Deficient GATA6-CXCR7 signaling leads to bicuspid aortic valve.

The cardiac outflow tract (OFT) transiently links the ventricles to the aortic sac and forms the arterial valves. Abnormalities in these valves, such as bicuspid aortic valve (BAV), are common congenital anomalies. GATA6-inactivating variants cause cardiac OFT defects and BAV, but their mechanisms are unclear. We generated Gata6STOP/+ mice using CRISPR-Cas9, which show highly penetrant BAV (70%) and membranous ventricular septal defects (43%). These mice exhibited decreased proliferation and increased ISL1-positive progenitor cells in the OFT, indicating abnormal cardiovascular differentiation. Gata6 deletion with the Mef2cCre driver line recapitulated Gata6STOP/+ phenotypes, indicating a cell-autonomous role for Gata6 in the second heart field. Gata6STOP/+ mice showed reduced OFT length and caliber, associated with deficient cardiac neural crest cell contribution, which may cause valvulo-septal defects. RNA-sequencing analysis showed depletion in pathways related to cell proliferation and migration, highlighting Cxcr7 (also known as Ackr3) as a candidate gene. Reduced mesenchymal cell migration and invasion were observed in Gata6STOP/+ OFT tissue. CXCR7 agonists reduced mesenchymal cell migration and increased invasion in wild-type but not in Gata6STOP/+ explants, indicating the GATA6-dependent role of CXCR7 in OFT development and its potential link to BAV.

Heterogeneous expression of the atypical chemokine receptor ACKR3 in glioblastoma patient-derived tissue samples and cell cultures.

Glioblastoma (GBM) is the most aggressive glial tumor of the adult brain, associated with invariably fatal outcome, and a deeper understanding of the underlying malignant mechanisms is necessary to address the current therapeutic failure. We previously demonstrated the role of the CXCL12/CXCR4 axis in GBM cell migration and resistance to ionizing radiation. The atypical chemokine receptor ACKR3, responsible for CXCL12 scavenging, was previously suggested as additional important player in the context of GBM. Following validation of the detection tools, we observed that ACKR3 is expressed within GBM patient tumor tissue, distributed in diverse cell types. In contrast to CXCR4, ACKR3 expression in patient-derived stem-like cells (GSCs) remains however low, while ACKR3 gene expression by tumor cells appears to be modulated by the in-vivo environment. Using overexpression models, we also showed that in vitro ACKR3 had no significant direct effect on cell proliferation or invasion. Altogether, these results suggest that in vitro ACKR3 plays a minor role in malignant GBM cell biology and that its expression is possibly regulated by in-vivo influences. The subtle and multifaceted functions ACKR3 could exert in GBM should therefore only be tackled within a comprehensive tumor microenvironment considering tumoral but also non-tumoral cells.

The SDF-1/CXCR4 axis is involved in adipose-derived stem cell migration.

BACKGROUND: Intravenous injection of adipose-derived stem cells (ADSCs) can improve the urinary function of stress urinary incontinence (SUI) model rats and C-X-C chemokine receptor type 4 (CXCR4)-positive ADSCs are found in urethral tissues. The CXCR4 ligand stromal cell-derived factor-1 (SDF-1) is highly expressed in urinary incontinence model rats. In this study, we investigated the involvement of the SDF-1/CXCR4 axis in the homing of ADSCs. METHODS: ADSCs were isolated from rats and purified. The levels of CXCR4 and CXCR7 were determined by western blot analysis and immunofluorescence assays following stimulation with SDF-1. Hypoxia conditioning was performed to treat the cells in vitro, following which the messenger RNA (mRNA) and protein level of SDF-1, CXCR4, and CXCR7 were estimated. RESULTS: We found that CXCR4 and CXCR7 were expressed in ADSCs at passage zero (P0), P1, and P3, and the expression of both increased after SDF-1 stimulation. The level of expression of the mRNAs and proteins of SDF-1, CXCR4, and CXCR7 in ADSCs was higher after hypoxic conditioning. We then knocked down CXCR4 or CXCR7 using small interfering RNAs and found that the mRNA levels of CXCR4 and CXCR7 were considerably downregulated in the si-CXCR4/7-transfected cells. We also found that the SDF-1/CXCR4 axis was required for the migration of ADSCs. The phosphorylation levels of Janus kinase (JAK), protein kinase B (AKT), and extracellular regulated protein kinase significantly increased in SDF-1-stimulated ADSCs. However, the migration of ADSCs was suppressed when the corresponding specific inhibitors were used to block JAK and AKT signaling or silence CXCR4, whereas no significant change was observed in the migratory ability of ADSCs when the ERK pathway was blocked or CXCR7 was silenced. CONCLUSIONS: The SDF-1/CXCR4 axis is involved in the migration of ADSCs and may play a role in the migrate of ADSCs in SUI.

Conformational dynamics underlying atypical chemokine receptor 3 activation.

Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon ß-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of ß-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.

Crosstalk between CXCL12/CXCR4/ACKR3 and the STAT3 Pathway.

The reciprocal modulation between the CXCL12/CXCR4/ACKR3 axis and the STAT3 signaling pathway plays a crucial role in the progression of various diseases and neoplasms. Activation of the CXCL12/CXCR4/ACKR3 axis triggers the STAT3 pathway through multiple mechanisms, while the STAT3 pathway also regulates the expression of CXCL12. This review offers a thorough and systematic analysis of the reciprocal regulatory mechanisms between the CXCL12/CXCR4/ACKR3 signaling axis and the STAT3 signaling pathway in the context of diseases, particularly tumors. It explores the potential clinical applications in tumor treatment, highlighting possible therapeutic targets and novel strategies for targeted tumor therapy.

CXCR7 activation evokes the anti-PD-L1 antibody against glioblastoma by remodeling CXCL12-mediated immunity.

The interaction between glioblastoma cells and glioblastoma-associated macrophages (GAMs) influences the immunosuppressive tumor microenvironment, leading to ineffective immunotherapies. We hypothesized that disrupting the communication between tumors and macrophages would enhance the efficacy of immunotherapies. Transcriptomic analysis of recurrent glioblastoma specimens indicated an enhanced neuroinflammatory pathway, with CXCL12 emerging as the top-ranked gene in secretory molecules. Single-cell transcriptome profiling of naïve glioblastoma specimens revealed CXCL12 expression in tumor and myeloid clusters. An analysis of public glioblastoma datasets has confirmed the association of CXCL12 with disease and PD-L1 expression. In vitro studies have demonstrated that exogenous CXCL12 induces pro-tumorigenic characteristics in macrophage-like cells and upregulated PD-L1 expression through NF-κB signaling. We identified CXCR7, an atypical receptor for CXCL12 predominantly present in tumor cells, as a negative regulator of CXCL12 expression by interfering with extracellular signal-regulated kinase activation. CXCR7 knockdown in a glioblastoma mouse model resulted in worse survival outcomes, increased PD-L1 expression in GAMs, and reduced CD8+ T-cell infiltration compared with the control group. Ex vivo T-cell experiments demonstrated enhanced cytotoxicity against tumor cells with a selective CXCR7 agonist, VUF11207, reversing GAM-induced immunosuppression in a glioblastoma cell-macrophage-T-cell co-culture system. Notably, VUF11207 prolonged survival and potentiated the anti-tumor effect of the anti-PD-L1 antibody in glioblastoma-bearing mice. This effect was mitigated by an anti-CD8ß antibody, indicating the synergistic effect of VUF11207. In conclusion, CXCL12 conferred immunosuppression mediated by pro-tumorigenic and PD-L1-expressing GAMs in glioblastoma. Targeted activation of glioblastoma-derived CXCR7 inhibits CXCL12, thereby eliciting anti-tumor immunity and enhancing the efficacy of anti-PD-L1 antibodies.

Multiplex Detection of Fluorescent Chemokine Binding to CXC Chemokine Receptors by NanoBRET.

NanoLuc-mediated bioluminescence resonance energy transfer (NanoBRET) has gained popularity for its ability to homogenously measure ligand binding to G protein-coupled receptors (GPCRs), including the subfamily of chemokine receptors. These receptors, such as ACKR3, CXCR4, CXCR3, play a crucial role in the regulation of the immune system, are associated with inflammatory diseases and cancer, and are seen as promising drug targets. The aim of this study was to optimize NanoBRET-based ligand binding to NLuc-ACKR3 and NLuc-CXCR4 using different fluorescently labeled chemokine CXCL12 analogs and their use in a multiplex NanoBRET binding assay of two chemokine receptors at the same time. The four fluorescent CXCL12 analogs (CXCL12-AZD488, -AZD546, -AZD594, -AZD647) showed high-affinity saturable binding to both NLuc-ACKR3 and NLuc-CXCR4, with relatively low levels of non-specific binding. Additionally, the binding of all AZDye-labeled CXCL12s to Nluc receptors was inhibited by pharmacologically relevant unlabeled chemokines and small molecules. The NanoBRET binding assay for CXCL10-AZD488 binding to Nluc-CXCR3 was also successfully established and successfully employed for the simultaneous measurement of the binding of unlabeled small molecules to NLuc-CXCR3 and NLuc-CXCR4. In conclusion, multiplexing the NanoBRET-based competition binding assay is a promising tool for testing unlabeled (small) molecules against multiple GPCRs simultaneously.

Structural basis for selectivity and antagonism in extracellular GPCR-nanobodies.

G protein-coupled receptors (GPCRs) are pivotal therapeutic targets, but their complex structure poses challenges for effective drug design. Nanobodies, or single-domain antibodies, have emerged as a promising therapeutic strategy to target GPCRs, offering advantages over traditional small molecules and antibodies. However, an incomplete understanding of the structural features enabling GPCR-nanobody interactions has limited their development. In this study, we investigate VUN701, a nanobody antagonist targeting the atypical chemokine receptor 3 (ACKR3). We determine that an extended CDR3 loop is required for ACKR3 binding. Uncommon in most nanobodies, an extended CDR3 is prevalent in GPCR-targeting nanobodies. Combining experimental and computational approaches, we map an inhibitory ACKR3-VUN701 interface and define a distinct conformational mechanism for GPCR inactivation. Our results provide insights into class A GPCR-nanobody selectivity and suggest a strategy for the development of these new therapeutic tools.

Protective Effect of CXCR7 Against Hypoxia/Reoxygenation Injury in Renal Tubular Epithelial Cells.

Acute kidney injury (AKI) is a multifactorial syndrome with complex pathophysiology and prognosis. Ischaemia‒reperfusion injury (IRI) is a major cause of induced AKI. The aim of this study was to investigate the effect of upregulated CXCR7 expression on renal tubular epithelial cell apoptosis induced by hypoxia/reoxygenation (H/R). HK-2 cells were divided into three groups: control group (pcDNA3.1), hypoxia/reoxygenation + pcDNA3.1 group (H/R+pcDNA3.1) and CXCR7 overexpression + hypoxia/reoxygenation group (H/R+ Flag-CXCR7). Protein levels of renal tubular epithelial cell injury-, apoptosis- and autophagy-related markers were assessed by qRT‒PCR, Western blotting, flow cytometry (FCM), immunofluorescence and transmission electron microscopy (TEM). In addition, HK-2 cells were treated with the autophagy inhibitor 3-MA and divided into 3 groups: control group, 3-MA + pcDNA3.1 group, and 3-MA + Flag-CXCR7 group. Changes in autophagy and apoptosis in renal tubule epithelial cells were assessed by Western blotting and FCM. Compared with those in the control group, the protein and mRNA expression levels of CXCR7 in HK-2 cells were significantly lower under H/R conditions. Under H/R conditions, CXCR7 overexpression in HK-2 cells significantly downregulated the expression of NGAL. Moreover, CXCR7 overexpression significantly decreased H/R-induced cleaved PARP-1 and cleaved Caspase 3 levels, increased the level of the antiapoptotic protein BCL-2 and the autophagy-related molecules ATG5 and LC3B II, and significantly inhibited the expression of P62. Autophagy flow and TEM also showed that CXCR7 significantly promoted autophagy. CXCR7 significantly alleviated the 3-MA-induced inhibition of autophagy and increase in apoptosis. Upregulated CXCR7 expression can inhibit renal tubular epithelial cell apoptosis and damage by regulating autophagy. In conclusion, CXCR7 is a promising target for the prevention and/or treatment of AKI.

Bilayer lipids modulate ligand binding to atypical chemokine receptor 3.

Chemokine receptors belong to the large class of G protein-coupled receptors (GPCRs) and are involved in a number of (patho)physiological processes. Previous studies highlighted the importance of membrane lipids for modulating GPCR structure and function. However, the underlying mechanisms of how lipids regulate GPCRs are often poorly understood. Here, we report that anionic lipid bilayers increase the binding affinity of the chemokine CXCL12 for the atypical chemokine receptor 3 (ACKR3) by modulating the CXCL12 binding kinetics. Notably, the anionic bilayer favors CXCL12 over the more positively charged chemokine CXCL11, which we explained by bilayer interactions orienting CXCL12 but not CXCL11 for productive ACKR3 binding. Furthermore, our data suggest a stabilization of active ACKR3 conformations in anionic bilayers. Taken together, the described regulation of chemokine selectivity of ACKR3 by the lipid bilayer proposes an extended version of the classical model of chemokine binding including the lipid environment of the receptor.

Disruption of placental ACKR3 impairs growth and hematopoietic development of offspring.

ACKR3 scavenges and degrades the stem cell recruiting chemokine CXCL12, which is essential for proper embryonic and, in particular, haematopoietic development. Here, we demonstrate strong expression of ACKR3 on trophoblasts. Using a maternally administered pharmacological blocker and Cre-mediated genetic approaches, we demonstrate that trophoblast ACKR3 is essential for preventing movement of CXCL12 from the mother to the embryo, with elevated plasma CXCL12 levels being detected in embryos from ACKR3-blocker-treated mothers. Mice born to mothers treated with the blocker are lighter and shorter than those born to vehicle-treated mothers and, in addition, display profound anaemia associated with a markedly reduced bone marrow haematopoietic stem cell population. Importantly, although the haematopoietic abnormalities are corrected as mice age, our studies reveal a postnatal window during which offspring of ACKR3-blocker-treated mice are unable to mount effective inflammatory responses to inflammatory/infectious stimuli. Overall, these data demonstrate that ACKR3 is essential for preventing CXCL12 transfer from mother to embryo and for ensuring properly regulated CXCL12 control over the development of the haematopoietic system.

CXC chemokine receptor 7 ameliorates renal fibrosis by inhibiting ß-catenin signaling and epithelial-to-mesenchymal transition in tubular epithelial cells.

Renal fibrosis is a common feature of various chronic kidney diseases. However, the underlying mechanism remains poorly understood. The CXC chemokine receptor (CXCR) family plays a role in renal fibrosis; however, the detailed mechanisms have not been elucidated. In this study, we investigated the potential role of CXCR7 in mediating renal fibrosis. CXCR7 expression is decreased in unilateral ischemia-reperfusion injury (UIRI) and unilateral ureteral obstruction mouse models. Furthermore, CXCR7 was specifically expressed primarily in the Lotus Tetragonolobus Lectin-expressing segment of tubules, was slightly expressed in the peanut agglutinin-expressing segment, and was barely expressed in the Dolichos biflorus agglutinin-expressing segment. Administration of pFlag-CXCR7, an overexpression plasmid for CXCR7, significantly inhibited the activation of ß-catenin signaling and protected against the progression of epithelial-to-mesenchymal transition (EMT) and renal fibrosis in a UIRI mouse model. Using cultured HKC-8 cells, we found that CXCR7 significantly downregulated the expression of active ß-catenin and fibrosis-related markers, including fibronectin, Collagen I, and α-SMA. Furthermore, CXCR7 significantly attenuated TGF-ß1-induced changes in ß-catenin signaling, EMT and fibrosis. These results suggest that CXCR7 plays a crucial role in inhibiting the activation of ß-catenin signaling and the progression of EMT and renal fibrosis. Thus, CXCR7 could be a novel therapeutic target for renal fibrosis.

G protein­mediated EGFR transactivation is a common mechanism through which the CXCL12 receptors, CXCR4 and CXCR7, control human cancer cell migration.

C­X­C motif chemokine 12 (CXCL12) promotes metastasis of several tumors by affecting cell migration and invasion via its receptors, C­X­C chemokine receptor type (CXCR)4 and CXCR7. Current therapeutic approaches focus on the selective inactivation of either CXCR4 or CXCR7 in patients with cancer. Alternative strategies may emerge from the analysis of downstream events that mediate the migratory effects of CXCL12 in cancer cells. While CXCR4 activates cell signaling through both G proteins and arrestins, CXCR7 is believed to preferentially signal through arrestins. The present study analyzed the CXCL12­dependent chemotaxis of A549, C33A, DLD­1, MDA­MB­231 and PC­3 cells, in which either the activity of G proteins, EGFR or Src kinase was inhibited pharmacologically or the expression of arrestins was inhibited by RNA interference. The results demonstrated that CXCL12­induced migration of A549, C33A, DLD­1, MDA­MB­231 and PC­3 cells was attenuated by the Gαi/o­inhibitor pertussis toxin (PTX), but was unaffected by small interfering RNA­mediated gene silencing of ß­arrestin1/2. In particular, the sensitivity of DLD­1 migration to PTX was unexpected, as it is solely dependent on the non­classical chemokine receptor, CXCR7. Furthermore, chemotactic responses to CXCL12 were additionally prevented by inhibiting EGFR activity via AG1478 and Src kinase activity via Src inhibitor­1. In conclusion, the results of the present study suggest that G protein­ and Src­dependent transactivation of EGFR is a common mechanism through which CXCL12­bound CXCR4 and/or CXCR7 control cancer cell migration and metastasis. These findings highlight EGFR as a potential therapeutic target that interferes with CXCL12­induced cancer expansion.

Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system.

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust ßarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced ßarr-coupling compared to CXCR7. These two receptors induce distinct ßarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for ßarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery.

G protein activation via chemokine (C-X-C motif) receptor 4 and α1b -adrenoceptor is ligand and heteromer-dependent.

It is unknown whether heteromerization between chemokine (C-X-C motif) receptor 4 (CXCR4), atypical chemokine receptor 3 (ACKR3) and α1b -adrenoceptor (α1b -AR) influences effects of the CXCR4/ACKR3 agonist chemokine (C-X-C motif) ligand 12 (CXCL12) and the noncognate CXCR4 agonist ubiquitin on agonist-promoted G protein activation. We provide biophysical evidence that both ligands stimulate CXCR4-mediated Gαi activation. Unlike CXCL12, ubiquitin fails to recruit ß-arrestin. Both ligands differentially modulate the conformation of CXCR4:ACKR3 heterodimers and its propensity to hetero-trimerize with α1b -AR. CXCR4:ACKR3 heterodimerization reduces the potency of CXCL12, but not of ubiquitin, to activate Gαi. Ubiquitin enhances phenylephrine-stimulated α1b -AR-promoted Gαq activation from hetero-oligomers comprising CXCR4. CXCL12 enhances phenylephrine-stimulated α1b -AR-promoted Gαq activation from CXCR4:α1b -AR heterodimers and reduces phenylephrine-stimulated α1b -AR-promoted Gαq activation from ACKR3 comprising heterodimers and trimers. Our findings suggest heteromer and ligand-dependent functions of the receptor partners.

[Correlation between macrophage chemotaxis and disease severity in patients with knee osteoarthritis].

OBJECTIVE: To investigate the enhancement of macrophage chemotaxis in patients with knee osteoarthritis (KOA) and its correlation with the disease severity. METHODS: Eighty patients with KOA admitted from July 2019 to June 2022 were enrolled as the observation group and divided into 29 cases of moderate group, 30 cases of severe group and 21 cases of extremely severe group. At the same time, 30 healthy subjects were included as the control group. The gene expressions of NF-κB, CXC chemokine receptor 7 (CXCR7) and CXC chemokine ligand 12 (CXCL12) in macrophages of each group were analyzed. Visual analogue scale(VAS) was used to evaluate the degree of joint pain. Joint function was evaluated by knee Joint Society Scoring system(KSS). Finally, data analysis was carried out. RESULTS: The expression levels of NF-κB, CXCR7 and CXCL12 in moderate group, severe group and extreme recombination group were higher than those in control group. The VAS, the expression of NF-κB, CXCR7 and CXCL12 in the severe group and the extreme recombination group were higher than those in the moderate group, whereas KSS was lower than that in the moderate group. The VAS, expression levels of NF-κB, CXCR7 and CXCL12 in the extremely severe group were higher than those in the severe group, and KSS was lower than that in the severe group (all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with VAS score, but negatively correlated with KSS(all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with the severity of disease. After excluding the influence of traditional factors (gender, age and disease duration), multiple linear regression analysis further showed that the expression levels of NF-κB, CXCR7 and CXCL12 were still positively correlated with the severity of disease(all P<0.01). CONCLUSION: The chemotaxis of macrophages in patients with KOA increased with the aggravation of the disease, and was related to the degree of pain and function impairment.

Chemokine receptor CXCR7 activates Aurora Kinase A and promotes neuroendocrine prostate cancer growth.

CXCR7 is an atypical chemokine receptor that recruits ß-arrestin (ARRB2) and internalizes into clathrin-coated intracellular vesicles where the complex acts as a scaffold for cytoplasmic kinase assembly and signal transduction. Here, we report that CXCR7 was elevated in the majority of prostate cancer (PCa) cases with neuroendocrine features (NEPC). CXCR7 markedly induced mitotic spindle and cell cycle gene expression. Mechanistically, we identified Aurora Kinase A (AURKA), a key regulator of mitosis, as a novel target that was bound and activated by the CXCR7-ARRB2 complex. CXCR7 interacted with proteins associated with microtubules and golgi, and, as such, the CXCR7-ARRB2-containing vesicles trafficked along the microtubules to the pericentrosomal golgi apparatus, where the complex interacted with AURKA. Accordingly, CXCR7 promoted PCa cell proliferation and tumor growth, which was mitigated by AURKA inhibition. In summary, our study reveals a critical role of CXCR7-ARRB2 in interacting and activating AURKA, which can be targeted by AURKA inhibitors to benefit a subset of patients with NEPC.

Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells.

Atypical chemokine receptor 3 (ACKR3) is a scavenger of the chemokines CXCL11 and CXCL12 and of several opioid peptides. Additional evidence indicates that ACKR3 binds two other non-chemokine ligands, namely the peptide hormone adrenomedullin (AM) and derivatives of the proadrenomedullin N-terminal 20 peptide (PAMP). AM exhibits multiple functions in the cardiovascular system and is essential for embryonic lymphangiogenesis in mice. Interestingly, AM-overexpressing and ACKR3-deficient mouse embryos both display lymphatic hyperplasia. Moreover, in vitro evidence suggested that lymphatic endothelial cells (LECs), which express ACKR3, scavenge AM and thereby reduce AM-induced lymphangiogenic responses. Together, these observations have led to the conclusion that ACKR3-mediated AM scavenging by LECs serves to prevent overshooting AM-induced lymphangiogenesis and lymphatic hyperplasia. Here, we further investigated AM scavenging by ACKR3 in HEK293 cells and in human primary dermal LECs obtained from three different sources in vitro. LECs efficiently bound and scavenged fluorescent CXCL12 or a CXCL11/12 chimeric chemokine in an ACKR3-dependent manner. Conversely, addition of AM induced LEC proliferation but AM internalization was found to be independent of ACKR3. Similarly, ectopic expression of ACKR3 in HEK293 cells did not result in AM internalization, but the latter was avidly induced upon co-transfecting HEK293 cells with the canonical AM receptors, consisting of calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP)2 or RAMP3. Together, these findings indicate that ACKR3-dependent scavenging of AM by human LECs does not occur at ligand concentrations sufficient to trigger AM-induced responses mediated by canonical AM receptors.

Stem-like signatures in human meningioma cells are under the control of CXCL11/CXCL12 chemokine activity.

BACKGROUND: Meningiomas are mainly benign brain tumors, although about 20% of histologically benign cases are clinically aggressive and recur after resection. We hypothesize that meningioma brain invasiveness and recurrence may be related to the presence of cancer stem cells and their high responsiveness to the CXCL12-CXCR4/CXCR7 chemokine axis. The aim of this study was to isolate meningioma stem cells from human samples, characterize them for biological features related to malignant behavior, and to identify the role of CXCR4/CXCR7 in these processes. METHODS: Meningioma stem cells were isolated from patient-derived primary cultures in stem cell-permissive conditions, and characterized for phenotype, self-renewal, proliferation and migration rates, vasculogenic mimicry (VM), and in vivo tumorigenesis, in comparison with differentiated meningioma cells and stem-like cells isolated from normal meninges. These cell populations were challenged with CXCL12 and CXCL11 and receptor antagonists to define the chemokine role in stem cell-related functions. RESULTS: Stem-like cells isolated from meningioma cultures display higher proliferation and migration rates, and VM, as compared to meningioma non-stem cells or cells isolated from normal meninges and were the only tumorigenic population in vivo. In meningioma cells, these stem-like functions were under the control of the CXCR4/CXCR7 chemokine axis. CONCLUSIONS: We report a role for CXCL11 and CXCL12 in the control of malignant features in stem-like cells isolated from human meningioma, providing a possible basis for the aggressive clinical behavior observed in subsets of these tumors. CXCR4/CXCR7 antagonists might represent a useful approach for meningioma at high risk of recurrence and malignant progression.

Exploring the CXCR4/CXCR7/CXCL12 Axis in Primary Desmoid Tumors.

BACKGROUND: Desmoid tumors have an extremely variable natural history. The uncertainty behind desmoid behavior reflects the complexity, which subtends its development and non-linear advancement. Apart from Wnt- ßcatenin mutation, estrogen receptors, and COX-2 overexpression, little is known about the ability of desmoids to grow and recur while being unable to metastasize. Several tumors have been shown to express the CXCR4/CXCR7/CXCL12 axis, whose functions are essential for tumoral development. AIMS: This study aimed to investigate the expression of the CXCR4/CXCR7/CXCL12 axis in primary desmoid tumors and discuss the potential role of this key-signaling as an antiangiogenic therapeutic strategy. METHODS: In this study, 3 µm-thick consecutive sections from each formalin-fixed and paraffin-embedded tissue block were treated with mouse monoclonal antibodies developed against CD34, CXCR4, CXCR7, and CXCL12. RESULTS: Two distinct vessel populations: CXCR4+ and CXCR4- vessels, have been found. Similarly, chemokine receptor CXCR7 expression in the entire desmoid tumor series positively stained a portion of tumor-associated vessels, identifying two distinct subpopulations of vessels: CXCR7+ and CXCR7- vessels. All 8 neoplastic tissue samples expressed CXCL12. Immunohistochemical positivity was identified in both stromal and endothelial vascular cells. Compared to CXCR4 and CXCR7, the vast majority of tumor-associated vessels were found to express this chemokine. CONCLUSION: It is the first time, as per our knowledge, that CXCR4/CXCR7/CXCL12 axis expression has been identified in a desmoid type-fibromatosis series. CXCL12 expression by neoplastic cells, together with CXCR4 and CXCR7 expression by a subgroup of tumor-associated vessels, was detected in all desmoid tumor tissue samples examined. Since chemokines are known contributors to neovascularization, CXCR4/CXCR7/CXCL12 axis may play a role in angiogenesis in this soft-tissue tumor histotype, thereby supporting its growth.