued by direct pharmacological inhibition of the interaction of CXCL12 with its receptor CXCR4 using the drug AMD3100. These data suggest that there are diabetes-induced changes in bone marrow physiology and microanatomy and point to a potential intervention to overcome poor HSPC mobilization in diabetic patients.

New G-protein-coupled receptor structures provide insights into the recognition of CXCL12 and HIV-1 gp120 by CXCR4

The G protein-coupled receptor (GPCR) superfamily consists of thousands of integral membrane proteins that exert a wide variety of physiological functions and account for a large portion of the drug targets identified so far. However, structural knowledge of GPCRs is scarce, with crystal structures determined for only a few members including b1 and b2 adrenergic receptors, adenosine receptor, rhodopsin, and dopamine D3 receptor [1]. Recently, Wu et al. published in Science the structure of CXCR4, which is the first structure of the 19 known chemokine receptors of the GPCR superfamily [2]. The widely expressed chemokine receptor CXCR4 is exclusively activated by CXCL12 (also known as SDF-1). The CXCL12/CXCR4 signaling plays important functional roles in various biological processes such as cell trafficking, cell survival, and proliferation, and is essential for the development (reviewed in [3,4]). The CXCL12/CXCR4 axis has also been indicated to be critical for the homing of CXCR4-expressing tumor cells to tissues secreting CXCL12 such as the bone marrow, lung, liver, and lymph nodes, and their survival and proliferation in the new environment [3,4]. Therefore, the CXCL12/CXCR4 pathway is an important drug target for various types of cancers [5], and one small-molecule inhibitor of the pathway, namely Plerixafor has been approved by the US Food and Drug Administration for the treatment of non-Hodgkin lymphoma and multiple myeloma together with granulocyte-colony stimulating factor. In addition, CXCR4 also functions as a co-receptor for the entry of X4 HIV-1 strains and is a validated pharmaceutical target for modulating the viral entry of HIV-1 into T cells [6]. Precise structural information is valuable for design and development of new regimens against diseases. Although CXCR4 is an important drug target, by the time Wu et al. published their results, only one structure of CXCL12 in complex with a peptide derived from the sequence of the N-terminal 38 residues of CXCR4 (p38) was reported [7]. Although the CXCL12-p38 structure elucidated the structural basis for the interaction of CXCL12 with the N-terminus of CXCR4 [7], the structure of CXCR4 is still largely unknown, and the other regions of CXCR4 that potentially interact with CXCL12 has not been identified. Moreover, the dimeric CXCL12-p38 structure might only represent one of the dynamic oligomeric states of the complex. For the fusion of HIV-1, CCR5 is a co-receptor for CD4 for R5 HIV-1 strains, which is also a chemokine receptor of the GPCR superfamily. The structure of the CCR5 N-terminus (residues 2–15) in complex with HIV-1 gp120 and CD4 had been determined recently [8]; however, a similar structure of CXCR4 was not available yet. In the recent paper by Wu et al., five crystal structures of human CXCR4 variants in complexes with a smallmolecule isothiourea derivative, namely 1T15, and with a cyclic peptide inhibitor, namely CVX15 at resolution of 2.5–3.2 A are reported [2]. Comparison of the CXCR4 structures with the previously reported GPCR structures reveals that in CXCR4 the overall architecture of the canonical bundle of seven transmembrane helices is divergent from that in the other GPCR structures with multiple profound differences, underscoring the necessity of obtaining precise structural information with experimentally determined structures rather than constructed homology models. On the extracellular side of CXCR4, the conserved disulfide bonds between Cys28 of the N-terminal segment and Cys274 at the tip of helix VII, and between Cys109 and Cys186 of extracellular loop 2 (ECL2) are found to stabilize the N-terminal segment and ECL2 to form the entrance to the ligand-binding pocket, which explains their critical roles in the ligand binding. On the intracellular side, the intracellular part of helix VII is one turn shorter compared with the other GPCR structures, and helix VIII is missing with the C-terminus taking an extended conformation. The CXCR4 mutant (CXCR4-3) carrying a T240P mutation Acta Biochim Biophys Sin 2011, 43: 337–338 | a The Author 2011. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmr026. Advance Access Publication 31 March 2011

CXCL12 and CXCR4 in bone marrow physiology

This article discusses the multiple roles of CXCL12 and its receptor, CXCR4, in bone marrow (BM) hematopoietic stem cell (HSC) development and regulation. CXCL12 interaction with CXCR4 results in effects as varied as cell migration, proliferation and survival or apoptosis. The selective signaling pathways that mediate these varied outcomes are summarized briefly. The CXCL12/CXCR4 pair is crucially involved in homing and repopulation of HSCs in the specific BM niches. Mechanisms of HSC mobilization to the peripheral circulation in response to physiological requests and therapeutic stimulations, as well as recent data on the novel receptor for CXCL12, CXCR7, are reviewed.