Cell Surface Expression Of CXCR4 Research Articles

CXCR4 Enriched T Regulatory Cells Preferentially Home to Bone Marrow and Decrease Inflammation

Background and aim Adoptive therapy with T regulatory cells (Tregs) is emerging as an important therapeutic strategy for several autoimmune and inflammatory diseases. Optimal homing and payload delivery of these anti-inflammatory cells remain a cornerstone of optimizing their therapeutic efficacy. CXCR4 cell surface expression has been shown to be critical for homing of hematopoietic cells to the bone marrow (BM). Also, CXCR4/CXCL12 axis plays a major role in the pathogenesis of BM inflammatory disorders including myelofibrosis. We hypothesize that enrichment of CXCR4 on the Tregs cell surface will enable their trafficking to BM and ensure a homogenous delivery. Methods/results/conclusion Magnetic enrichment of CD25 + cells was performed on umbilical cord blood on day 0, followed by dual enrichment on CXCR4 using CRANE TM (cord blood Tregs, activated and enriched) process on day 3 or 4 (Fig 1A). The enrichment process led to increased number of Tregs expressing CXCR4 on their cell surface (Fig 1B; Treg CXCR4) as well as a CXCR4 hi Treg cells when compared to Treg control (Fig 1C, p<0.0001; n=16). In a Transwell TM system, a higher number of Treg CXCR4 migrated to the bottom well in chemotactic response to SDF1α at 15 minutes as compared to the Treg control cells. Such migration was inhibited by CXCR4 antagonist, AMD3100 (Fig 1D). The migration superiority of Treg CXCR4 was maintained at 30 and 60 min (Fig 1E). In order to understand in vivo transit kinetics, Treg cells were injected into NSG mice and organs were harvested at 12 and 24 hours (Fig 1F). Treg CXCR4 showed preferential homing to BM (Fig 1G), where a higher expression of CXCR4 and CXCL12 than control, was observed (Fig 1H). Other markers including CD62L (homing and suppression), CD39 & CD73 (conversion of ATP to immunosuppressive adenosine), and CXCR5 (Follicular Treg) were also increased in Treg CXCR4 recipients. A decrease in biomarkers including TGFα and TNFβ (inflammation), IL-13 (fibrosis) (Fig 1I) with an increase in GM-CSF (increase Treg potency) (Fig 1J), was observed in Treg CXCR4 recipients with a corresponding decrease in TGFβ family (hematopoietic suppressor) (Fig 1K). We conclude that Treg CXCR4 shows superior homing to BM and is currently being studied in a clinical setting to treat myelofibrosis with suboptimal response to ruxolitinib (NCT05423691).

PIM3 kinase promotes tumor metastasis in hepatoblastoma by upregulating cell surface expression of chemokine receptor cxcr4.

Patients presenting with metastatic hepatoblastoma have limited treatment options and survival rates as low as 25%. We previously demonstrated that Proviral Integration site in Maloney murine leukemia virus 3 (PIM3) kinase promotes tumorigenesis and cancer cell stemness in hepatoblastoma. In this study, we assessed the role of PIM3 kinase in promoting hepatoblastoma metastasis. We utilized a tail vein injection model of metastasis to evaluate the effect of CRISPR/Cas9-mediated PIM3 knockout, stable overexpression of PIM3, and pharmacologic PIM inhibition on the formation of lung metastasis. In vivo studies revealed PIM3 knockout impaired the formation of lung metastasis: 5 out of 6 mice injected with wild type hepatoblastoma cells developed lung metastasis while none of the 7 mice injected with PIM3 knockout hepatoblastoma cells developed lung metastasis. PIM3 overexpression in hepatoblastoma increased the pulmonary metastatic burden in mice and mechanistically, upregulated the phosphorylation and cell surface expression of CXCR4, a key receptor in the progression of cancer cell metastasis. CXCR4 blockade with AMD3100 decreased the metastatic phenotype of PIM3 overexpressing cells, indicating that CXCR4 contributed to PIM3's promotion of hepatoblastoma metastasis. Clinically, PIM3 expression correlated positively with CXCR4 expression in primary hepatoblastoma tissues. In conclusion, we have shown PIM3 kinase promotes the metastatic phenotype of hepatoblastoma cells through upregulation of CXCR4 cell surface expression and these findings suggest that targeting PIM3 kinase may provide a novel therapeutic strategy for metastatic hepatoblastoma.

Inhibition of Pim Kinase Supports the Neural Stem Cell Phenotype in Neuroblastoma

INTRODUCTION: Relapse rates for high-risk neuroblastoma exceed 50%. Expression of the serine-threonine protein kinase, PIM, is associated with cancer progression and is negatively correlated with neural stemness markers in neural progenitor cells. Because promoting cellular differentiation is a component of neuroblastoma therapy, and PIM kinase negatively affects neural stemness, we aimed to determine whether PIM affected the neural stem cell phenotype in neuroblastoma. METHODS: We queried the Kocak (R2) gene database to compare PIM1 and the neural stemness marker, Nanog, in neuroblastoma. For in vitro studies, SK-N-BE(2), human neuroblastoma cells and human neuroblastoma patient–derived xenograft (PDX06) cells were treated with siRNA to achieve PIM1 kinase knockdown or with AZD1208 for PIM inhibition. Abundance of mRNA for the neural stemness marker, Nanog, was detected with quantitative reverse transcription polymerase chain reaction, and protein expression was confirmed with immunoblotting. Flow cytometry detected cell surface expression of CD133 and CXCR4, 2 additional neural stemness markers. Data were reported as mean ± SEM, with p ≤ 0.05 considered significant. RESULTS: R2 database revealed a negative correlation between PIM1 and Nanog (Fig. 1A). Protein expression of Nanog was increased following siRNA knockdown of PIM1 (Fig. 1B). AZD1208 increased the abundance of Nanog mRNA and resultant protein expression (Fig. 1C). AZD1208 treatment significantly increased cell surface expression of CD133 and CXCR4 (Fig. 1D).Figure 1CONCLUSION: Neuroblastoma cells treated with PIM inhibition dedifferentiated to a more neuronal stem cell phenotype as demonstrated by increased expression of neuronal stemness markers. These findings support further studies exploring the clinical implications of PIM kinase inhibition as an adjunct to neuroblastoma therapy.

MIF/CXCR4 signaling axis contributes to survival, invasion, and drug resistance of metastatic neuroblastoma cells in the bone marrow microenvironment

BackgroundThe bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets.MethodsPatient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O2) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo.ResultsWe identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model.ConclusionsOur findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB.

Cell surface expression of GRP78 and CXCR4 is associated with childhood high-risk acute lymphoblastic leukemia at diagnostics

Acute lymphocytic leukemia is the most common type of cancer in pediatric individuals. Glucose regulated protein (GRP78) is an endoplasmic reticulum chaperone that facilitates the folding and assembly of proteins and regulates the unfolded protein response pathway. GRP78 has a role in survival of cancer and metastasis and cell-surface associated GRP78 (sGRP78) is expressed on cancer cells but not in normal cells. Here, we explored the presence of sGRP78 in pediatric B-ALL at diagnosis and investigated the correlation with bona fide markers of leukemia. By using a combination of flow cytometry and high multidimensional analysis, we found a distinctive cluster containing high levels of sGRP78, CD10, CD19, and CXCR4 in bone marrow samples obtained from High-risk leukemia patients, which was absent in the compartment of Standard-risk leukemia. We confirmed that sGRP78+CXCR4+ blood-derived cells were more frequent in High-risk leukemia patients. Finally, we analyzed the dissemination capacity of sGRP78 leukemia cells in a model of xenotransplantation. sGRP78+ cells emigrated to the bone marrow and lymph nodes, maintaining the expression of CXCR4. Testing the presence of sGRP78 and CXCR4 together with conventional markers may help to achieve a better categorization of High and Standard-risk pediatric leukemia at diagnosis.

CXCR4 Mediates Enhanced Cell Migration in CALM-AF10 Leukemia.

Leukemia transformed by the CALM-AF10 chromosomal translocation is characterized by a high incidence of extramedullary disease, central nervous system (CNS) relapse, and a poor prognosis. Invasion of the extramedullary compartment and CNS requires leukemia cell migration out of the marrow and adherence to the cells of the local tissue. Cell adhesion and migration are increasingly recognized as contributors to leukemia development and therapeutic response. These processes are mediated by a variety of cytokines, chemokines, and their receptors, forming networks of both secreted and cell surface factors. The cytokines and cytokine receptors that play key roles in CALM-AF10 driven leukemia are unknown. We find high cell surface expression of the cytokine receptor CXCR4 on leukemia cells expressing the CALM-AF10 oncogenic protein, contributing to the migratory nature of this leukemia. Our discovery of altered cytokine receptor expression and function provides valuable insight into the propagation and persistence of CALM-AF10 driven leukemia.

DC-SIGN binding to mannosylated B-cell receptors in follicular lymphoma down-modulates receptor signaling capacity

In follicular lymphoma (FL), surface immunoglobulin (sIg) carries mandatory N-glycosylation sites in the variable regions, inserted during somatic hypermutation. These glycosylation sites are tumor-specific, indicating a critical function in FL. Added glycan unexpectedly terminates at high mannose (Mann) and confers capability for sIg-mediated interaction with local macrophage-expressed DC-SIGN lectin resulting in low-level activation of upstream B-cell receptor signaling responses. Here we show that despite being of low-level, DC-SIGN induces a similar downstream transcriptional response to anti-IgM in primary FL cells, characterized by activation of pathways associated with B-cell survival, proliferation and cell–cell communication. Lectin binding was also able to engage post-transcriptional receptor cross-talk pathways since, like anti-IgM, DC-SIGN down-modulated cell surface expression of CXCR4. Importantly, pre-exposure of a FL-derived cell line expressing sIgM-Mann or primary FL cells to DC-SIGN, which does not block anti-IgM binding, reversibly paralyzed the subsequent Ca2+ response to anti-IgM. These novel findings indicate that modulation of sIg function occurs in FL via lectin binding to acquired mannoses. The B-cell receptor alternative engagement described here provides two advantages to lymphoma cells: (i) activation of signaling, which, albeit of low-level, is sufficient to trigger canonical lymphoma-promoting responses, and (ii) protection from exogenous antigen by paralyzing anti-IgM-induced signaling. Blockade of this alternative engagement could offer a new therapeutic strategy.

Combined Blockage of E-Selectin and CXCR4 (GMI-1359) Enhances Anti-Leukemia Effect of FLT3 Inhibition (Sorafenib) and Protects Hematopoiesis in Pre-Clinical AML Models

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Nakao et al., Leukemia 1996). FLT3 inhibitors like sorafenib efficiently eliminate circulating leukemia blasts, but frequently not in the bone marrow (BM), which suggests a protective effect of the BM niche for leukemic stem cell survival (Zhang et al., JNCI 2008). The homing of AML cells in BM is mediated chiefly by the adhesion to E-selectin on endothelial cells (ECs) and by CXCR4-directed cellular migration to stromal CXCL12 (SDF1) sources (Chien et al., Blood 2013; Peled and Tavor, Theranostics 2013). In many respects, BM homing signals are shared between leukemia and hematopoietic stem cells (HSCs). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and mesenchymal stem cells, reduced the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cellularity in the BM in vivo (Zhang et al., Can Res suppl 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in mice with FLT3-mutated leukemia (Zhang et al., Blood suppl 2015). In the present study, we sought to evaluate dual E-selectin/CXCR4 blockage in the context of FLT3 inhibition by sorafenib in vivo, and to better understand the underlying mechanism. We compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. Resistant cells expressed 1.7 to ~5.6-fold higher levels of total E-selectin ligand detected by a soluble E-selectin reagent, and 10-fold higher levels of CXCR4. In addition, BM-mimetic hypoxia culture profoundly upregulated the cell surface expression of E-selectin ligands and CXCR4 on leukemia cells. We evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft (PDX) model harboring FLT3-ITD and WT1 mutations. We observed that addition of GMI-1359 to sorafenib greatly reduced leukemia cellularity compared to sorafenib alone, and as much as by 92%, 82%, 69% and 45% in, respectively, liver, lung, spleen and BM (Fig. 1) as compared with vehicle-treated mice (p < 0.05). As expected, the number of circulating leukemia cells transiently increased. The GMI-1359/sorafenib combination improved mouse survival (median survival 138.5 versus 109, 87 and 126 days for the GMI-1359/sorafenib versus vehicle, GMI-1359 and sorafenib, respectively, p < 0.001). Using intravital 2-photon microscopy, we observed AML cell behavior in calvarial BM and their response to acute GMI-1359 bolus infusion. Remarkably, AML cell mobility began to increase in the BM microenvironment as soon as 20 min after treatment (Fig. 2), followed by intravasation and cellular outflow through the BM capillary vasculature over the next 2-4 hours. Moreover, although BM homing signals are thought to be shared between leukemia and HSCs, the combination therapy improved hematopoiesis parameters compared to sorafenib alone. In particular, this important effect was associated with increased numbers of megakaryocytes (2.1-fold), myelocytes (2.1-fold), and erythrocytes (7.1-fold) in BM (p < 0.01). The underlying mechanism(s) of hematopoiesis protection by GMI-1359 are under investigation. Conclusion: Co-inhibition of E-selectin/CXCR4 enhances the anti-leukemia efficacy of FLT3 inhibition and preserves hematopoiesis in the BM in a PDX model of AML. Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zal:Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

IPSC-derived endothelial cell response to hypoxia via SDF1a/CXCR4 axis facilitates incorporation to revascularize ischemic retina.

Ischemic retinopathies are major causes of blindness worldwide. Local hypoxia created by loss of vascular supply leads to tissue injury and aberrant neovascularization in the retina. There is a great need for therapies that enhance revascularization of hypoxic neuroretinal tissue. To test the therapeutic feasibility of human-induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) for the treatment of ischemic retinopathies, we compared the angiogenic potential of hiPSC-ECs with mature human retinal endothelial cells (HRECs) in response to hypoxia. hiPSC-ECs formed more robust and complex vascular networks in collagen gels, whereas HRECs displayed minimal sprouting. The cells were further tested in the mouse oxygen-induced retinopathy (OIR) model. Retinas with hiPSC-EC injection showed colocalization with host vessels, whereas HRECs lacked such responses. hiPSC-ECs markedly reduced vaso-obliteration and pathological neovascularization. This beneficial effect of hiPSC-ECs was explained by the stromal cell-derived factor-1a (SDF1a)/CXCR4 axis; hiPSC-ECs exhibited much higher cell-surface expression of CXCR4 than HRECs and greater chemotaxis toward SDF1a-embedded 3D collagen hydrogel. Furthermore, treatment with neutralizing antibody to CXCR4 abolished recruitment of hiPSCs in the OIR model. These findings suggest superior angiogenic potential of hiPSC-ECs under hypoxia and underscore the importance of SDF1a/CXCR4 in the reparative function of hiPSC-ECs in ischemic diseases.

Inhibition of Anti-Apoptotic Mcl-1 Exerts Anti-Leukemia Activity through Modulation of Leukemia-Stromal Interactions and Metabolic Functions in AML

Inhibition of Anti-Apoptotic Mcl-1 Exerts Anti-Leukemia Activity through Modulation of Leukemia-Stromal Interactions and Metabolic Functions in AML

SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.

Intrathymic Notch3 and CXCR4 combinatorial interplay facilitates T-cell leukemia propagation

Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying “pre-leukemic” cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4+CD8+ thymocytes, possibly contributing to “pre-leukemic” cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4+CD8+ cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4+CD8+ cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4+CD8+ cells and such an expression is regulated by Notch3 through β-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4+CD8+ cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4+CD8+ cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of “pre-leukemic” CD4+CD8+ cells, by a “thymus-autonomous” mechanism.

PO-393 Notch3 and CXCR4 cross-signalling sustains acute T-cell leukaemia progression

IntroductionAcute T-cell lymphoblastic leukaemia (T-ALL) is a childhood cancer, characterised by infiltration of immature T-cells in bone marrow. Notch hyperactivation is a major driver of T-ALL development where CXCL12/CXCR4 axis plays an important role in T-ALL maintenance. In thymus the lympho-stromal communication drives progressive maturation of T-cells. Notch receptors regulate T-cell fate choices, dominating early steps of thymocyte maturation. In T-cell differentiation, Notch3, in association with pre-TCR and chemochine receptor CXCR4, govern the transition from double negative (DN) to double positive (DP) thymocytes. Previously, our laboratory demonstrated the lymphomagenic potential of Notch3 by creating a transgenic mouse model (N3-ICtg), characterised by the constitutive activation of the intracellular domain (IC) of Notch3 receptor (N3-IC) in immature thymocytes. In order to investigate the oncogenic cross-talk between Notch3 and CXCR4 in T-ALL progression, we analysed DP T-cells in different lymphoid compartments of N3-ICtg mice.Material and methodsFreshly isolated cells from thymus, blood and bone marrow of N3-ICtg and WT mice were analysed by flow cytometry in order to verify the presence of DP T-cells and their cell-surface expression of CXCR4 and Notch3 receptors. Experiments in TALL1, a human T-ALL leukemic CD3+/CD4+/CD8+ cell line characterised by the activation of Notch3 and high expression of CXCR4, were also performed. TALL1 cells were treated with γ-secretase inhibitor (GSI) or their gene expression of Notch3 was silenced and then analysed by flow cytometry, RT-PCR and western blot. Statistical interpretation of the results was performed.Results and discussionsDP-gated thymocytes obtained by N3-ICtg mice have shown a high co-expression of Notch3 and CXCR4 and a high migratory ability induced by SDF-1. An anomalous percentage representation of these DP T-cells at different ages in circulating blood, spleen and bone morrow may suggest an interaction between CXCR4 and Notch3 in T-ALL cell propagation. Experiments in human TALL1 cell line with Notch3 targeted inhibition suggest a modulated expression of CXCR4 through a β-arrestin1-mediated mechanism. CXCR4-antagonists treatment will further elucidate the molecular crosstalk between the two receptors.ConclusionNotch3 abnormal pathway, through boosting the expression of CXCR4 on cell-surface, may play a role in DP T-cells egress from thymus, and define a possible mechanism of ’pre-leukemic-cells’ dissemination.

The MRPS18-2 protein levels correlate with prostate tumor progression and it induces CXCR4-dependent migration of cancer cells

We have earlier found abnormal expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2, S18-2) in endometrial cancer, compared to the expression in hyperplasia and in normal endometrium. Here we report that expression of S18-2 was increased with disease progression in clinical specimens of prostate cancer (PCa). The level of induction of epithelial to mesenchymal cell transition (EMT) correlated with the expression level of S18-2 in PCa cell lines. Moreover, cells acquired increased ability of migration upon S18-2 overexpression, as was evaluated in zebrafish embryo model and in trans-well assay. We found that this is due to increased CXCR4 cell surface expression. Neutralizing CXCR4 protein or abrogating S18-2 expression in cells significantly reduced their migratory ability directed toward CXCL12. The mRNA expression of TWIST2, encoding one of transcription factors that induce EMT upon CXCR4 increase, positively correlated with the S18-2 protein level. Together, these data suggest that the S18-2 protein induces EMT through the TWIST2/E-cadherin signalling and, consequently, CXCR4-mediated migration of PCa cells.

Evaluation of somatostatin, CXCR4 chemokine and endothelin A receptor expression in a large set of paragangliomas.

Paragangliomas are predominantly benign tumors, but in some cases invasive growth and also metastasis are observed. Given the limited number of nonsurgical treatment options, novel target structures for diagnostics and therapy of this tumor entity are urgently needed.In the present study, expression of all five somatostatin receptor (SST) subtypes, chemokine receptor CXCR4 and endothelin receptor type A (ETA) was assessed by means of immunohistochemistry in a total of 66 paraffin-embedded paraganglioma samples from 55 patients. The stainings were rated by means of the Immunoreactive Score and correlated to clinical data and to succinate dehydrogenase subunit B (SDHB) expression.SST2A was by far the most prominent receptor in the paragangliomas investigated. It was present in 89% of the tumors at a high intensity, followed by SST5, SST3, SST1 and SST4, which were detected in 47%, 35%, 35% and 13% of the samples, respectively. SDHB positive tumors exhibited significantly higher SST2A and SST3 expression as compared to SDHB negative cases. There was no correlation between SST and Ki-67 expression or grading of the tumors and no difference in SST expression between primary tumors and metastases. Cell surface expression of CXCR4 and ETA was detected only in few samples. On tumor capillaries, however, exceptionally strong staining for these two receptors was noticed in the vast majority of the tumors.In conclusion, paragangliomas are well suited for SST2A-based diagnostics and treatment modalities. An indirect targeting of these highly vascularized tumors via CXCR4 or ETA may also represent a promising future strategy.

Abstract 947: CXCR4 in combination with immune check point inhibition in ovarian cancer mouse model demonstrates potential for novel therapeutic strategies

Abstract Chemokine CXCL12 and its receptor CXCR4 are the most highly expressed chemokine axis in serous ovarian cancer. CXCR4 promotes tumor growth, angiogenesis, and metastasis. High CXCR4 expression is associated with poor survival, making it an attractive therapeutic target. The objective of this study was to establish an immune competent murine ovarian cancer model with high CXCR4 expression and to test single agent CXCR4 blockade and combination therapy to optimize emerging treatment strategies for future clinical trials. CXCR4 expression was evaluated by flow cytometry in ID8, ID8-VEGF, IE9-MP1, BR5, BR5-Akt, BR5-C2k, and BR5-Kras mouse ovarian cancer cell lines as well as IE9-MP1 tumor samples. Subsequently an exploratory study was undertaken utilizing the ID8 immune competent mouse model to assess the efficacy of AMD3100, the only commercially available CXCR4 inhibitor, alone and in combination with bevacizumab and anti-PD-1. Immune analysis of circulating lymphocytes by flow cytometry was performed during treatment to monitor changes of immune cell subsets. Surface expression of CXCR4 receptor was minimal in ID8 (1.6%), ID8-VEGF (0.9%), IE9-MP1 (1.8%), BR5 (0.9%), BR5-Akt (1.2%), BR5-C2k (2.3%), and BR5-Kras (1.1%), however intracellular staining revealed widespread CXCR4 expression in ID8 (99.6%), ID8-VEGF (99.7%) BR5 (96.7%), BR5-Akt (94.3%), BR5-C2k (98.2%), and BR5-Kras (98.7%) cell lines. Using the ID8 intraperitoneal ovarian cancer mouse model in C57BL/6J mice, treatment with immunotherapy significantly increased cell surface expression of CXCR4 from 2% in the control group to 79.6-91.8% in the treatment group. Treatment with AMD3100 as a single agent showed no improvement in survival over control. However, AMD3100 in combination with anti-PD-1 therapy significantly improved survival. The addition of bevacizumab demonstrated no further survival benefit and bevacizumab as a single agent showed no improvement in survival over control, either alone or in combination with anti-PD-1. Analysis of circulating lymphocytes revealed a trend toward higher CD4 to CD8 ratio in mice who were treated with anti-PD1 therapy. Targeting the CXCL12-CXCR4 axis in combination with other immunotherapies such as immune checkpoint inhibitors is a novel therapeutic strategy which may provide benefit over single-agent immunotherapy and warrant investigation in a clinical trial. Citation Format: Kristen Starbuck, Robert McGray, Samar Masoumi-Moghaddam, Ariel Francois, Kunle Odunsi, Emese Zsiros. CXCR4 in combination with immune check point inhibition in ovarian cancer mouse model demonstrates potential for novel therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 947. doi:10.1158/1538-7445.AM2017-947

CAMP Signaling Enhances HIV-1 Long Terminal Repeat (LTR)-directed Transcription and Viral Replication in Bone Marrow Progenitor Cells.

CD34+ hematopoietic progenitor cells have been shown to be susceptible to HIV-1 infection, possibly due to a low-level expression of CXCR4, a coreceptor for HIV-1 entry. Given these observations, we have explored the impact of forskolin on cell surface expression of CXCR4 in a cell line model (TF-1). The elevation of intracellular cyclic adenosine monophosphate (cAMP) by forskolin through adenylyl cyclase (AC) resulted in transcriptional upregulation of CXCR4 with a concomitant increase in replication of the CXCR4-utilizing HIV-1 strain IIIB. Transient expression analyses also demonstrated an increase in CXCR4-, CCR5-, and CXCR4-/CCR5-utilizing HIV-1 (LAI, YU2, and 89.6, respectively) promoter activity. Studies also implicated the protein kinase A (PKA) pathway and the downstream transcription factor CREB-1 in interfacing with cAMP response elements located in the CXCR4 and viral promoter. These observations suggest that the cAMP signaling pathway may serve as a regulator of CXCR4 levels and concomitantly of HIV-1 replication in bone marrow (BM) progenitor cells.

A 10 km time trial running bout acutely increases the number of angiogenic Tcells in the peripheral blood compartment of healthy males.

What is the central question of the study? Are CD31+ angiogenic T (TANG ) cells preferentially mobilized in response to acute exercise? What is the main finding and its importance? Our study reveals that TANG cells are redistributed into the circulation in response to acute strenuous exercise, but to a lesser extent than CD31- Tcells. Of the TANG cells mobilized, TANG cells expressing CXCR4 show greater redistribution compared with CXCR4- TANG cells. Stromal-derived factor 1-α does not appear to play a role in the redistribution of TANG cells expressing CXCR4. The results suggest that a single bout of strenuous exercise might provide a short vasculogenic window, which could benefit the vascular system by redistributing CD31+ TANG cells. CD31+ Tcells have been documented to possess vasculogenic properties and have been termed 'angiogenic T cells' (TANG cells). No study to date has fully characterized the effect of acute exercise on TANG cells. Twelve male participants aged 24-45years undertook a running 10km time trial, with peripheral blood samples taken before, immediately after and 1h postexercise for quantification of TANG cells and subsequent CXCR4 cell surface expression by flow cytometry. The TANG cells demonstrated a 102% increase in number in the peripheral circulation immediately postexercise compared with pre-exercise levels, followed by a large egress (50%) from the circulation in total TANG cells 1h postexercise. This was due to changes in both CD4+ and CD8+ TANG cells, with CD8+ TANG cells displaying greater ingress (123%) and egress (52%) compared with CD4+ TANG cells (ingress, 83%; egress, 37%). The cell surface expression intensity of CXCR4 was affected only on CD8+ TANG cells, with a significant increase in cell surface expression immediately postexercise versus pre-exercise levels. The CD31- Tcells displayed greater redistribution than CD31+ TANG cells (119versus 102%). CXCR4-expressing TANG cells showed greater response to acute exercise than CXCR4- cells, which was accompanied by large changes in CXCR4 ligand SDF-1α. The results show that acute exercise increases TANG cells in the circulation in response to an acute exercise stressor. Additionally, CXCR4 cell surface expression appears to be increased in response to exercise, which may result from the direct upregulation of CXCR4 on the T-cell surface or could be due to CD31+ Tcells being redistributed into the blood expressing greater levels of CXCR4.

Effects of short-term inflammatory and/or hypoxic pretreatments on periodontal ligament stem cells: in vitro and in vivo studies

In this study, we extensively screened the in vitro and in vivo effects of PDLSCs following short-term inflammatory and/or hypoxic pretreatments. We found that the 24-h hypoxic pretreatment of PDLSCs significantly enhanced cell migration and improved cell surface CXCR4 expression. In addition, hypoxia-pretreated PDLSCs exhibited improved cell colony formation and proliferation. Cells that were dually stimulated also formed more colonies compared to untreated cells but their proliferation did not increase. Importantly, the hypoxic pretreatment of PDLSCs enhanced cell differentiation as determined by elevated RUNX-2 and ALP protein expression. In this context, the inflammatory stimulus impaired cell OCN protein expression, while dual stimuli led to decreased RUNX-2 and OCN mRNA levels. Although preconditioning PDLSCs with inflammatory and/or hypoxic pretreatments resulted in no differences in the production of matrix proteins, hypoxic pretreatment led to the generation of thicker cell sheets; the inflammatory stimulus weakened the ability of cells to form sheets. All the resultant cell sheets exhibited clear bone regeneration following ectopic transplantation as well as in periodontal defect models; the amount of new bone formed by hypoxia-preconditioned cells was significantly greater than that formed by inflammatory stimulus- or dual-stimuli-treated cells or by nonpreconditioned cells. The regeneration of new cementum and periodontal ligaments was only identified in the hypoxia-stimulus and no-stimulus cell groups. Our findings suggest that PDLSCs that undergo short-term hypoxic pretreatment show improved cellular behavior in vitro and enhanced regenerative potential in vivo. The preconditioning of PDLSCs via combined treatments or an inflammatory stimulus requires further investigation.

High Cardiorespiratory Fitness Alleviates the Negative Impact of Increasing Sitting Time on CD31+ T-Cell Number

Sitting time and other forms of physical inactivity are associated with ill-effects on cardiovascular health. Short periods of physical inactivity result in reductions in insulin sensitivity as well as vascular function. Circulating CD31+ T-cells (TANG) possess significant pro-angiogenic capabilities and may contribute to maintenance of endothelial function. These cells can be influenced by cardiorespiratory fitness level (CRF) and thus may also be affected by physical inactivity. Therefore any effect of sitting time has on these cells needs to be investigated. PURPOSE: To determine the effects of sitting time on TANG number and to investigate if high levels of CRF attenuates any impact of sedentary behaviour on these cells. METHODS: Self-reported sitting time data was collected from 42 healthy males aged 31-65yrs. CRF was estimated in the participants through the use of a submaximal cycling ergometer test. TANG were quantified by flow cytometry in a resting blood sample, defined as co-expression of CD3 and CD31. These cells were further characterised using antibodies against CD4 and CD8. Cell surface expression of CXCR4 was also quantified. Linear regressions were performed to examine association between TANG and self-reported sitting time and CRF individually, and where appropriate, hierarchical multiple level regressions were performed. RESULTS: Self-reported sitting time was inversely associated with TANG number (r=-0.291, p<0.05). After correcting for VO2max (CRF), sitting time was no longer associated with these CD31+ T-cells (from t=-1.924, p=0.061 to t=-1.153, p=0.256). This was attributed to the positive impact of CRF on CD4+ TANG cells (from t=-1.838, p=0.074 to t=-1.144, p=0.260). Increasing levels of sitting time was associated with an increase in CXCR4 expression on CD4+ TANG cells, with no effect on CD8+ TANG cells. The effects of sitting time was observed in the absence of any effect on cardiometabiolic risk factors such as fasting glucose, blood pressure, cholesterol and IL-6. CONCLUSION: Sitting time is associated with a decreased level of CD31+ T-cells, however this effect is alleviated by correcting for CRF levels, indicating that maintaining high levels of aerobic fitness can attenuate negative impact of physical activity on this subset of circulating angiogenic cells.