18F-Radiolabeling and Evaluation of an AMD3465 Derivative for PET Imaging of CXCR4 in a Mouse Breast Tumor Model.

The exploration of pharmaceutically active agents and positron emission tomography (PET) tracers targeting CXCR4 has been a focal point in cancer research given its pivotal role in the development and progression of various cancers. While significant strides have been made in PET imaging with radiometal-labeled tracers, the landscape of 18F-labeled small molecule tracers remains relatively limited. Herein, we introduce a novel and promising derivative, [18F]SFB-AMD3465, as a targeted PET tracer for CXCR4. The compound was synthesized by modifying the pyridine ring of AMD3465, which was subsequently labeled with 18F using [18F]SFB. The study provides comprehensive insights into the design, synthesis, and biological evaluation of [18F]SFB-AMD3465. In vitro and in vivo assessments demonstrated the CXCR4-dependent, specific, and sensitive uptake of [18F]SFB-AMD3465 in the CXCR4-overexpressing 4T1 cell line and the corresponding xenograft-bearing mouse model. These findings contribute to bridging the gap in 18F-labeled PET tracers for CXCR4 and underscore the potential of [18F]SFB-AMD3465 as a PET radiotracer for in vivo CXCR4 imaging.

HIV-1 gp120-CXCR4 recognition probed with synthetic nanomolar affinity D-peptides containing fragments of gp120 V3 loop.

The human immunodeficiency virus type 1 (HIV-1) recognizes one of its principal coreceptors, the CXC chemokine receptor 4 (CXCR4) on the host cell via the third variable loop (V3 loop) of HIV-1 envelope glycoprotein gp120 during the viral entry process. Here, we investigated the stereochemical mechanism of the molecular recognition of HIV-1 gp120 V3 loop with coreceptor CXCR4 by using peptide probes containing important fragments of the V3 loop. The tip and base/stem fragments of the V3 loop critical for V3 loop function were linked individually with the fragment derived from another CXCR4's chemokine ligand, vMIP-II to generate nanomolar affinity peptide probes of the interactions of CXCR4-V3 loop fragments. When the amino acid residues of the V3 loop fragments in these combinational peptides were changed from L-to D-configurations, the resulting peptides remarkably retained or had even enhanced recognition by CXCR4 as shown by competitive ligand-receptor binding. The ability of these peptides, regardless of the different l- or d-amino acids used, in binding CXCR4 and antagonizing CXCR4 functions was demonstrated by their blockade of calcium influx, cell migration, and CXCR4 internalization triggered by the activation of CXCR4 signaling by its endogenous ligand SDF-1α. The structural mechanisms of CXCR4 interactions with these peptides were examined with site-directed mutagenesis and molecular modeling. These results indicate that CXCR4's interface with key segments of HIV-1 gp120 V3 loop is flexible in terms of stereospecificity of ligand-receptor interaction which may have implication on understanding the viral entry mechanism and how the virus evades immune detection with V3 loop mutations and retains effective recognition of the host cell's coreceptor.

68Ga-Pentixafor PET/CT Demonstrating In Vivo 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 in vivo whole-body disease burden of CXCR4 receptors. This approach thus holds promise for developing suitable radiotheranostics for lung cancers expressing these targets.

CXCR4 PET/MRI for follow-up of gastric mucosa-associated lymphoid tissue lymphoma after first-line Helicobacter pylori eradication.

Posttreatment evaluation of gastric mucosa-associated lymphoid tissue (MALT) lymphoma currently relies on esophagogastroduodenoscopy with histological assessment of biopsies. Overexpression of the G protein-coupled C-X-C chemokine receptor type 4 (CXCR4) has been previously observed in MALT lymphoma. The aim of this prospective study was to evaluate positron emission tomography (PET) with the novel CXCR4 tracer [68Ga]Pentixafor as a potential alternative to follow up biopsies for assessment of residual disease (noncomplete remission [CR]) after first-line Helicobacter pylori eradication. Forty-six post-H pylori eradication [68Ga]Pentixafor-PET/magnetic resonance imaging (MRI) examinations of 26 gastric MALT lymphoma patients, and 20 [68Ga]Pentixafor-PET/MRI examinations of 20 control group patients without lymphoma, were analyzed. In the MALT lymphoma group, time-matched gastric biopsies were used as reference standard and showed CR in 6 cases. Pooled examination-based accuracy, sensitivity, specificity, and positive and negative predictive values of [68Ga]Pentixafor-PET for detection of residual gastric MALT lymphoma at follow-up were 97.0%, 95.0%, 100.0%, 100.0%, and 92.9%, respectively. Maximum and mean PET standardized uptake values showed moderate correlation with immunohistochemistry-based CXCR4+ cell counts, with correlation coefficients of r = 0.51 and r = 0.52 (P = .008 and P = .006). In summary, CXCR4 imaging with [68Ga]Pentixafor-PET may represent a promising test for assessment of residual gastric MALT lymphomas after H pylori eradication.

Nonblinking carbon dots for imaging and tracking receptors on a live cell membrane.

Blinking occurs with nearly all fluorophores including organic dyes, fluorescent proteins, semiconductor quantum dots and carbon dots (CDs). We developed non-blinking and photoresistant fluorescent CDs by introducing multiple aromatic domains onto a single carbon dot and demonstrated their great potential for imaging and tracking of receptors on a live cell membrane.

Therapeutic effects of CXCR4+ subpopulation of transgene-free induced cardiosphere-derived cells on experimental myocardial infarction.

OBJECTIVES: Myocardial infarction (MI) is the most predominant type of cardiovascular diseases with high mortality and morbidity. Stem cell therapy, especially cardiac progenitor cell therapy, has been proposed as a promising approach for cardiac regeneration and MI treatment. Previously, we have successfully generated cardiac progenitor-like cells, induced cardiosphere (iCS), via somatic reprogramming. However, the genome integration characteristic of virus-based reprogramming approach hampered their therapeutic applications due to the risk of tumour formation. In the current study, we aim to establish a safer iCS generation strategy with transgene-free approaches. MATERIALS AND METHODS: Four transgene-free approaches for somatic reprogramming, including episome, minicircle, self-replicative RNA, and sendai virus, were compared, from the perspective of cardiac progenitor marker expression, iCS formation, and cardiac differentiation. The therapeutic effects were assessed in the mouse model of MI, from the perspective of survival rate, cardiac function, and structural alterations. RESULTS: The self-replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. In addition, the CXCR4 (C-X-C chemokine receptor 4) positive subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a safer delivery approach. CONCLUSION: Thus, the optimized strategy for iCS generation is safer and has more therapeutic potentials.

Vitamin D Regulates CXCL12/CXCR4 and Epithelial-to-Mesenchymal Transition in a Model of Breast Cancer Metastasis to Lung.

Vitamin D deficiency is associated with poor cancer outcome in humans, and administration of vitamin D or its analogs decreases tumor progression and metastasis in animal models. Using the mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) model of mammary cancer, we previously demonstrated a significant acceleration of carcinogenesis in animals on a low vitamin D diet and a reduction in spontaneous lung metastases when mice received vitamin D through perfusion. We investigate here the action mechanism for vitamin D in lung metastasis in the same non-immunodeficient model and demonstrate that it involves the control of epithelial to mesenchymal transition as well as interactions between chemokine C-X-C motif chemokine 12 (CXCL12) and its receptor C-X-C chemokine receptor type 4 (CXCR4). In vitro, 10-9M vitamin D treatment modifies the phenotype of MMTV-PyMT primary mammary tumor cells and significantly decreases their invasiveness and mammosphere formation capacity by 40% and 50%, respectively. Vitamin D treatment also inhibits phospho-signal transducer and activator of transcription 3 (p-STAT3), zinc finger E-box-binding homeobox 1 (Zeb1), and vimentin by 52%, 75%, and 77%, respectively, and increases E-cadherin by 87%. In vivo, dietary vitamin D deficiency maintains high levels of Zeb1 and p-STAT3 in cells from primary mammary tumors and increases CXCL12 expression in lung stroma by 64%. In lung metastases, vitamin D deficiency increases CXCL12/CXCR4 co-localization by a factor of 2.5. These findings indicate an involvement of vitamin D in mammary cancer "seed" (primary tumor cell) and "soil" (metastatic site) and link vitamin D deficiency to epithelial-to-mesenchymal transition (EMT), CXCL12/CXCR4 signaling, and accelerated metastasis, suggesting vitamin D repleteness in breast cancer patients could enhance the efficacy of co-administered therapies in preventing spread to distant organs.

Novel Peptide-Based PET Probe for Non-invasive Imaging of C-X-C Chemokine Receptor Type 4 (CXCR4) in Tumors.

The recently reported CXCR4 antagonist 3 (Ac-Arg-Ala-[DCys-Arg-2Nal-His-Pen]-CO2H) was investigated as a molecular scaffold for a CXCR4-targeted positron emission tomography (PET) tracer. Toward this end, 3 was functionalized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononanetriacetic acid (NOTA). On the basis of convincing affinity data, both tracers, [68Ga]NOTA analogue ([68Ga]-5) and [68Ga]DOTA analogue ([68Ga]-4), were evaluated for PET imaging in "in vivo" models of CHO-hCXCR4 and Daudi lymphoma cells. PET imaging and biodistribution studies revealed higher CXCR4-specific tumor uptake and high tumor/background ratios for the [68Ga]NOTA analogue ([68Ga]-5) than for the [68Ga]DOTA analogue ([68Ga]-4) in both in vivo models. Moreover, [68Ga]-4 and [68Ga]-5 displayed rapid clearance and very low levels of accumulation in all nontarget tissues but the kidney. Although the high tumor/background ratios observed in the mouse xenograft model could partially derive from the hCXCR4 selectivity of [68Ga]-5, our results encourage its translation into a clinical context as a novel peptide-based tracer for imaging of CXCR4-overexpressing tumors.

Influence of ß-D-mannuronic Acid, as a New Member of Non-steroidal Anti- Inflammatory Drugs Family, on the Expression Pattern of Chemokines and their Receptors in Rheumatoid Arthritis.

BACKGROUND: Based on the encouraging results of phase III clinical trial of ß-Dmannuronic acid (M2000) (as a new anti-inflammatory drug) in patients with RA, in this study, we aimed to evaluate the effects of this drug on the expression of chemokines and their receptors in PBMCs of RA patients. METHODS: PBMCs of RA patients and healthy controls were separated and the patients' cells were treated with low, moderate and high doses (5, 25 and 50 µg/mL) of M2000 and optimum dose (1 µg/mL) of diclofenac, as a control in RPMI-1640 medium. Real-time PCR was used for evaluating the mRNA expression of CXCR3, CXCR4, CCR2, CCR5 and CCL2/MCP-1. Cell surface expression of CCR2 was investigated using flow cytometry. RESULTS: CCR5 mRNA expression reduced significantly, after treatment of the patients' cells with all three doses of M2000 and optimum dose of diclofenac. CXCR3 mRNA expression was downregulated significantly followed by the treatment of these cells with moderate and high doses of M2000 and optimum dose of diclofenac. CXCR4 mRNA expression declined significantly after the treatment of these cells with moderate and high doses of M2000. CCL2 mRNA expression significantly reduced only followed by the treatment of these cells with a high dose of M2000, whereas, mRNA and cell surface expressions of CCR2 diminished significantly followed by the treatment of these cells with a high dose of M2000 and optimum dose of diclofenac. CONCLUSION: According to our results, M2000 through the down-regulation of chemokines and their receptors may restrict the infiltration of immune cells into the synovium.

How to distinguish between different cell lineages sharing common markers using combinations of double in-situ-hybridization and immunostaining in avian embryos: CXCR4-positive mesodermal and neural crest-derived cells.

Cell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18-HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail.

Clinical association of CXCR4 in primary tumor of papillary thyroid cancer and response to iodine-131 treatment.

OBJECT: Papillary thyroid cancer (PTC) has an excellent prognosis. However, patients with such, if refract to radioiodine treatment, increase recurrent and mortality rates. Tumor aggressiveness in primary tumor of PTC expresses CXCR4 chemokine receptor. Thus, CXCR4 expression of the tumor may predict response to radioiodine treatment. MATERIALS AND METHODS: Retrospective review of seventy-four PTC patients, treated with total/near-total thyroidectomy and radioiodine treatment at King Chulalongkorn Memorial Hospital from January 2007 to 2013, were classified as non-radioiodine-refractory (non-RAIR) or RAIR treatment response. All histopathologic diagnoses were reviewed and paraffin blocks were retrieved for CXCR4 immunostaining, determined by automated digital imaging analysis for intensity and extension. The scores were compared between primary tumour and adjacent normal thyroid tissue as well as between the tissue of non-RAIR and that of RAIR. Factors determining type of RAI response were analyzed. RESULTS: CXCR4 immunostaining scores of PTC is significantly higher than normal thyroid [2.03 (0.52) and 1.48 (0.75)] [mean (SD)] (P = 0.0001). CXCR4 immunostaining scores in RAIR are potentially higher than non-RAIR [1.95 (0.54) and 2.13 (0.47) (P = 0.149)]. Odds ratio of CXCR4 immunostaining score for predicting RAIR treatment is 1.99 (P = 0.150). CXCR4 immunostaining scores positively associate with tumor size (R = 0.298, P = 0.01); whereas no significant association with other clinicopathologic factors. CONCLUSION: Our data support the notion that CXCR4 are significantly expressed in PTC tumor over normal thyroid tissues. However, there is no clinical association with radioiodine treatment response.

In vitro study of SDF-1α-loaded injectable and thermally responsive hydrogels for adipose stem cell therapy by SDF-1/CXCR4 axis.

Stem cell-based approaches have become a promising therapeutic strategy for treating ischemic diseases. The aim of this study was to develop injectable hydrogel systems for the local release of stromal cell-derived factor-1α (SDF-1α) to recruit adipose stem cells (ASCs) that express CXCR4 to achieve stem cell therapy and therapeutic angiogenesis. Thermoresponsive and injectable chitosan (CS)/ß-glycerophosphate disodium salt pentahydrate (ßGP) hydrogels with different concentrations of hyaluronic acid (HA) were designed and fabricated to achieve local and sustained release of SDF-1α for ASC recruitment. Herein, the material structures, physical properties, gelation temperature, and gelation time of hydrogels with different compositions were determined. The incorporation of 0.9% HA in CS-based hydrogels not only enhanced the gelation time but also increased the strength of the hydrogels. In addition, the results revealed that the thermoresponsive and injectable CS/ßGP/HA hydrogels showed good biocompatibility. In addition, the in vitro release profiles showed that the hydrogels achieved sustained release of SDF-1α over 7 days and enhanced ASC migration. The results revealed that the hydrogels with HA enhanced the sustained release effect compared with the hydrogel without HA, indicating that the HA content regulated the physical and release properties of the injectable hydrogels. Therefore, thermoresponsive and injectable CS/ßGP/HA hydrogels may provide an alternative for treating ischemic diseases via SDF-1/CXCR4 axis for ASC recruitment and retention.

Clinical significance and in vitro cellular regulation of hypoxia mimicry on HIF-1α and downstream genes in canine appendicular osteosarcoma.

Cellular adaptation to a hypoxic microenvironment is essential for tumour progression and is largely mediated by HIF-1α and hypoxia-regulated factors, including CXCR4, VEGF-A and GLUT-1. In human osteosarcoma, hypoxia is associated with resistance to chemotherapy as well as with metastasis and poor survival, whereas little is known about its role in canine osteosarcoma (cOSA). This study aimed primarily to evaluate the prognostic value of several known hypoxic markers in cOSA. Immunohistochemical analysis for HIF-1α, CXCR4, VEGF-A and GLUT-1 was performed on 56 appendicular OSA samples; correlations with clinicopathological features and outcome was investigated. The second aim was to investigate the in vitro regulation of markers under chemically induced hypoxia (CoCl2). Two primary canine osteosarcoma cell lines were selected, and Western blotting, immunofluorescence and qRT-PCR were used to study protein and gene expression. Dogs with high-grade OSA (35.7%) were more susceptible to the development of metastases (P = 0.047) and showed high HIF-1α protein expression (P = 0.007). Moreover, HIF-1α overexpression (56%) was correlated with a shorter disease-free interval (DFI; P = 0.01), indicating that it is a reliable negative prognostic marker. The in vitro experiments identified an accumulation of HIF-1α in cOSA cells after chemically induced hypoxia, leading to a significant increase in GLUT-1 transcript (P = 0.02). HIF-1α might be a promising prognostic marker, highlighting opportunities for the use of therapeutic strategies targeting the hypoxic microenvironment in cOSA. These results reinforce the role of the dog as a comparative animal model since similar hypoxic mechanisms are reported in human osteosarcoma.

[Effectiveness of intraperitoneal chemotherapy for t4 colon cancer]. / Effektivnost' vnutribryushnoi khimioterapii pri rake obodochnoi kishki T4.

OBJECTIVE: To determine the effect of intraperitoneal chemotherapy (IPC) with mitomycin C on expression of intraperitoneal cancer cells markers in patients with T4 colon cancer. MATERIAL AND METHODS: For the period from January 2019 to April 2020, 65 patients with T4 colon cancer were included in prospective comparative study. There were 46 patients in the main group and 19 patients in the control group. In the main group, surgical procedure was followed by IPC with mitomycin C. No IPC was performed in the control group. An effectiveness of IPC was evaluated using CD133, CD24, CD26, CD44, CD184 markers expression in peritoneal lavages. RESULTS: Significant between-group differences were observed for CD133 (p=0.0168), CD24 (p=0.0455) and CD44 (p=0.0012). There was a tendency to decrease in the level of CD184 expression in both groups in the second lavage (p=0.0605). CONCLUSION: IPC in patients with T4 colon cancer can reduce the expression and proliferative potential of free cancer cells.

Automated Synthesis of Fluorine-18 Labeled CXCR4 Ligand via the Conjugation with Nicotinic Acid N-Hydroxysuccinimide Ester (6-[18F]SFPy).

The C-X-C motif chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled receptor that is overexpressed in numerous diseases, particularly in various cancers and is a powerful chemokine, attracting cells to the bone marrow niche. Therefore, CXCR4 is an attractive target for imaging and therapeutic purposes. The goal of this study is to develop an efficient, reproducible, and straightforward method to prepare a fluorine-18 labeled CXCR4 ligand. 6-[18F]Fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[18F]FPy-TFP) and nicotinic acid N-hydroxysuccinimide ester (6-[18F]SFPy) have been prepared using 'fluorination on the Sep-Pak' method. Conjugation of 6-[18F]SFPy or 6-[18F]FPy-TFP with the alpha-amino group at the N terminus of the protected T140 precursor followed by deprotection, yielded the final product 6-[18F]FPy-T140. The overall radiochemical yields were 6-17% (n = 15, decay-corrected) in a 90-min radiolabeling time with a radiochemical purity >99%. 6-[18F]FPy-T140 exhibited high specific binding and nanomolar affinity for CXCR4 in vitro, indicating that the biological activity of the peptide was preserved. For the first time, [18F]SFPy has been prepared using 'fluorination on the Sep-Pak' method that allows rapid automated synthesis of 6-[18F]FPy-T140. In addition to increased synthetic efficiency, this construct binds with CXCR4 in high affinity and may have potential as an in vivo positron emission tomography (PET) imaging agent. This radiosynthesis method should encourage wider use of this PET agent to quantify CXCR4 in both research and clinical settings.

Prediction of relapse in stage I testicular germ cell tumor patients on surveillance: investigation of biomarkers.

BACKGROUND: Better biomarkers for assessing risk of relapse in stage I testicular germ cell tumor patients are needed, to complement classical histopathological variables. We aimed to assess the prognostic value of previously suggested biomarkers, related to proliferation (MIB-1 and TEX19) and to immune microenvironment (CXCL12, CXCR4, beta-catenin and MECA-79) in a surveillance cohort of stage I testicular germ cell tumor patients. METHODS: A total of 70 patients were included. Survival analyses were performed, including Cox regression models. RESULTS: Patients with vascular invasion and elevated human chorionic gonadotropin levels showed significantly poorer relapse-free survival in multivariable analysis (hazard ratio = 2.820, 95% confidence interval 1.257-6.328; hazard ratio = 3.025, 95% confidence interval 1.345-6.808). Patients with no vascular invasion but with MIB-1 staining in > 50% tumor cells showed significantly shorter relapse-free survival (p = 0.042). TEX19 nuclear immunoexpression was confirmed in spermatogonial cells, and weak cytoplasmic immunoexpression was depicted in 15/70 tumors, not significantly impacting survival. CXCL12 immunoexpression in tumor cells did not associate with relapse, but non-seminoma patients exhibiting vascular invasion and CXCL12-positive stromal/inflammatory cells showed significantly improved relapse-free survival (p = 0.015). Exclusively nuclear immunoexpression of CXCR4 associated with better relapse-free survival (p = 0.032), but not after adjusting for vascular invasion. Patients with higher beta-catenin scores showed a tendency for poorer relapse-free survival (p = 0.056). MECA-79 immunoexpression was absent. CONCLUSIONS: The informative protein biomarkers (i.e., MIB-1, CXCL12, beta-catenin, and possibly CXCR4) may prove useful for risk-stratifying patients if validated in larger, multicentric and well-defined studies. Currently, classical histopathological features of testicular germ cell tumors remain key for relapse prediction.

Cytotoxic drugs in combination with the CXCR4 antagonist AMD3100 as a potential treatment option for pediatric rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. The prognosis of advanced stage RMS remains poor, and metastatic invasion is a major cause of treatment failure. Therefore, there is an urgent need for treatment alternatives focusing on metastatic invasion and drug resistance. The stromal cell­derived factor­1 (SDF­1)/chemokine receptor 4 (CXCR4) axis is a crucial factor for metastatic invasion in RMS. Clinical data has revealed that high CXCR4 expression is associated with a poor outcome and a high metastatic rate in several malignancies, including RMS. Thus, targeting CXCR4 in addition to classical chemotherapy may improve the effectiveness of RMS treatment. In the present study, flow cytometry and reverse transcription­quantitative PCR were used to assess the effects of the combined treatment with a CXCR4 antagonist and chemotherapy on CXCR4 expression in the embryonal RMS (RME) cell line RD and in the alveolar RMS (RMA) cell line RH30. The functional effect of CXCR4 expression on the migratory behavior of RMS cells was analyzed using Transwell assays. Treatment with cytotoxic agents modulated CXCR4 expression in RMS cells in a dose­, drug­ and cell line dependent manner; however, this was not observed in RD cells with vincristine. The expression levels of CXCR4 significantly increased the migratory behavior of RMA and did not affect RME cell migration towards stromal cell­derived factor­1α (SDF­1α). AMD3100 markedly reduced the migration of RH30 cells in the Transwell assays compared with SDF­1α alone, and the cytotoxic agents doxorubicin and vincristine increased this effect. The results of the combined treatment in RMS cells using the CXCR4 antagonist AMD3100 together with cytotoxic drugs demonstrated that this approach may be a promising alternative for the treatment of advanced stage pediatric RMS. The observed effects of circumventing metastatic invasion and drug resistance should be further investigated in vivo.

Association of CXCR4 Expression and Clinical Outcome in Different Subsets of De Novo Acute Myeloid Leukemia Patients.

BACKGROUND: Acute myeloid leukemia is a heterogeneous group of diseases characterized by the uncontrolled proliferation of hematopoietic stem cells (HSCs) and progenitor cells with a reduced capacity to differentiate into mature cells. CXC chemokine receptor (CXCR4) and its ligand stromal derived factor-1 (SDF-1/CXCL12) are important players involved in cross-talk between leukemia cells and the bone marrow (BM) microenvironment. The aim to study the association between the immunohistochemical CXCR4 expression and the clinical outcome of AML in adult Egyptian patients. METHODS: Fifty-eight patients suffering from AML were recruited for this study, with an age range from 18 to 60 years and presenting from January 2013 to March 2017. All patients were subjected to complete blood count, BM aspiration, immunophenotyping, BM trephine biopsy, immunohistochemical staining with CXCR4 McAb and cytogenetics when feasible. RESULTS: CXCR4 was widely expressed (55.2%) among the studied patients. There was a significant relationship between CXCR4 and patients' outcomes. Fifteen (71.4%) patients who died were CXCR4 positive. The estimated mean time until death among CXCR4 negative cases was 37.6 ± 4.04 months which was longer than that of CXCR4 positive cases who had mean of 20.04 ± 4.9 months p = 0.016. The risk for death among CXCR4 positive cases was higher than CXCR4 negative cases with hazard ratio (HR) = 2.147 (p = 0.048). CONCLUSIONS: These results suggest that CXCR4 was expressed in a subset of AML patients and was associated with poor prognosis. CXCR4 expression appears to be an independent prognostic factor for survival in a heterogeneous group of AML patients.

CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.

G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of ß-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels.