C-X-C Chemokine Receptor Type 4 Research Articles

A Novel lncRNA Mediates the Delayed Tooth Eruption of Cleidocranial Dysplasia.

Delayed eruption of permanent teeth is a common symptom of cleidocranial dysplasia (CCD). Previous studies have focused on the anomaly of osteogenesis resulting from mutations in the Runt-related transcription factor-2 gene (RUNX2). However, deficiencies in osteoclastogenesis and bone resorption, and the epigenetic regulation mediated by long non-coding (lnc)RNAs in CCD remain to be elucidated. Here, a novel osteoclast-specific lncRNA (OC-lncRNA) was identified during the osteoclast differentiation of RAW 264.7 cells transfected with a RUNX2 mutation expression cassette. We further confirmed that OC-lncRNA positively regulated osteoclastogenesis and bone resorption. The OC-lncRNA promoted the expression of CXC chemokine receptor type 3 (CXCR3) by competitively binding to microRNA (miR)-221-5p. The CXCR3–CXC-motif chemokine ligand 10 (CXCL10) interaction and nuclear factor-κB constituted a positive feedback that positively regulated osteoclastogenesis and bone resorption. These results demonstrate that OC-lncRNA-mediated osteoclast dysfunction via the OC-lncRNA–miR-221-5p–CXCR3 axis, which is involved in the process of delayed tooth eruption of CCD.

Potential role of CXCR4 in trastuzumab resistance in breast cancer patients

Despite the efficacy of trastuzumab in treating HER2-positive breast cancer patients, a significant proportion of patients relapse after treatment. The role of C-X-C chemokine receptor type 4 (CXCR4) in trastuzumab resistance was studied only in cell lines and the underlying mechanisms remain largely unclear. This study investigated the role of CXCR4 in trastuzumab resistance in breast cancer patients and explored the possible underlying mechanisms. The study was performed retrospectively on tissue samples from 62 breast cancer patients including 42 who were treated with trastuzumab and chemotherapy and 20 who received chemotherapy alone in adjuvant setting. Expression levels of CXCR4 and its regulators hypoxia-inducible factor 1-alpha (HIF-1α), tristetraprolin (TTP), human antigen R (HuR), itchy E3 ubiquitin protein ligase (ITCH), miR-302a and miR-494 were determined and their associations with tumor recurrence and disease-free survival were analyzed. In trastuzumab-treated patients, high CXCR4 expression was associated with recurrence and was an independent predictor of progression risk after therapy. CXCR4 correlated positively with its transcriptional regulator, HIF-1α, and negatively with its post-translational regulator, ITCH. HIF-1α, HuR and ITCH were significantly associated with clinical outcome. In chemotherapy-treated patients, neither CXCR4 nor any of its regulators were associated with recurrence or predicted disease progression risk after chemotherapy. In conclusion, this study suggests a potential role for CXCR4 in recurrence after trastuzumab-based therapy in human breast cancer that could be mediated, at least in part, by hypoxia and/or decreased ubiquitination. These findings highlight the potential utility of CXCR4 as a promising target for enhancing trastuzumab therapeutic outcome.

IMMUNOHISTOCHEMICAL ANALYSIS OF CXCL12 AND CXCR4 IN SYNDROMIC AND NONSYNDROMIC ODONTOGENIC KERATOCYSTS

Objectives To evaluate the immunoexpression of chemokine CXCL12 and C-X-C chemokine receptor type 4 (CXCR4) in odontogenic keratocysts associated with Gorlin-Goltz syndrome (SOKCs) and nonsyndromic odontogenic keratocysts (NSOKCs). Study Design The percentages of cytoplasmic (CXCL12 and CXCR4) and nuclear (CXCR4) staining in epithelial and fibrous capsule cells of 22 SOKCs and 22 NSOKCs were determined. The results were analyzed statistically using the nonparametric Mann-Whitney test and Spearman correlation test. Results Higher cytoplasmic expression of CXCL12 was observed in the epithelial lining and fibrous capsule of NSOKCs compared to SOKCs (P < .001). No statistically significant differences in the cytoplasmic expression of CXCR4 were observed between SOKCs and NSOKCs (P > .05). Compared to SOKCs, NSOKCs exhibited higher nuclear expression of CXCR4 in the epithelial lining and lower immunoexpression in the fibrous capsule (P < .05). In the epithelial lining of SOKCs, a positive correlation was observed between cytoplasmic and nuclear expression of CXCR4 (P = .003). In the fibrous capsule of SOKCs and NSOKCs, cytoplasmic and nuclear expressions of CXCR4 were positively correlated (P < .001). Conclusions The results suggest a potential involvement of CXCL12 and CXCR4 in the development of OKCs. The heterogenous expression of these proteins in SOKCs and NSOKCs may reflect differences in their pathogenesis and biological behavior. To evaluate the immunoexpression of chemokine CXCL12 and C-X-C chemokine receptor type 4 (CXCR4) in odontogenic keratocysts associated with Gorlin-Goltz syndrome (SOKCs) and nonsyndromic odontogenic keratocysts (NSOKCs). The percentages of cytoplasmic (CXCL12 and CXCR4) and nuclear (CXCR4) staining in epithelial and fibrous capsule cells of 22 SOKCs and 22 NSOKCs were determined. The results were analyzed statistically using the nonparametric Mann-Whitney test and Spearman correlation test. Higher cytoplasmic expression of CXCL12 was observed in the epithelial lining and fibrous capsule of NSOKCs compared to SOKCs (P < .001). No statistically significant differences in the cytoplasmic expression of CXCR4 were observed between SOKCs and NSOKCs (P > .05). Compared to SOKCs, NSOKCs exhibited higher nuclear expression of CXCR4 in the epithelial lining and lower immunoexpression in the fibrous capsule (P < .05). In the epithelial lining of SOKCs, a positive correlation was observed between cytoplasmic and nuclear expression of CXCR4 (P = .003). In the fibrous capsule of SOKCs and NSOKCs, cytoplasmic and nuclear expressions of CXCR4 were positively correlated (P < .001). The results suggest a potential involvement of CXCL12 and CXCR4 in the development of OKCs. The heterogenous expression of these proteins in SOKCs and NSOKCs may reflect differences in their pathogenesis and biological behavior.

IMMUNOHISTOCHEMICAL ANALYSIS OF C-X-C CHEMOKINE RECEPTOR TYPE 4 IN ODONTOGENIC KERATOCYSTS AND DENTIGEROUS CYSTS

<h3>Objectives</h3> To evaluate the immunohistochemical expression of C-X-C chemokine receptor type 4 (CXCR4) in odontogenic keratocysts (OKCs) and dentigerous cysts (DCs). <h3>Study Design</h3> The percentages (cytoplasmic and nuclear) of CXCR4-positive cells in the epithelial lining and fibrous capsule of 15 OKCs and 15 DCs were assessed. The results were analyzed statistically using the nonparametric Mann-Whitney test and Spearman correlation test. <h3>Results</h3> Cytoplasmic and nuclear expressions of CXCR4 were observed in all cases of OKCs and DCs. Lower cytoplasmic expression of this chemokine receptor was found in the epithelial lining and fibrous capsule of OKCs compared to DCs (<i>P</i> < .005). Analysis of nuclear immunoexpression of CXCR4 revealed lower percentages of positive cells in the fibrous capsule of OKCs compared to DCs (<i>P</i> = .003). In the epithelial lining of DCs, a positive correlation was observed between cytoplasmic and nuclear expression of CXCR4 (<i>P</i> = .034). In the fibrous capsule of OKCs and DCs, cytoplasmic and nuclear expressions of CXCR4 were positively correlated (<i>P</i> < .005). <h3>Conclusions</h3> The results suggest a potential role of CXCR4 in the pathogenesis of OKCs and DCs. This chemokine receptor may be more important for the development of odontogenic cysts with less aggressive biological behavior.

Disease Progression of WHIM Syndrome in an International Cohort of 66 Pediatric and Adult Patients

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS) is a combined immunodeficiency caused by gain-of-function mutations in the C-X-C chemokine receptor type 4 (CXCR4) gene. We characterize a unique international cohort of 66 patients, including 57 (86%) cases previously unreported, with variable clinical phenotypes. Of 17 distinct CXCR4 genetic variants within our cohort, 11 were novel pathogenic variants affecting 15 individuals (23%). All variants affect the same CXCR4 region and impair CXCR4 internalization resulting in hyperactive signaling. The median age of diagnosis in our cohort (5.5 years) indicates WHIM syndrome can commonly present in childhood, although some patients are not diagnosed until adulthood. The prevalence and mean age of recognition and/or onset of clinical manifestations within our cohort were infections 88%/1.6 years, neutropenia 98%/3.8 years, lymphopenia 88%/5.0 years, and warts 40%/12.1 years. However, we report greater prevalence and variety of autoimmune complications of WHIM syndrome (21.2%) than reported previously. Patients with versus without family history of WHIM syndrome were diagnosed earlier (22%, average age 1.3 years versus 78%, average age 5 years, respectively). Patients with a family history of WHIM syndrome also received earlier treatment, experienced less hospitalization, and had less end-organ damage. This observation reinforces previous reports that early treatment for WHIM syndrome improves outcomes. Only one patient died; death was attributed to complications of hematopoietic stem cell transplantation. The variable expressivity of WHIM syndrome in pediatric patients delays their diagnosis and therapy. Early-onset bacterial infections with severe neutropenia and/or lymphopenia should prompt genetic testing for WHIM syndrome, even in the absence of warts.

Isoimperatorin down-regulates epithelial mesenchymal transition through modulating NF-κB signaling and CXCR4 expression in colorectal and hepatocellular carcinoma cells

Worldwide, metastatic spread of tumor is the major cause of cancer-related deaths. There are many anti-metastatic agents that can target metastasis-related pathways, but there are relatively few studies on agents targeting C-X-C chemokine receptor type 4 (CXCR4) signaling axis. Here, we have investigated whether Isoimperatorin (IIPT), derived from Angelica dahurica can modulate CXCR4 signaling axis-related epithelial-to-mesenchymal transition (EMT) and tumor metastasis process. We evaluated the influence of IIPT on CXCR4, HER2, MMPs, EMT markers, and NF-κB signaling pathway by using Western blot analysis. The cellular invasion and migration were observed by Boyden chamber and wound healing assays. IIPT has a down-regulatory effect on CXCR4, HER2, and MMP-9/2. On the contrary, imperatorin (IPT) as compared to IIPT did not alter the expression of CXCR4. IIPT down-regulated the protein levels and RNA levels of mesenchymal markers, twist, snail, and enhanced the levels of different epithelial markers. IIPT also inhibited cell migration, invasion, and proliferation. Furthermore, IIPT negatively regulated constitutive NF-κB activation and inhibited the translocation of phospho-p65 and p65 into the nuclei. IIPT can potentially function as a novel anti-metastatic agent by inhibiting EMT and metastasis process via inhibition of NF-κB activation and CXCR4 expression in colorectal and hepatocellular carcinoma cells.

Bone Marrow Mesenchymal Stem Cells (BMSC) Carried with miR-338 Improve BMSC Homing in Intestinal Mucosa of Inflammatory Bowel Disease (IBD) Rats Through Increasing C-X-C Chemokine Receptor Type 4 (CXCR-4)

This study assessed whether bone marrow mesenchymal stem cells (BMSC) carried with miR-338 improve its homing in intestinal mucosa of IBD rats. Fifty SD rats were equally and randomly assigned into control set, model set, miR-338 set, CXCR-4 Inhibitor set and positive control set followed by analysis of miR-338 expression, body weight, DAI grade, pathological changes of distal colonic tissue, ultra-microstructure of intestinal epithelium, level of IL-8, IL-10, TGF-β1, VEGF, and CXCR4, as well as the relation between miR-338 and CXCR-4. miR-338 in decorated BMSC was significantly upregulated. Body weight in model set and CXCR-4 Inhibitor set was lowest followed by miR-338 set and positive control set. However, opposite results were obtained for DAI grade. IL-8 level was highest and IL-10 was lowest in model set and CXCR-4 Inhibitor set. The BMSC carried with miR-338 exerted recovery action on the intestinal mucosa of IBD rats and inhibited the activity of inflammatory cells. In conclusion, BMSC carried with miR-338 develops recovery action on the intestinal mucosa of IBD rats possibly through regulation of CXCR4, indicating that miR-338 might be a target for the treatment of IBD.

Enhanced migration and immunoregulatory capacity of BMSCs mediated by overexpression of CXCR4 and IL-35

Bone marrow-derived mesenchymal stem cells (BMSCs) have been widely studied for their applications in immunoregulation and tissue repair. However, the therapeutic effects of BMSCs in the body are limited, partly due to the low homing efficiency of BMSCs to affected parts. The stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis is well known to play an essential role in the homing of BMSCs. Interleukin 35 (IL-35) is a newly discovered cytokine confirmed to inhibit overactivated immune function and have a good therapeutic effect on autoimmune diseases. In this study, we innovatively developed dual gene modification of BMSCs by transducing CXCR4 and IL-35 and found that the migration and immunomodulatory activity of genetically engineered BMSCs were significantly enhanced compared to their natural counterparts. These results suggest that BMSCs modified by dual overexpression of CXCR4 and IL-35 may provide a potential treatment strategy for autoimmune diseases.

The effect of reparixin on survival in patients at high riskfor in-hospital mortality: a meta-analysis of randomized trials.

IntroductionA great number of anti-inflammatory drugs have been suggested in the treatment of SARS-CoV-2 infection. Reparixin, a non-competitive allosteric inhibitor of the CXCL8 (IL-8) receptors C-X-C chemokine receptor type 1 (CXCR1) and C-X-C chemokine receptor type 2 (CXCR2), has already been tried out as a treatment in different critical settings. Due to the contrasting existing literature, we decided to perform the present meta-analysis of randomized controlled trials (RCTs) to investigate the effect of the use of reparixin on survival in patients at high risk for in-hospital mortality.MethodsWe created a search strategy to include any human RCTs performed with reparixin utilization in patients at high risk for in-hospital mortality, excluding oncological patients. Two trained, independent authors searched PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) for appropriate studies. Furthermore, references of review articles and included RCTs were screened to identify more studies. No language restrictions were enforced. To assess the risk of bias of included trials, the Revised Cochrane risk-of-bias tool for randomized trials (RoB 2) was used.ResultsOverall, six studies were included and involved 406 patients (220 received reparixin and 186 received the comparator). The all-cause mortality in the reparixin group was significantly lower than that in the control group [5/220 (2.3%) in the reparixin group vs. 12/186 (6.5%) in the control group, odds ratio = 0.33 (95% confidence interval 0.12 to 0.96), p-value for effect 0.04, p for heterogeneity 0.20, I 2 = 36%]. In addition, no difference in the rate of pneumonia, sepsis, or non-serious infections was shown between the two groups.ConclusionOur meta-analysis of randomized trials suggests that short-term inhibition of CXCL8 activity improved survival in patients at high risk for in-hospital mortality without increasing the risk of infection.Meta-analysis registrationPROSPERO, identifier CRD42021254467.

Emerging Roles of the Atypical Chemokine Receptor 3 (ACKR3) in Cardiovascular Diseases.

Chemokines, and their receptors play a crucial role in the pathophysiology of cardiovascular diseases (CVD). Chemokines classically mediate their effects by binding to G-protein-coupled receptors. The discovery that chemokines can also bind to atypical chemokine receptors (ACKRs) and initiate alternative signaling pathways has changed the paradigm regarding chemokine-related functions. Among these ACKRs, several studies have highlighted the exclusive role of ACKR3, previously known as C-X-C chemokine receptor type 7 (CXCR7), in CVD. Indeed, ACKR3 exert atheroprotective, cardioprotective and anti-thrombotic effects through a wide range of cells including endothelial cells, platelets, inflammatory cells, fibroblasts, vascular smooth muscle cells and cardiomyocytes. ACKR3 functions as a scavenger receptor notably for the pleiotropic chemokine CXCL12, but also as a activator of different pathways such as β-arrestin-mediated signaling or modulator of CXCR4 signaling through the formation of ACKR3-CXCR4 heterodimers. Hence, a better understanding of the precise roles of ACKR3 may pave the way towards the development of novel and improved therapeutic strategies for CVD. Here, we summarize the structural determinant characteristic of ACKR3, the molecules targeting this receptor and signaling pathways modulated by ACKR3. Finally, we present and discuss recent findings regarding the role of ACKR3 in CVD.

Dual Stromal Targeting Sensitizes Pancreatic Adenocarcinoma for Anti-Programmed Cell Death Protein 1 Therapy

The stroma in pancreatic ductal adenocarcinoma (PDAC) contributes to its immunosuppressive nature and therapeutic resistance. Herein we sought to modify signaling and enhance immunotherapy efficacy by targeting multiple stromal components through both intracellular and extracellular mechanisms. A murine liver metastasis syngeneic model of PDAC was treated with focal adhesion kinase inhibitor (FAKi), anti-programmed cell death protein 1 (PD-1) antibody, and stromal hyaluronan (HA) degradation by PEGylated recombinant human hyaluronidase (PEGPH20) to assess immune and stromal modulating effects of these agents and their combinations. The results showed that HA degradation by PEGPH20 and reduction in phosphorylated FAK expression by FAKi leads to improved survival in PDAC-bearing mice treated with anti-PD-1 antibody. HA degradation in combination with FAKi and anti-PD-1 antibody increases T-cell infiltration and alters T-cell phenotype toward effector memory T cells. FAKi alters the expression of T-cell modulating cytokines and leads to changes in T-cell metabolism and increases in effector T-cell signatures. HA degradation in combination with anti-PD-1 antibody and FAKi treatments reduces granulocytes, including granulocytic- myeloid-derived suppressor cells and decreases C-X-C chemokine receptor type 4 (CXCR4)-expressing myeloid cells, particularly the CXCR4-expressing granulocytes. Anti-CXCR4 antibody combined with FAKi and anti-PD-1 antibody significantly decreases metastatic rates in the PDAC liver metastasis model. This represents the first preclinical study to identify synergistic effects of targeting both intracellular and extracellular components within the PDAC stroma and supports testing anti-CXCR4 antibody in combination with FAKi as a PDAC treatment strategy.

Suppressing the activity of CXCR4 down-regulates the expression of renal fibrosis related genes in primary glomerular cells.

BackgroundC-X-C chemokine receptor type 4 (CXCR4) has a certain effect on renal fibrosis, and there are few specific studies in cells. We want to investigate the impact of suppressing CXCR4 activity on the expression of renal fibrosis-related genes in primary glomerular endothelial cells, mesangial cells, and podocytes.MethodsImmunofluorescence assays were used to determine the purity of isolated glomerular endothelial cells, mesangial cells, and podocytes. CXCR4 knockdown cell lines were established by transfecting the short hairpin (sh)RNA against CXCR4. T140 and AMD3100 were used to inhibit the activity of CXCR4. LY294002 was used to inhibit the activity of phosphoinositide 3-kinase (PI3K). The mRNA expression of CXCR4 was determined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The protein expression of CXCR4, collagen IV, matrix metallopeptidase (MMP)-9, PI3K, Rac1, and vascular cell adhesion protein 1 (VCAM-1) was evaluated by Western blot analysis.ResultsHigh purity was observed on isolated primary glomerular endothelial cells and podocytes. However, the purity of isolated mesangial cells was relatively low. The mRNA expression of CXCR4 was significantly suppressed by the transfection of shRNA. Compared to control cells, the expression of CXCR4, collagen IV, MMP-9, PI3K, Rac1, and VCAM-1 were dramatically downregulated in cell lines transfected with shRNA against CXCR4. Furthermore, cell lines treated with T140, AMD3100, or LY294002 also showed downregulated expression of these proteins compared to untreated cells. No significant differences were observed in the protein expression of these proteins between control cells and cells transfected with the shRNA negative control (NC).ConclusionsSuppressing the activity of CXCR4 downregulated the expression of renal fibrosis-related genes in primary glomerular cells, even under a non-inflammatory state.

CD44 potentiates hepatocellular carcinoma migration and extrahepatic metastases via the AKT/ERK signaling CXCR4 axis.

BackgroundCell adhesion molecule cluster of differentiation 44 (CD44) plays a significant role in cancer cell local invasion, intravasation, migration, and the establishment of metastatic lesions. However, little is known about the underlying mechanism of how CD44 regulates hepatocellular carcinoma (HCC) extrahepatic metastasis (EHM).MethodsThe expression of CD44 in HCC tissues and cell lines was detected through western blot and immunohistochemistry (IHC). Through gain- and loss-of-function assays, we examined the oncogenic roles of CD44 in regulating HCC cell growth and metastasis in vitro and in vivo. To identify the potential mechanism, we employed quantitative real-time polymerase chain reaction, and western blot.ResultsIn this study, CD44 was highly expressed in HCC cells and HCC-patient specimens that exhibited high malignancy potential. The overall survival (OS) was worse and the cumulative recurrence rate was higher in HCC patients with CD44 overexpression than those with low levels of CD44 expression. Our in-vitro and in-vivo experiments showed that CD44 downregulation reduced HCC cell colony formation, migration, and invasion, and HCC tumor growth and metastasis, and that the pro-metastasis effect of CD44 was mediated by the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling-chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) axis. The reported capacity of CD44 to induce CXCR4 expression and increase the propensity of tumors to invade and metastasize to distant organs is consistent with the aggressive clinical characteristics of HCCs.ConclusionsCD44 could represent a future therapeutic target for EHM.

Production of human embryonic kidney 293T cells stably expressing C-X-C chemokine receptor type 4 (CXCR4) as a screening tool for anticancer lead compound targeting CXCR4

AimThe C-X-C chemokine-receptor type 4 (CXCR4) is an emerging target for cancer drug discovery due to its high expression in cancer cells. The present study aimed to produce CXCR4 overexpressing HEK293T cells for a non-radioactive binding assay as a platform to identify drug candidates targeting CXCR4. Main methodsHEK293T cells stably expressing human CXCR4 were constructed by transfection of CXCR4 plasmids from the human CXCR4 gene. The CXCR4 overexpressing HEK293T cells were obtained by fluorescence-activated sorting and verified by conducting the competition binding assay of a known CXCR4 inhibitor, AMD3100 (plerixafor), to determine the IC50 value against monoclonal anti-human CD184 (hCD184) antibody tagged with fluorescence probe, phycoerythrin (PE). The non-radioactive binding assay using CXCR4 overexpressing HEK293T cells and PE-anti hCD184 was applied as a platform for identifying the target of natural compounds that exhibited cytotoxicity against cancer cell lines. Key findingsThe CXCR4 overexpressing HEK293T cells were produced with high expression (99.8%). The IC50 value of plerixafor determined by fluorescence tagged antibody-based competition assay using our developed cells agree with previously reported values using a radioligand binding assay. We observed no significant displacement of bound PE-anti-hCD184 by the test natural compounds which could be due to non-specific binding to other functional targets or organelles, low potency of the natural compounds, or binding to CXCR4 at deeper pockets. SignificanceThe verified non-radioactive binding assay can serve as an alternative screening tool for anticancer lead compounds targeting CXCR4 and an essential tool for proof of mechanism study in the drug discovery.

Molecular effects of genistein, as a potential anticancer agent, on CXCR-4 and VEGF pathway in acute lymphoblastic leukemia.

Vascular endothelial growth factor (VEGF) is one of the angiogenic mediators that can be secreted by leukemic cells and plays an important role in tumor invasion and metastasis. Another important agent contributing to the relapse of ALL is C-X-C chemokine receptor type-4 (CXCR-4), expression of this receptor in cancer cells has been related to metastasis. It has been identified that genistein-a soy-derived isoflavonoid-has anti-angiogenesis functions. We aimed to show the effects of this compound on VEGF and CXCR-4 in Acute lymphoblastic leukemia (ALL) cell models. The cytotoxicity of Genistein was measured using the MTS colorimetric assay. After being treated with Genistein, the expression of VEGF in mRNA and protein levels was measured in MOLT-4 and Jurkat cells. We also used flow cytometry assay to determine the expression of CXCR-4 in cell surfaces. We found that Genistein decreased cell viability in two cell models while was more effective on MOLT-4 cells. After Genistein-treatment, surface expression levels of CXCR-4 were decreased, while VEGF secretion and mRNA expression levels were increased in MOLT-4 and Jurkat cells. The results suggest that Genistein may not be a reliable choice for the treatment of ALL; however, this different identified pattern can be useful for the recognition of VEGF and CXCR-4 modulators and thus for planning new treatments for leukemia and other VEGF related disorders.

68Ga-Pentixafor PET/CT Demonstrating InVivo CXCR4 Receptor Overexpression in Rare Lung Malignancies: Correlation with Histologic and Histochemical Findings.

68Ga-pentixafor PET/CT imaging allows noninvasive assessment of C-X-C chemokine receptor type 4 (CXCR4) expression in various malignancies, but its use in rare lung cancer variants has not been reported. Methods: 68Ga-pentixafor PET/CT imaging was performed on 6 patients (3 men, 3 women; mean age, 57.0 ± 16.8 y) with suspected lung masses. Whole-body PET/CT images were acquired 1 h after intravenous injection of 148.0-185.0 MBq of the tracer. PET/CT images were reconstructed and analyzed. The image findings were correlated with histopathologic and quantitative (CXCR4) fluorescence-activated cell sorting analysis. Results: Histopathologic diagnosis of hemangioendothelioma, sarcomatoid carcinoma, and hemangiopericytoma was confirmed in 1 patient each. Lung metastasis was diagnosed in the remaining 3 of 6 patients with primary sarcoma (n = 1), renal cell carcinoma (n = 1), and unknown primary (n = 1). Increased uptake in the primary lung mass, with an SUVmax of 3.0, 6.34, and 13.0, was noted in the hemangiopericytoma, sarcomatoid carcinoma and hemangioendothelioma cases, respectively. The mean SUVmax, mean fluorescence intensity, and percentage of stained cells were highest in hemangioendothelioma. Among 3 patients with lung metastases, the highest SUVmax, 9.5, was in the primary sarcoma patient. Conclusion: 68Ga-pentixafor selectively targets the invivo whole-body disease burden of CXCR4 receptors. This approach thus holds promise for developing suitable radiotheranostics for lung cancers expressing these targets.

C-X-C Chemokine Receptor Type 4 (CXCR4) and Programmed Death-Ligand 1 (PD-L1) Expression are Prognostic Biomarkers for Papillary Thyroid Carcinoma

Chemokines and immunomodulatory factors involve in tumor development. Papillary thyroid carcinoma (PTC) is considered to start from dendritic cell infiltration and then produce immunomodulatory factors. In this study, CXCR4 and PD-L1 biomarkers were used to explore their prognostic role in PTC survival. Confocal microscopy detected the transfection efficiency in tumor cells. 42 PTC patients and thyroiditis patients (control) were enrolled to measure the expressions of CXCR4 and PD-L1. Multi-factor analysis analyzed the effect of combined CXCR4 and PD-L1 expression on ROC. The two groups had no differences in the baseline characteristics. CTXCR4 and PD-L1 level in PTC patients was significantly higher than control. CXCR4 was lowly expressed in thyroid cancer tissue and PD-L1 was highly expressed in serological samples. Compared with single measurement, the combined detection of CXCR4 and PD-L1 showed more ROC area. In conclusion, reduced CXCR4 and increased PD-L1 level is found in thyroid cancer and their level might be used as predictive markers for PTC to improve the curative effect.

Regulation of Endothelial Cell function by Integration of Gαi and β‐arrestin signaling at Atypical Chemokine Receptor 3

Atypical Chemokine receptor 3 (ACKR3), also known as C‐X‐C chemokine receptor type 7 (CXCR7), is a G protein‐coupled receptor (GPCR) implicated in several physiological processes including leukocyte trafficking, cancer, and angiogenesis. Commonly co‐expressed with C‐X‐C chemokine receptor type 4 (CXCR4), ACKR3 is classified as an β‐arrestin‐biased “scavenger” receptor due to its lack of G‐protein signaling and its ability to efficiently internalize and degrade its ligands, CXCL11 and CXCL12. However, much is still unknown regarding ACKR3 signaling mechanisms and its implications on cellular physiology. Here we describe ongoing studies to characterize the mechanisms that underlie ACKR3 signaling and endothelial cell function. We demonstrated two ACKR3 specific agonists, WW36 and WW38, promote proliferation and migration of human umbilical vein endothelial cells (HUVECs). Surprisingly, we found that ACKR3‐promoted proliferation and migration was sensitive to pertussis toxin (PTX), suggesting the involvement of Gαi/o proteins at a receptor which does not canonically signal via G proteins. We also observed similar sensitivity to PTX on ERK activation in HUVECs. We hypothesized that these results may in part be explained by the recently described coordination of Gαi and β‐arrestin (βarr) to promote Gαi:βarr complex‐specific downstream signaling. In support of this hypothesis, we demonstrated the formation of a Gαi:βarr complex upon ACKR3 activation in a dose‐dependent manner. To characterize the necessary components in Gαi and βarr, we first developed active proximal polar core mutant and proximal finger loop βarr mutant and tested these at the prototypical class B Vasopressin 2 Receptor. Both mutants lead to considerable decrease in complex formation with the finger loop mutation nearly abolishing our split signal. Within the Gαi subfamily, we observed considerable differences in the ability to form complex, with Gαi2 and Gαi3 promoting less complex formation than Gαi1 and Gαo. We developed several Gαi mutants, hypothesizing that residues that differ between Gαi isoforms may impact Gαi:βarr formation. Using NanoBRET we demonstrated that these mutants impair complex formation, suggesting that they may be involved either via direct contacts with βarr or impacting Gαi conformation such that complex formation is no longer as favorable. These studies reveal a novel role for Gαi:βarr complexes in mediating ACKR3 function that contribute to endothelial cell biology. Additionally, these findings support an emerging paradigm in which G proteins and βarr coordinate their signaling downstream of GPCRs.

The role of ERK2 in regulating germinal center B cell fate decisions

Abstract The regulation of B cell activation, differentiation, and survival is a crucial control point for the maintenance of B cell tolerance in the germinal center (GC) and involves the precise coordination of several complex intracellular signaling pathways. Dysregulation of these pathways can lead to the generation and persistence of autoreactive memory B cells and antibody-secreting cells (ASCs), resulting in autoimmune disease. The mitogen-activated protein kinase (MAPK) pathway is central to signals required for GC B cell selection and differentiation, such as those received through the B cell receptor (BCR) and C-X-C chemokine receptor type 4 (CXCR4), respectively. Extracellular signal-regulated kinases 1 and 2 (ERK1/2), important effectors of this pathway, play a major role in B cell activation and differentiation. Though ERK2 specifically regulates the pro-apoptotic protein, Bmf, its role in regulating GC B cell fate is unknown. Our results show that peripheral Erk2-deficient B cells express DEC205, which is associated with follicular T helper cell interactions and CD80, which is involved in co-stimulation and immunoglobulin class switching. Thus, we hypothesize that ERK2 regulates several aspects of GC B cell selection. Our results elucidate key molecular mechanisms underlying the role of ERK2 in B cell differentiation, potentially offering new approaches to treating B cell-driven autoimmune diseases. Supported by grant R21AI44731 from the NIH/NIAID (TGF), and partially funded by the UTSA president’s distinguished researcher fellowship and NIH/NIGMS RISE grant GM60655 (A.N.).

Visfatin inhibits colon cancer cell apoptosis and decreases chemosensitivity to 5‑FU by promoting the SDF‑1/CXCR4/Akt axis.

5-Fluorouracil (5-FU) is the preferred chemotherapeutic drug used in the treatment of colon cancer; however, drug resistance affects its clinical efficacy. Visfatin, an adipokine that promotes tumour development, has the potential to increase resistance to chemotherapy. The present study aimed to verify the effects of visfatin on the sensitivity of colon cancer cells to 5-FU and to elucidate the potential mechanisms involved. Tissue microarrays (TMAs) were used to analyse visfatin differential expression in normal colon and colon cancer tissues, and the data were further validated in vitro. Cell Counting Kit-8, clone formation, caspase-3/7 activity assays, as well as other analyses were used to verify the effects of visfatin on sensitivity to 5-FU. TMA and correlation analyses were used to predict and verify the correlation between visfatin and stromal cell-derived factor-1 (SDF-1). Rescue experiments and PI3K/Akt inhibitors were used to verify the role of the visfatin/SDF-1/Akt axis in the sensitivity of colon cancer cells to 5-FU. Visfatin was found to be highly expressed in colon cancer tissues and cell lines. Moreover, visfatin knockdown increased apoptosis, reduced cell proliferation and enhanced the chemosensitivity of DLD-1 and SW48 cells to 5-FU. A positive correlation between visfatin and SDF-1 was observed, with the knockdown of visfatin enhancing cell sensitivity to 5-FU chemotherapy by targeting the SDF-1/C-X-C chemokine receptor type 4 (CXCR4) axis. Furthermore, the Akt signalling pathway downstream of SDF-1/CXCR4 proved to be critical in the decreased sensitisation of colon cancer cells to 5-FU induced by visfatin. On the whole, the present study demonstrates that visatin can potentially decrease colon cancer cell apoptosis, promote proliferation and decrease colon cancer cell sensitivity to 5-FU via the visfatin/SDF-1/Akt axis.