Pamoic acid is an inhibitor of HMGB1·CXCL12 elicited chemotaxis and reduces inflammation in murine models of Pseudomonas aeruginosa pneumonia.

BACKGROUND: High-mobility group box 1 protein (HMGB1) is an ubiquitous nuclear protein that once released in the extracellular space acts as a Damage Associated Molecular Pattern and promotes inflammation. HMGB1 is significantly elevated during Pseudomonas aeruginosa infections and has a clinical relevance in respiratory diseases such as Cystic Fibrosis (CF). Salicylates are HMGB1 inhibitors. To address pharmacological inhibition of HMGB1 with small molecules, we explored the therapeutic potential of pamoic acid (PAM), a salicylate with limited ability to cross epithelial barriers. METHODS: PAM binding to HMGB1 and CXCL12 was tested by Nuclear Magnetic Resonance Spectroscopy using chemical shift perturbation methods, and inhibition of HMGB1·CXCL12-dependent chemotaxis was investigated by cell migration experiments. Aerosol delivery of PAM, with single or repeated administrations, was tested in murine models of acute and chronic P. aeruginosa pulmonary infection in C57Bl/6NCrlBR mice. PAM efficacy was evaluated by read-outs including weight loss, bacterial load and inflammatory response in lung and bronco-alveolar lavage fluid. RESULTS: Our data and three-dimensional models show that PAM is a direct ligand of both HMGB1 and CXCL12. We also showed that PAM is able to interfere with heterocomplex formation and the related chemotaxis in vitro. Importantly, PAM treatment by aerosol was effective in reducing acute and chronic airway murine inflammation and damage induced by P. aeruginosa. The results indicated that PAM reduces leukocyte recruitment in the airways, in particular neutrophils, suggesting an impaired in vivo chemotaxis. This was associated with decreased myeloperoxidase and neutrophil elastase levels. Modestly increased bacterial burdens were recorded with single administration of PAM in acute infection; however, repeated administration in chronic infection did not affect bacterial burdens, indicating that the interference of PAM with the immune system has a limited risk of pulmonary exacerbation. CONCLUSIONS: This work established the efficacy of treating inflammation in chronic respiratory diseases, including bacterial infections, by topical delivery in the lung of PAM, an inhibitor of HMGB1.

Sphingomyelin Depletion Inhibits CXCR4 Dynamics and CXCL12-Mediated Directed Cell Migration in Human T Cells.

Sphingolipids, ceramides and cholesterol are integral components of cellular membranes, and they also play important roles in signal transduction by regulating the dynamics of membrane receptors through their effects on membrane fluidity. Here, we combined biochemical and functional assays with single-particle tracking analysis of diffusion in the plasma membrane to demonstrate that the local lipid environment regulates CXCR4 organization and function and modulates chemokine-triggered directed cell migration. Prolonged treatment of T cells with bacterial sphingomyelinase promoted the complete and sustained breakdown of sphingomyelins and the accumulation of the corresponding ceramides, which altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under these conditions CXCR4 retained some CXCL12-mediated signaling activity but failed to promote efficient directed cell migration. Our data underscore a critical role for the local lipid composition at the cell membrane in regulating the lateral mobility of chemokine receptors, and their ability to dynamically increase receptor density at the leading edge to promote efficient cell migration.

Systemic Delivery of an Adjuvant CXCR4-CXCL12 Signaling Inhibitor Encapsulated in Synthetic Protein Nanoparticles for Glioma Immunotherapy.

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4+ monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

C­X­C receptor 7 agonist acts as a C­X­C motif chemokine ligand 12 inhibitor to ameliorate osteoclastogenesis and bone resorption.

The C­X­C receptor (CXCR) 7 agonist, VUF11207, is a chemical compound that binds specifically to CXCR7, and negatively regulates C­X­C motif chemokine ligand 12 (CXCL12) and CXCR4­induced cellular events. Lipopolysaccharide (LPS) can induce inflammatory cytokines and pathological bone loss. LPS also induces expression of CXCL12, enhancing sensitivity to receptor activator of NF­κB ligand (RANKL) and tumor necrosis factor­α (TNF­α) in vivo. RANKL and TNF­α induce the differentiation of osteoclasts into osteoclast precursors and bone resorption. The current study was performed to examine the effects of a CXCR7 agonist on osteoclastogenesis and bone resorption induced by LPS in vivo. In addition, the mechanisms underlying these in vivo effects were investigated by in vitro experiments. Eight­week­old male C57BL/6J mice were subcutaneously injected over the calvariae with LPS alone or LPS and CXCR7 agonist. After sacrifice, the number of osteoclasts and the bone resorption area were measured. In vitro experiments were performed to investigate the effects of CXCL12 and CXCR7 agonist on osteoclastogenesis induced by RANKL and TNF­α. Mice injected with LPS and CXCR7 agonist showed significantly reduced osteoclastogenesis and bone resorption compared with mice injected with LPS alone. Moreover, the CXCR7 agonist inhibited CXCL12 enhancement of RANKL­ and TNF­α­induced osteoclastogenesis in vitro. Thus, CXCR7 agonist inhibited LPS­induced osteoclast­associated cytokines, such as RANKL and TNF­α, as well as RANKL­ and TNF­α­induced osteoclastogenesis in vitro by modulating CXCL12­mediated enhancement of osteoclastogenesis. In conclusion, CXCR7 agonist reduced CXCL12­mediated osteoclastogenesis and bone resorption.

Combined inhibition of CXCL12 and PD-1 in MSS colorectal and pancreatic cancer: modulation of the microenvironment and clinical effects.

BACKGROUND: Immunotherapy in microsatellite stable colorectal or pancreatic cancer has not shown promising results. It has been hypothesized that targeting immunosuppressive molecules like SDF1-alpha/CXCL12 could contribute to immunotherapy and animal models showed promising results on T cell activation and migration in combination with immune checkpoint inhibition. METHODS: Here, we describe the successful application of anti-CXCL12 (NOX-A12) in patients with advanced stage pretreated metastatic colorectal and pancreatic cancer (OPERA trial). The treatment consisted of 2 weeks of anti-CXCL12 monotherapy with NOX-A12 followed by combination therapy with pembrolizumab (n=20 patients) until progression or intolerable toxicity had occurred. RESULTS: The treatment was safe and well tolerated with 83.8% grade I/II, 15.5% grade III and 0.7% grade V adverse events. Of note, for a majority of patients, time on trial treatment was prolonged compared with their last standard treatment preceding trial participation. Systematic serial biopsies revealed distinct patterns of modulation. Tissue and clinical responses were associated with Th1-like tissue reactivity upon CXCL12 inhibition. A downregulation of a cytokine cassette of interleukin (IL)-2/IL-16/CXCL-10 was associated with tumor resistance and furthermore linked to a rare, CXCL12-associated CD14+CD15+promonocytic population. T cells showed aggregation and directed movement towards the tumor cells in responding tissues. Serum analyses detected homogeneous immunomodulatory patterns in all patients, regardless of tissue responses. CONCLUSIONS: We demonstrate that the combination of CXCL12 inhibition and checkpoint inhibition is safe and grants further exploration of synergistic combinatorial strategies.

Combination of dociparstat sodium (DSTAT), a CXCL12/CXCR4 inhibitor, with azacitidine for the treatment of hypomethylating agent refractory AML and MDS.

Leukemia stem cells utilize cell adhesion molecules like CXCR4/CXCL12 to home to bone marrow stromal niches where they are maintained in a dormant, protected state. Dociparstat sodium (DSTAT, CX-01) is a low anticoagulant heparin with multiple mechanisms of action, including inhibition of the CXCR4/CXCL12 axis, blocking HMGB1, and binding platelet factor 4 (PF-4). We conducted a pilot study adding DSTAT to azacitidine for patients with AML or MDS unresponsive to or relapsed after prior hypomethylating agent therapy, hypothesizing that DSTAT may improve response rates. Twenty patients were enrolled, with a median of 2 prior lines of therapy and 6 cycles of prior hypomethylating agents. Among fifteen patients evaluable for response, there was 1 complete remission, and 3 marrow complete remissions, for a response rate of 27 % among evaluable patients (20 % overall). Hematologic improvement was observed in 5 additional patients. The median overall survival for all enrolled patients was 205 days (95 % CI 119-302). While cytopenias and infections were common, these were not out of proportion to what would be expected in this population of patients undergoing treatment with azacitidine alone. In summary, this trial demonstrated the feasibility of combining DSTAT with azacitidine, with several responses observed, suggesting this combination warrants further study.

Systematic Development of Peptide Inhibitors Targeting the CXCL12/HMGB1 Interaction.

During inflammatory reactions, the production and release of chemotactic factors guide the recruitment of selective leukocyte subpopulations. The alarmin HMGB1 and the chemokine CXCL12, both released in the microenvironment, can form a heterocomplex, which exclusively acts on the chemokine receptor CXCR4, enhancing cell migration, and in some pathological conditions such as rheumatoid arthritis exacerbates the immune response. An excessive cell influx at the inflammatory site can be diminished by disrupting the heterocomplex. Here, we report the computationally driven identification of the first peptide (HBP08) binding HMGB1 and selectively inhibiting the activity of the CXCL12/HMGB1 heterocomplex. Furthermore, HBP08 binds HMGB1 with the highest affinity reported so far (Kd of 0.8 ± 0.4 µM). The identification of this peptide represents an important step toward the development of innovative pharmacological tools for the treatment of severe chronic inflammatory conditions characterized by an uncontrolled immune response.

CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies.

Chemokines are chemotactic cytokines that promote cancer growth, metastasis, and regulate resistance to chemotherapy. Stromal cell-derived factor 1 (SDF1) also known as C-X-C motif chemokine 12 (CXCL12), a prognostic factor, is an extracellular homeostatic chemokine that is the natural ligand for chemokine receptors C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or cluster of differentiation 184 (CD184) and chemokine receptor type 7 (CXCR7). CXCR4 is the most widely expressed rhodopsin-like G protein coupled chemokine receptor (GPCR). The CXCL12-CXCR4 axis is involved in tumor growth, invasion, angiogenesis, and metastasis in colorectal cancer (CRC). CXCR7, recently termed as atypical chemokine receptor 3 (ACKR3), is amongst the G protein coupled cell surface receptor family that is also commonly expressed in a large variety of cancer cells. CXCR7, like CXCR4, regulates immunity, angiogenesis, stem cell trafficking, cell growth and organ-specific metastases. CXCR4 and CXCR7 are expressed individually or together, depending on the tumor type. When expressed together, CXCR4 and CXCR7 can form homo- or hetero-dimers. Homo- and hetero-dimerization of CXCL12 and its receptors CXCR4 and CXCR7 alter their signaling activity. Only few drugs have been approved for clinical use targeting CXCL12-CXCR4/CXCR7 axis. Several CXCR4 inhibitors are in clinical trials for solid tumor treatment with limited success whereas CXCR7-specific inhibitors are still in preclinical studies for CRC. This review focuses on current knowledge of chemokine CXCL12 and its receptors CXCR4 and CXCR7, with emphasis on targeting the CXCL12-CXCR4/CXCR7 axis as a treatment strategy for CRC.

Cancer-Associated Fibroblasts in Mycosis Fungoides Promote Tumor Cell Migration and Drug Resistance through CXCL12/CXCR4.

Cancer cells are known to reprogram normal fibroblasts into cancer-associated fibroblasts (CAFs) to act as tumor supporters. The presence and role of CAFs in mycosis fungoides (MF), the most common type of cutaneous T-cell lymphoma, are unknown. This study sought to characterize CAFs in MF and their cross talk with the lymphoma cells using primary fibroblast cultures from punch biopsies of patients with early-stage MF and healthy subjects. MF cultures yielded significantly increased levels of FAPα, a CAF marker, and CAF-associated genes and proteins: CXCL12 (ligand of CXCR4 expressed on MF cells), collagen XI, and matrix metalloproteinase 2. Cultured MF fibroblasts showed greater proliferation than normal fibroblasts in ex vivo experiments. A coculture with MyLa cells (MF cell line) increased normal fibroblast growth, reduced the sensitivity of MyLa cells to doxorubicin, and enhanced their migration. Inhibiting the CXCL12/CXCR4 axis increased doxorubicin-induced apoptosis of MyLa cells and reduced MyLa cell motility. Our data suggest that the fibroblasts in MF lesions are more proliferative than fibroblasts in normal skin and that CAFs protect MF cells from doxorubicin-induced cell death and increase their migration through the secretion of CXCL12. Reversing the CAF-mediated tumor microenvironment in MF may improve the efficiency of anticancer therapy.

Discovery of Potential Chemical Probe as Inhibitors of CXCL12 Using Ligand-Based Virtual Screening and Molecular Dynamic Simulation.

CXCL12 are small pro-inflammatory chemo-attractant cytokines that bind to a specific receptor CXCR4 with a role in angiogenesis, tumor progression, metastasis, and cell survival. Globally, cancer metastasis is a major cause of morbidity and mortality. In this study, we targeted CXCL12 rather than the chemokine receptor (CXCR4) because most of the drugs failed in clinical trials due to unmanageable toxicities. Until now, no FDA approved medication has been available against CXCL12. Therefore, we aimed to find new inhibitors for CXCL12 through virtual screening followed by molecular dynamics simulation. For virtual screening, active compounds against CXCL12 were taken as potent inhibitors and utilized in the generation of a pharmacophore model, followed by validation against different datasets. Ligand based virtual screening was performed on the ChEMBL and in-house databases, which resulted in successive elimination through the steps of pharmacophore-based and score-based screenings, and finally, sixteen compounds of various interactions with significant crucial amino acid residues were selected as virtual hits. Furthermore, the binding mode of these compounds were refined through molecular dynamic simulations. Moreover, the stability of protein complexes, Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and radius of gyration were analyzed, which led to the identification of three potent inhibitors of CXCL12 that may be pursued in the drug discovery process against cancer metastasis.

CXCL12/CXCR4 Axis-Targeted Dual-Functional Nano-Drug Delivery System Against Ovarian Cancer.

INTRODUCTION: Traditional chemotherapy for ovarian cancer is limited due to drug resistance and systemic side effects. Although various targeted drug delivery strategies have been designed to enhance drug accumulation at the tumor site, simply improvement of targeting capability has not consistently led to satisfactory outcomes. Herein, AMD3100 was selected as the targeting ligand because of its high affinity to chemokine receptor 4 (CXCR4), which was highly expressed on ovarian cancer cells. Moreover, the AMD3100 has been proved having blockage capability of stromal cell-derived factor 1 (SDF-1 or CXCL12)/CXCR4 axis and to be a sensitizer of chemotherapeutic therapy. We designed a dual-functional targeting delivery system by modifying paclitaxel (PTX)-loaded PEGylation bovine serum albumin (BSA) nanoparticles (NPs) with AMD3100 (AMD-NP-PTX), which can not only achieve specific tumor-targeting efficiency but also enhance the therapeutic outcomes. METHODS: AMD3100 was chemically modified to Mal-PEG-NHS followed by reacting with BSA, then AMD-NP-PTX was synthesized and characterized. The targeting efficiency of AMD-NP was evaluated both in vitro and in vivo. The anticancer effect of AMD-NP-PTX was determined on Caov3 cells and ovarian cancer-bearing nude mice. Finally, the potential therapeutic mechanism was studied. RESULTS: AMD-NP-PTX was synthesized successfully and well characterized. Cellular uptake assay and in vivo imaging experiments demonstrated that NPs could be internalized by Caov3 cells more efficiently after modification of AMD3100. Furthermore, the AMD-NP-PTX exhibited significantly enhanced inhibition effect on tumor growth and metastasis compared with PTX, NP-PTX and free AMD3100 plus NP-PTX both in vitro and in vivo, and demonstrated improved safety profile. We also confirmed that AMD-NP-PTX worked through targeting CXCL12/CXCR4 axis, thereby disturbing its downstream signaling pathways including epithelial-mesenchymal transition (EMT) processes and nuclear factor κB (NF-κB) pathway. CONCLUSION: The AMD-NP-PTX we designed would open a new avenue for dual-functional NPs in ovarian cancer therapy.

Electroacupuncture Alleviates Mechanical Allodynia in a Rat Model of Complex Regional Pain Syndrome Type-I via Suppressing Spinal CXCL12/CXCR4 Signaling.

Complex regional pain syndrome (CRPS) results in chronic and excruciating pain in patients. Conventional therapies lack effectiveness, rendering it one of the most difficult to treat neurological conditions.. Electroacupuncture (EA) is an effective alternative therapy for pain relief. Here, we investigated whether EA exerts analgesic effect on a rat model of CRPS type-I (CRPS-I) and related mechanisms. The rat chronic postischemic pain (CPIP) model was established to mimic CRPS-I. 100Hz EA exerted robust and persistent antiallodynic effect on CPIP model compared with 2 Hz EA or sham EA. EA markedly suppressed the overexpression of CXCL12/CXCR4 in spinal cord dorsal horn (SCDH) of CPIP model, leading to substantial decrease in neuronal and glial cell activities in SCDH. Pharmacological blocking CXCR4 mimicked EA-induced antiallodynic effect and related cellular events in SCDH, whereas exogenous CXCL12 abolished EA's effect. CXCR4 signaling resulted in ERK activation in SCDH, contributing to mechanical allodynia of CPIP model rats, whereas EA markedly reduced ERK activation. Therefore, we demonstrated that EA interferes with CXCL12/CXCR4 signaling in SCDH and downstream ERK pathway to exert robust antiallodynic effect on an animal model of CRPS-I. Our work suggests that EA may be a potential therapeutic option for CRPS-I in clinic. PERSPECTIVE: Our work identified that EA exerts robust antiallodynic effect on an animal model of CRPS-I, via mechanisms involving inhibition of CXCL12/CXCR4 signaling. EA further attenuates downstream neuronal and glial cell activation and ERK pathway in SCDH. This work suggests that EA may be a potential therapeutic option for CRPS-I management in clinic.

CXCL12 Attracts Bone Marrow-Derived Cells to Uterine Leiomyomas.

Uterine leiomyomas, also known as fibroids or myomas, are a common benign gynecologic tumor found in women of reproductive age. Though advances have been made in understanding leiomyomas, the etiology and pathogenesis of this disease are not fully characterized. Current evidence supports a role of putative human uterine stem/progenitor cells in the onset of uterine disease such as uterine myomas. In this study, we report that increased expression of CXCL12 in leiomyomas recruits bone marrow-derived cells (BMDCs) that may contribute to leiomyoma growth. Tissue was collected from leiomyomas or control myometrium from women with or without leiomyomas. qRT-PCR analysis showed increased expression of CXCL12 and decreased CXCR4 expression in the leiomyoma and myometrium of women with leiomyoma compared with normal myometrium. Increased CXCL12 protein secretion from cultured myoma cells was confirmed by ELISA. Further, we found that BMDCs migration was increased toward leiomyoma conditioned medium compared with conditioned medium from normal myometrium. CXCR4 antagonist AMD3100 completely blocked this migration. Engraftment of BMDCs significantly increased in myoma of mouse uteri treated with CXCL12 compared with placebo. We conclude that CXCL12 may play a role in leiomyomas growth by attracting bone marrow-derived cells to leiomyoma. Therefore, CXCL12 and its receptors are novel targets for leiomyoma therapy.

Amide-sulfamide modulators as effective anti-tumor metastatic agents targeting CXCR4/CXCL12 axis.

Breast cancer is the most frequently diagnosed malignancy and the second common cause of death in women worldwide. High mortality in breast cancer is frequently associated with metastatic progression rather than the primary tumor itself. It has been recently identified that the CXCR4/CXCL12 axis plays a pivotal role in breast cancer metastasis, especially in directing metastatic cancer cells to CXCL12-riched organs and tissues. Herein, taking the amide-sulfamide as the lead structure, the second-round structural modifications to the sulfamide structure were performed to obtain more active CXCR4 modulators against tumor metastasis. Both in vivo and in vitro experiments illustrated that compound IIIe possessed potent CXCR4 binding affinity, excellent anti-metastatic and anti-angiogenetic activity against breast cancer. More importantly, in a mouse breast cancer lung metastasis model, compound IIIe exerted a significant inhibitory effect on breast cancer metastasis. Taken together, all these positive results demonstrated that developing of CXCR4 modulators is a promising strategy to mediate breast cancer metastasis.

The role of PKC and PKD in CXCL12 directed prostate cancer migration.

Cancer metastasis is the cause of most cancer related deaths and many cancers are becoming resistant to current therapies. An alternative approach is to investigating signalling pathways that cause cancer cell migration such as chemokine signalling pathways. Such downstream signalling proteins are PKC and PKD. Therefore, we investigated the role of these two proteins in CXCL12 mediated PC3 prostate cancer migration. Whereas PKC and PKD inhibitors do not affect the release of calcium in PC3 prostate cancer cells, both are involved in migration, particularly inhibition of the atypical PKC isoform PKCζ causes the greatest reduction in PC3 cell migration. Classical and/or Novel PKC isoform inhibition changes the shape of the PC3 cells, they show a more rounded morphology, whereas PKD inhibition causes prostate cancer cell to elongate. PKCζ inhibition causes the enlargement of PC3 area possibly due to dysregulated actin cytoskeletal control. These results highlight the importance of verifying which signalling proteins, in which cell and in which chemokine signalling cascade enable cancer cellular migration.

Hypoxic preconditioning improves the survival and neural effects of transplanted mesenchymal stem cells via CXCL12/CXCR4 signalling in a rat model of cerebral infarction.

The treatment of neural deficiency after cerebral infarction is challenging, with limited therapeutic options. The transplantation of mesenchymal stem cells (MSCs) to the ischemic penumbra is a potential therapeutic approach. In the present study, a cerebral infarction model was generated by performing middle cerebral artery occlusion (MCAO) in SD rats. The expression of CXCR4 increased, and the number of MSCs migrating to the peri-infarct area was higher in rats transplanted with preconditioned MSCs than in rats transplanted with untreated MSCs. The rate of apoptosis, as evaluated by TUNEL staining and immunoblotting assays, was reduced in rats receiving preconditioned MSCs. A significant amelioration of neural regeneration and improved neurological function were observed in rats injected with preconditioned MSCs compared with those injected with untreated MSCs. However, the application of an siRNA targeting CXCL12 significantly inhibited the protective role of preconditioned MSCs against apoptosis and promoted the migration of MSCs to the ischemic area, leading to impaired neuronal regeneration and limited recovery of neuronal function. Hypoxic preconditioning of MSCs prior to transplantation suppressed apoptosis and increased their migration abilities, leading to the promotion of neuronal regeneration and improvement in neural function after transplantation. This preconditioning strategy may be considered as a potential approach for the modification of MSCs prior to cell transplantation therapy in patients with cerebral infarction. SIGNIFICANCE OF THE STUDY: We found that hypoxic preconditioning of MSCs improved their ability to promote neuronal regeneration and the recovery of neuronal function. Moreover, we showed that CXCR4 inhibited apoptosis, improved cell homing, and promoted neuronal differentiation, without influencing angiogenesis. Our study provides a relatively safe preconditioning method for potential use for cell transplantation therapy in ischemic cerebral infarction. The results presented here will facilitate the development of novel strategies and techniques to improve the tolerance and migration ability of transplanted cells for the treatment of cerebral infarction sequelae.

CCL2/MCP-1 and CXCL12/SDF-1 blockade by L-aptamers improve pancreatic islet engraftment and survival in mouse.

The blockade of pro-inflammatory mediators is a successful approach to improve the engraftment after islet transplantation. L-aptamers are chemically synthesized, nonimmunogenic bio-stable oligonucleotides that bind and inhibit target molecules conceptually similar to antibodies. We aimed to evaluate if blockade-aptamer-based inhibitors of C-C Motif Chemokine Ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and C-X-C Motif Chemokine Ligand 12/stromal cell-derived factor-1 (CXCL12/SDF-1) are able to favor islet survival in mouse models for islet transplantation and for type 1 diabetes. We evaluated the efficacy of the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12 on islet survival in a syngeneic mouse model of intraportal islet transplantation and in a multiple low doses of streptozotocin (MLD-STZ) diabetes induction model. Moreover, we characterized intrahepatic infiltrated leukocytes by flow cytometry before and 3 days after islet infusion in presence or absence of these inhibitors. The administration for 14 days of mNOX-E36 and NOX-A12 significantly improved islet engraftment, either compound alone or in combination. Intrahepatic islet transplantation recruited CD45+ leucocytes and more specifically CD45+/CD11b+ mono/macrophages; mNOX-E36 and NOX-A12 treatments significantly decreased the recruitment of inflammatory monocytes, CD11b+ /Ly6Chigh /CCR2+ and CD11b+ /Ly6Chigh /CXCR4+ cells, respectively. Additionally, both L-aptamers significantly attenuated diabetes progression in the MLD-STZ model. In conclusion, CCL2/MCP-1 and CXCL12/SDF-1 blockade by L-aptamers is an efficient strategy to improve islet engraftment and survival.

Dual-action CXCR4-targeting liposomes in leukemia: function blocking and drug delivery.

CXC chemokine receptor 4 (CXCR4) is overexpressed by a broad range of hematological disorders, and its interaction with CXC chemokine ligand 12 (CXCL12) is of central importance in the retention and chemoprotection of neoplastic cells in the bone marrow and lymphoid organs. In this article, we describe the biological evaluation of a new CXCR4-targeting and -antagonizing molecule (BAT1) that we designed and show that, when incorporated into a liposomal drug delivery system, it can be used to deliver cancer therapeutics at high levels to chronic lymphocytic leukemia (CLL) cells. CXCR4 targeting and antagonism by BAT1 were demonstrated alone and following its incorporation into liposomes (BAT1-liposomes). Antagonism of BAT1 against the CXCR4/CXCL12 interaction was demonstrated through signaling inhibition and function blocking: BAT1 reduced ERK phosphorylation and cell migration to levels equivalent to those seen in the absence of CXCL12 stimulation (P < .001). Specific uptake of BAT1-liposomes and delivery of a therapeutic cargo to the cell nucleus was seen within 3 hours of incubation and induced significantly more CLL cell death after 24 hours than control liposomes (P = .004). The BAT1 drug-delivery system is modular, versatile, and highly clinically relevant, incorporating elements of proven clinical efficacy. The combined capabilities to block CXCL12-induced migration and intracellular signaling while simultaneously delivering therapeutic cargo mean that the BAT1-liposome drug-delivery system could be a timely and relevant treatment of a range of hematological disorders, particularly because the therapeutic cargo can be tailored to the disease being treated.

Targeting CXCL12/CXCR4 and myeloid cells to improve the therapeutic ratio in patient-derived cervical cancer models treated with radio-chemotherapy.

BACKGROUND: The CXCL12/CXCR4 chemokine pathway is involved in cervical cancer pathogenesis and radiation treatment (RT) response. We previously reported that radiochemotherapy (RTCT) and concurrent administration of the CXCR4 inhibitor plerixafor improved primary tumour response. The aims of this study were to determine optimal sequencing of RTCT and plerixafor, the mechanisms responsible for improved response and the effect of plerixafor on late intestinal toxicity. METHODS: Orthotopic cervical cancer xenografts were treated with RTCT (30 Gy in 2 Gy fractions and cisplatin) with or without concurrent, adjuvant or continuous plerixafor. The endpoints were growth delay and molecular and immune cell changes at the end of treatment. Late intestinal toxicity was assessed by histologic examination of the rectum 90 days after a single 20 Gy fraction. RESULTS: RTCT increased CXCL12/CXCR4 signalling and the intratumoral accumulation of myeloid cells; the addition of plerixafor mitigated these effects. All of the RTCT and plerixafor arms showed prolonged tumour growth delay compared to RTCT alone, with the adjuvant arm showing the greatest improvement. Plerixafor also reduced late intestinal toxicity. CONCLUSION: Adding Plerixafor to RTCT blunts treatment-induced increases in CXCL12/CXCR4 signalling, improves primary tumour response and reduces intestinal side effects. This combination warrants testing in future clinical trials.

Bone-derived Nestin-positive mesenchymal stem cells improve cardiac function via recruiting cardiac endothelial cells after myocardial infarction.

BACKGROUND: Bone-derived mesenchymal stem cell (BMSC) transplantation has been reported to be effective for the treatment of ischemic heart disease, but whether BMSCs are the optimal cell type remains under debate. Increasing numbers of studies have shown that Nestin, an intermediate filament protein, is a potential marker for MSCs, which raises the question of whether Nestin+ cells in BMSCs may play a more crucial role in myocardial repair. METHODS: Nestin+ cells were isolated using flow cytometry by gating for CD45- Ter119- CD31- cells from the compact bone of Nestin-GFP transgenic mice, expressing GFP driven by the Nestin promoter. Colony-forming and proliferative curve assays were conducted to determine the proliferative capacity of these cells, while qRT-PCR was used to analyze the mRNA levels of relative chemokines and growth factors. Cardiac endothelial cell (CEC) recruitment was assessed via a transwell assay. Moreover, permanent ligation of the left anterior descending (LAD) coronary artery was performed to establish an acute myocardial infarction (AMI) mouse model. After cell transplantation, conventional echocardiography was conducted 1 and 4 weeks post-MI, and hearts were harvested for hematoxylin-and-eosin (HE) staining and immunofluorescence staining 1 week post-MI. Further evaluation of paracrine factor levels and administration of a neutralizing antibody (TIMP-1, TIMP-2, and CXCL12) or a CXCR4 antagonist (AMD3100) in MI hearts were performed to elucidate the mechanism involved in the chemotactic effect of Nestin+ BMSCs in vivo. RESULTS: Compared with Nestin- BMSCs, a greater proliferative capacity of Nestin+ BMSCs was observed, which further exhibited moderately high expression of chemokines instead of growth factors. More CEC recruitment in the Nestin+ BMSC-cocultured group was observed in vitro, while this effect was obviously abolished after treatment with neutralizing antibodies against TIMP-1, TIMP-2, or CXCL12, and more importantly, blocking the CXCL12/CXCR4 axis with a AMD3100 significantly reduced the chemotactic effect of Nestin+ BMSCs. After transplantation into mice exposed to myocardial infarction (MI), Nestin+ BMSC-treated mice showed significantly improved survival and left ventricular function compared with Nestin- BMSC-treated mice. Moreover, endogenous CECs were markedly increased, and chemokine levels were significantly higher, in the infarcted border zone with Nestin+ BMSC treatment. Meanwhile, neutralization of each TIMP-1, TIMP-2, or CXCL12 in vivo could reduce the left ventricular function at 1 and 4 weeks post-MI; importantly, the combined use of these three neutralizing antibodies could make a higher significance on cardiac function. Finally, blocking the CXCL12/CXCR4 axis with AMD3100 significantly reduced the left ventricular function and greatly inhibited Nestin+ BMSC-induced CEC chemotaxis in vivo. CONCLUSIONS: These results suggest that Nestin+ BMSC transplantation can improve cardiac function in an AMI model by recruiting resident CECs to the infarcted border region via the CXCL12/CXCR4 chemokine pathway. And we demonstrated that Nestin+BMSC-secreted TIMP-1/2 enhances CXCL12(SDF1α)/CXCR4 axis-driven migration of endogenous Sca-1+ endothelial cells in ischemic heart post-AMI. Taken together, our results show that Nestin is a useful marker for the identification of functional BMSCs and indicate that Nestin+ BMSCs could be a better therapeutic candidate for cardiac repair.