Global stability and parameter analysis reinforce therapeutic targets of PD-L1-PD-1 and MDSCs for glioblastoma.

Glioblastoma (GBM) is an aggressive primary brain cancer that currently has minimally effective treatments. Like other cancers, immunosuppression by the PD-L1-PD-1 immune checkpoint complex is a prominent axis by which glioma cells evade the immune system. Myeloid-derived suppressor cells (MDSCs), which are recruited to the glioma microenviroment, also contribute to the immunosuppressed GBM microenvironment by suppressing T cell functions. In this paper, we propose a GBM-specific tumor-immune ordinary differential equations model of glioma cells, T cells, and MDSCs to provide theoretical insights into the interactions between these cells. Equilibrium and stability analysis indicates that there are unique tumorous and tumor-free equilibria which are locally stable under certain conditions. Further, the tumor-free equilibrium is globally stable when T cell activation and the tumor kill rate by T cells overcome tumor growth, T cell inhibition by PD-L1-PD-1 and MDSCs, and the T cell death rate. Bifurcation analysis suggests that a treatment plan that includes surgical resection and therapeutics targeting immune suppression caused by the PD-L1-PD1 complex and MDSCs results in the system tending to the tumor-free equilibrium. Using a set of preclinical experimental data, we implement the approximate Bayesian computation (ABC) rejection method to construct probability density distributions that estimate model parameters. These distributions inform an appropriate search curve for global sensitivity analysis using the extended fourier amplitude sensitivity test. Sensitivity results combined with the ABC method suggest that parameter interaction is occurring between the drivers of tumor burden, which are the tumor growth rate and carrying capacity as well as the tumor kill rate by T cells, and the two modeled forms of immunosuppression, PD-L1-PD-1 immune checkpoint and MDSC suppression of T cells. Thus, treatment with an immune checkpoint inhibitor in combination with a therapeutic targeting the inhibitory mechanisms of MDSCs should be explored.

CCR2 inhibition reduces tumor myeloid cells and unmasks a checkpoint inhibitor effect to slow progression of resistant murine gliomas.

Immunotherapy directed at the PD-L1/PD-1 axis has produced treatment advances in various human cancers. Unfortunately, progress has not extended to glioblastoma (GBM), with phase III clinical trials assessing anti-PD-1 monotherapy failing to show efficacy in newly diagnosed and recurrent tumors. Myeloid-derived suppressor cells (MDSCs), a subset of immunosuppressive myeloid derived cells, are known to infiltrate the tumor microenvironment of GBM. Growing evidence suggests the CCL2-CCR2 axis is important for this process. This study evaluated the combination of PD-1 blockade and CCR2 inhibition in anti-PD-1-resistant gliomas. CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CD11b+/Ly6Chi/PD-L1+ MDSCs within established gliomas decreased with a concomitant increase in overall CCR2+ cells and MDSCs within bone marrow of CCR2-deficient mice. The CCR2 antagonist CCX872 increased median survival as a monotherapy in KR158 glioma-bearing animals and further increased median and overall survival when combined with anti-PD-1. Additionally, combination of CCX872 and anti-PD-1 prolonged median survival time in 005 GSC GBM-bearing mice. In both models, CCX872 decreased tumor associated MDSCs and increased these cells within the bone marrow. Examination of tumor-infiltrating lymphocytes revealed an elevated population, increased IFNγ expression, indicating enhanced cytolytic activity, as well as decreased expression of exhaustion markers in CD4+ and CD8+ T cells following combination treatment. These data establish that combining CCR2 and PD-1 blockade extends survival in clinically relevant murine glioma models and provides the basis on which to advance this combinatorial treatment toward early-phase human trials.

Detection of primary cilia in human glioblastoma.

Glioblastoma (GBM) is the most common malignant adult brain tumor and carries a poor prognosis due to primary and acquired resistance. While many cellular features of GBM have been documented, it is unclear if cells within these tumors extend a primary cilium, an organelle whose associated signaling pathways may regulate proliferation, migration, and survival of neural precursor and tumor cells. Using immunohistochemical and electron microscopy (EM) techniques, we screened human GBM tumor biopsies and primary cell lines for cilia. Immunocytochemical staining of five primary GBM cell lines revealed that between 8 and 25 % of the cells in each line possessed gamma tubulin-positive basal bodies from which extended acetylated, alpha-tubulin-positive axonemes. EM analyses confirmed the presence of cilia at the cell surface and revealed that their axonemes contained organized networks of microtubules, a structural feature consistent with our detection of IFT88 and Arl13b, two trafficked cilia proteins, along the lengths of the axonemes. Notably, cilia were detected in each of 23 tumor biopsies (22 primary and 1 recurrent) examined. These cilia were distributed across the tumor landscape including regions proximal to the vasculature and within necrotic areas. Moreover, ciliated cells within these tumors co-stained with Ki67, a marker for actively dividing cells, and ZEB1, a transcription factor that is upregulated in GBM and linked to tumor initiation, invasion, and chemoresistance. Collectively, our data show that subpopulations of cells within human GBM tumors are ciliated. In view of mounting evidence supporting roles of primary cilia in tumor initiation and propagation, it is likely that further study of the effects of cilia on GBM tumor cell function will improve our understanding of GBM pathogenesis and may provide new directions for GBM treatment strategies.

Glioma-derived M-CSF and IL-34 license M-MDSCs to suppress CD8+ T cells in a NOS-dependent manner.

Glioblastoma (GBM) is the most common malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a subset of myeloid cells, expressing monocytic (M)-MDSC markers and dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate the TME. This study evaluated the mechanism of CCR2+/CX3CR1+ M-MDSC differentiation and T cell suppressive function in murine glioma models. We determined that bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Glioma secreted CSF1R ligands M-CSF and IL-34 were identified as key drivers of M-MDSC differentiation while adenosine and iNOS pathways were implicated in M-MDSC suppression of T cells. Mining a human GBM spatial RNAseq database revealed a variety of different pathways that M-MDSCs utilize to exert their suppressive function that are driven by complex niches within the microenvironment. These data provide a more comprehensive understanding of the mechanism of M-MDSCs in glioblastoma.

Optimal control of combination immunotherapy for a virtual murine cohort in a glioblastoma-immune dynamics model.

The immune checkpoint inhibitor anti-PD-1, commonly used in cancer immunotherapy, has not been successful as a monotherapy for the highly aggressive brain cancer glioblastoma. However, when used in conjunction with a CC-chemokine receptor-2 (CCR2) antagonist, anti-PD-1 has shown efficacy in preclinical studies. In this paper, we aim to optimize treatment regimens for this combination immunotherapy using optimal control theory. We extend a treatment-free glioblastoma-immune dynamics ODE model to include interventions with anti-PD-1 and the CCR2 antagonist. An optimized regimen increases the survival of an average mouse from 32 days post-tumor implantation without treatment to 111 days with treatment. We scale this approach to a virtual murine cohort to evaluate mortality and quality of life concerns during treatment, and predict survival, tumor recurrence, or death after treatment. A parameter identifiability analysis identifies five parameters suitable for personalizing treatment within the virtual cohort. Sampling from these five practically identifiable parameters for the virtual murine cohort reveals that personalized, optimized regimens enhance survival: 84% of the virtual mice survive to day 100, compared to 60% survival in a previously studied experimental regimen. Subjects with high tumor growth rates and low T cell kill rates are identified as more likely to die during and after treatment due to their compromised immune systems and more aggressive tumors. Notably, the MDSC death rate emerges as a long-term predictor of either disease-free survival or death.

KR158 spheres harboring slow-cycling cells recapitulate GBM features in an immunocompetent system.

Glioblastoma (GBM) poses a significant challenge in clinical oncology due to its aggressive nature, heterogeneity, and resistance to therapies. Cancer stem cells (CSCs) play a critical role in GBM, particularly in treatment-resistance and tumor relapse, emphasizing the need to comprehend the mechanisms regulating these cells. Also, their multifaceted contributions to the tumor-microenvironment (TME) underline their significance, driven by their unique properties. This study aimed to characterize glioblastoma stem cells (GSCs), specifically slow-cycling cells (SCCs), in an immunocompetent murine GBM model to explore their similarities with their human counterparts. Using the KR158 mouse model, we confirmed that SCCs isolated from this model exhibited key traits and functional properties akin to human SCCs. KR158 murine SCCs, expanded in the gliomasphere assay, demonstrated sphere forming ability, self-renewing capacity, positive tumorigenicity, enhanced stemness and resistance to chemotherapy. Together, our findings validate the KR158 murine model as a framework to investigate GSCs and SCCs in GBM-pathology, and explore specifically the SCC-immune system communications, understand their role in disease progression, and evaluate the effect of therapeutic strategies targeting these specific connections.

KR158 Spheres Harboring Slow-Cycling Cells Recapitulate High-Grade Glioma Features in an Immunocompetent System.

Glioblastoma (GBM) poses a significant challenge in clinical oncology due to its aggressive nature, heterogeneity, and resistance to therapies. Cancer stem cells (CSCs) play a critical role in GBM, particularly in treatment resistance and tumor relapse, emphasizing the need to comprehend the mechanisms regulating these cells. Also, their multifaceted contributions to the tumor microenvironment (TME) underline their significance, driven by their unique properties. This study aimed to characterize glioblastoma stem cells (GSCs), specifically slow-cycling cells (SCCs), in an immunocompetent murine GBM model to explore their similarities with their human counterparts. Using the KR158 mouse model, we confirmed that SCCs isolated from this model exhibited key traits and functional properties akin to human SCCs. KR158 murine SCCs, expanded in the gliomasphere assay, demonstrated sphere forming ability, self-renewing capacity, positive tumorigenicity, enhanced stemness and resistance to chemotherapy. Together, our findings validate the KR158 murine model as a framework to investigate GSCs and SCCs in GBM pathology, and explore specifically the SCC-immune system communications, understand their role in disease progression, and evaluate the effect of therapeutic strategies targeting these specific connections.

Systemically targeting monocytic myloid-derrived suppressor cells using dendrimers and their cell-level biodistribution kinetics.

The focus of nanoparticles in vivo trafficking has been mostly on their tissue-level biodistribution and clearance. Recent progress in the nanomedicine field suggests that the targeting of nanoparticles to immune cells can be used to modulate the immune response and enhance therapeutic delivery to the diseased tissue. In the presence of tumor lesions, monocytic-myeloid-derived suppressor cells (M-MDSCs) expand significantly in the bone marrow, egress into peripheral blood, and traffic to the solid tumor, where they help maintain an immuno-suppressive tumor microenvironment. In this study, we investigated the interaction between PAMAM dendrimers and M-MDSCs in two murine models of glioblastoma, by examining the cell-level biodistribution kinetics of the systemically injected dendrimers. We found that M-MDSCs in the tumor and lymphoid organs can efficiently endocytose hydroxyl dendrimers. Interestingly, the trafficking of M-MDSCs from the bone marrow to the tumor contributed to the deposition of hydroxyl dendrimers in the tumor. M-MDSCs showed different capacities of endocytosing dendrimers of different functionalities in vivo. This differential uptake was mediated by the unique serum proteins associated with each dendrimer surface functionality. The results of this study set up the framework for developing dendrimer-based immunotherapy to target M-MDSCs for cancer treatment.

DR5 disulfide bonding as a sensor and effector of protein folding stress.

New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.

A model of GAG/MIP-2/CXCR2 interfaces and its functional effects.

MIP-2/CXCL2 is a murine chemokine related to human chemokines that possesses the Glu-Leu-Arg (ELR) activation motif and activates CXCR2 for neutrophil chemotaxis. We determined the structure of MIP-2 to 1.9 Å resolution and created a model with its murine receptor CXCR2 based on the coordinates of human CXCR4. Chemokine-induced migration of cells through specific G-protein coupled receptors is regulated by glycosaminoglycans (GAGs) that oligomerize chemokines. MIP-2 GAG-binding residues were identified that interact with heparin disaccharide I-S by NMR spectroscopy. A model GAG/MIP-2/CXCR2 complex that supports a 2:2 complex between chemokine and receptor was created. Mutants of these disaccharide-binding residues were made and tested for heparin binding, in vitro neutrophil chemotaxis, and in vivo neutrophil recruitment to the mouse peritoneum and lung. The mutants have a 10-fold decrease in neutrophil chemotaxis in vitro. There is no difference in neutrophil recruitment between wild-type MIP-2 and mutants in the peritoneum, but all activity of the mutants is lost in the lung, supporting the concept that GAG regulation of chemokines is tissue-dependent.

Hypoxia-induced endothelial CX3CL1 triggers lung smooth muscle cell phenotypic switching and proliferative expansion.

Distal arterioles with limited smooth muscles help maintain the high blood flow and low pressure in the lung circulation. Chronic hypoxia induces lung distal vessel muscularization. However, the molecular events that trigger alveolar hypoxia-induced peripheral endothelium modulation of vessel wall smooth muscle cell (SMC) proliferation and filling of nonmuscular areas are unclear. Here, we investigated the role of CX3CL1/CX3CR1 system in endothelial-SMC cross talk in response to hypoxia. Human lung microvascular endothelial cells responded to alveolar oxygen deficiency by overproduction of the chemokine CX3CL1. The CX3CL1 receptor CX3CR1 is expressed by SMCs that are adjacent to the distal endothelium. Hypoxic release of endothelial CX3CL1 induced SMC phenotypic switching from the contractile to the proliferative state. Inhibition of CX3CR1 prevented CX3CL1 stimulation of SMC proliferation and monolayer expansion. Furthermore, CX3CR1 deficiency attenuated spiral muscle expansion, distal vessel muscularization, and pressure elevation in response to hypoxia. Our findings indicate that the capillary endothelium relies on the CX3CL1-CX3CR1 axis to sense alveolar hypoxia and promote peripheral vessel muscularization. These results have clinical significance in the development of novel therapeutics that target mechanisms of distal arterial remodeling associated with pulmonary hypertension induced by oxygen deficiency that is present in people living at high altitudes and patients with obstructive lung diseases.

The Dynamics of Tumor-Infiltrating Myeloid Cell Activation and the Cytokine Expression Profile in a Glioma Resection Site during the Post-Surgical Period in Mice.

Glioblastoma is the most aggressive brain cancer and is highly infiltrated with cells of myeloid lineage (TIM) that support tumor growth and invasion. Tumor resection is the primary treatment for glioblastoma; however, the activation state of TIM at the site of tumor resection and its impact on glioma regrowth are poorly understood. Using the C57BL/6/GL261 mouse glioma implantation model, we investigated the state of TIM in the tumor resection area during the post-surgical period. TIM isolated from brain tissue at the resection site were analyzed at 0, 1, 4, 7, 14, and 21 days after tumor resection. An increase in expression of CD86 during the first 7 days after surgical resection and then upregulation of arginase 1 from the 14th to 21st days after resection were detected. Cytokine expression analysis combined with qRT-PCR revealed sustained upregulation of IL4, IL5, IL10, IL12, IL17, vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein 1 (MCP1/CCL2) in TIM purified from regrown tumors compared with primary implanted tumors. Flow cytometry analysis revealed increased CD86+/CD206+ population in regrown tumors compared with primary implanted tumors. Overall, we found that TIM in primary implanted tumors and tumors regrown after resection exhibited different phenotypes and cytokine expression patterns.

Glioma-derived CCL2 and CCL7 mediate migration of immune suppressive CCR2+/CX3CR1+ M-MDSCs into the tumor microenvironment in a redundant manner.

Glioblastoma (GBM) is the most common and malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized in part by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive, hematopoietic cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a potent subset of myeloid cells, expressing monocytic (M)-MDSC markers, distinguished by dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate into the TME. This study evaluated the T cell suppressive function and migratory properties of CCR2+/CX3CR1+ MDSCs. Bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Recombinant and glioma-derived CCL2 and CCL7 induce the migration of CCR2+/CX3CR1+ MDSCs with similar efficacy. KR158B-CCL2 and -CCL7 knockdown murine gliomas contain equivalent percentages of CCR2+/CX3CR1+ MDSCs compared to KR158B gliomas. Combined neutralization of CCL2 and CCL7 completely blocks CCR2-expressing cell migration to KR158B cell conditioned media. CCR2+/CX3CR1+ cells are also reduced within KR158B gliomas upon combination targeting of CCL2 and CCL7. High levels of CCL2 and CCL7 are also associated with negative prognostic outcomes in GBM patients. These data provide a more comprehensive understanding of the function of CCR2+/CX3CR1+ MDSCs and the role of CCL2 and CCL7 in the recruitment of these immune suppressive cells and further support the significance of targeting this chemokine axis in GBM.

Chemokine receptor CXCR3 promotes growth of glioma.

Human glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The poor prognosis and minimally successful treatments of GBM indicates a need to identify new therapeutic targets. In this study, we examined the role of CXCR3 in glioma progression using the GL261 murine model of malignant glioma. Intracranial GL261 tumors express CXCL9 and CXCL10 in vivo. Glioma-bearing CXCR3-deficient mice had significantly shorter median survival time and reduced numbers of tumor-infiltrated natural killer and natural killer T cells as compared with tumor-bearing wild-type (WT) mice. In contrast, pharmacological antagonism of CXCR3 with NBI-74330 prolonged median survival times of both tumor-bearing WT and CXCR3-deficient mice when compared with vehicle-treated groups. NBI-74330 treatment did not impact tumor infiltration of lymphocytes and microglia. A small percentage of GL261 cells were identified as CXCR3(+), which was similar to the expression of CXCR3 in several grade IV human glioma cell lines (A172, T98G, U87, U118 and U138). When cultured as gliomaspheres (GS), the human and murine lines increased CXCR3 expression; CXCR3 expression was also found in a primary human GBM-derived GS. Additionally, CXCR3 isoform A was expressed by all lines, whereas CXCR3-B was detected in T98G-, U118- and U138-GS cells. CXCL9 or CXCL10 induced in vitro glioma cell growth in GL261- and U87-GS as well as inhibited cell loss in U138-GS cells and this effect was antagonized by NBI-74330. The results suggest that CXCR3 antagonism exerts a direct anti-glioma effect and this receptor may be a potential therapeutic target for treating human GBM.

Modulation of the chemokine/chemokine receptor axis as a novel approach for glioma therapy.

Chemokines are a large subfamily of cytokines known for their ability to facilitate cell migration, most notably leukocytes, throughout the body. Chemokines are necessary for a functioning immune system in both health and disease and have received considerable attention for their roles in orchestrating temporal-spatial regulation of immune cell populations in cancer. Gliomas comprise a group of common central nervous system (CNS) primary tumors that are extremely challenging to treat. Immunotherapy approaches for highly malignant brain tumors offer an exciting new avenue for therapeutic intervention but so far, have seen limited successful clinical outcomes. Herein we focus on important chemokine/chemokine receptor systems in the regulation of pro- and anti-tumor mechanisms, highlighting potential therapeutic advantages of modulating these systems in malignant gliomas and other cancers.

Hematopoietic- and neurologic-expressed sequence 1 (Hn1) depletion in B16.F10 melanoma cells promotes a differentiated phenotype that includes increased melanogenesis and cell cycle arrest.

The Hematopoietic- and neurologic-expressed sequence 1 (Hn1) gene encodes a small protein that is highly conserved among species. Hn1 expression is upregulated in regenerating neural tissues, including the axotomized adult rodent facial motor nerve and dedifferentiating retinal pigment epithelial cells of the Japanese newt. It is also expressed in numerous tissues during embryonic development as well as in regions of the adult brain that exhibit high plasticity. Hn1 has also been reported as a marker for human ovarian carcinoma and it is expressed in high-grade human gliomas. This study was directed toward understanding the function of Hn1 in a murine melanoma cell line. Hn1 mRNA and protein were identified in B16.F10 cells and in tumors formed from these cells. Inhibition of Hn1 protein expression with siRNA increased melanogenesis. Hn1-depleted cells expressed higher levels of the melanogenic proteins tyrosinase and Trp2 and an increased interaction between actin and Rab27a. The in vitro cell growth rate of Hn1-depleted cells was significantly reduced due to G1/S cell cycle arrest. This was consistent with a reduction in the phosphorylation of retinoblastoma protein as well as lower levels of p27 and increased expression of p21. Decreased expression of c-Met, the receptor for hepatocyte growth factor, was also detected in the Hn1-depleted cells, however HGF-dependent stimulation of phosphorylated-ERK was unaffected. Hn1 depletion also led to increased basal levels of phosphorylated p38 MAPK, while basal ERK phosphorylation was reduced. Moreover, Hn1-depleted cells had reduced expression of transcription factors MITF and USF-1, and increased expression of TFE3. These data, coupled with reports on Hn1 expression in regeneration and development, suggest that Hn1 functions as a suppressor of differentiation in cells undergoing repair or proliferation.

Adult immuno-oncology: using past failures to inform the future.

In oncology, "immunotherapy" is a broad term encompassing multiple means of utilizing the patient's immune system to combat malignancy. Prominent among these are immune checkpoint inhibitors, cellular therapies including chimeric antigen receptor T-cell therapy, vaccines, and oncolytic viruses. Immunotherapy for glioblastoma (GBM) has had mixed results in early trials. In this context, the past, present, and future of immune oncology for the treatment of GBM was discussed by clinical, research, and thought leaders as well as patient advocates at the first annual Remission Summit in 2019. The goal was to use current knowledge (published and unpublished) to identify possible causes of treatment failures and the best strategies to advance immunotherapy as a treatment modality for patients with GBM. The discussion focuses on past failures, current limitations, failure analyses, and proposed best practices moving forward.

CX3CL1 and CX3CR1 in the GL261 murine model of glioma: CX3CR1 deficiency does not impact tumor growth or infiltration of microglia and lymphocytes.

Human glioblastoma multiforme (GBM) is the most malignant form of human brain tumors. A characteristic of GBM is the marked presence of tumor infiltrated microglia/macrophages and lymphocytes. The goal of this study was directed toward understanding the role of the chemokine system CX3CL1 and its receptor CX3CR1 in the GL261 murine model of malignant glioma. In situ hybridization analysis identified CX3CL1 and CX3CR1 expression in GL261 tumors. The impact of CX3CR1 deletion on the growth of intracranial GL261 gliomas and associated immune cell infiltration was evaluated in CX3CR1 gene-disrupted C57BL/6 mice. A slight increase in the tumor growth rate in CX3CR1-/- mice was evident with similar numbers of microglia and CD4+, CD8+, FoxP3+, or Ly49G2+ lymphocytes within tumors established in CX3CR1 +/- and -/- mice. These data indicate that CX3CR1 has little or no effects on either gliomagenesis or the migration of microglia and lymphocytes into GL261 tumors.

Nitric oxide cytoskeletal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes.

Stromal-derived factor-1 (SDF-1) is a critical chemokine for endothelial progenitor cell (EPC) recruitment to areas of ischemia, allowing these cells to participate in compensatory angiogenesis. The SDF-1 receptor, CXCR4, is expressed in developing blood vessels as well as on CD34+ EPCs. We describe that picomolar and nanomolar concentrations of SDF-1 differentially influence neovascularization, inducing CD34+ cell migration and EPC tube formation. CD34+ cells isolated from diabetic patients demonstrate a marked defect in migration to SDF-1. This defect is associated, in some but not all patients, with a cell surface activity of CD26/dipeptidyl peptidase IV, an enzyme that inactivates SDF-1. Diabetic CD34+ cells also do not migrate in response to vascular endothelial growth factor and are structurally rigid. However, incubating CD34+ cells with a nitric oxide (NO) donor corrects this migration defect and corrects the cell deformability. In addition, exogenous NO alters vasodilator-stimulated phosphoprotein and mammalian-enabled distribution in EPCs. These data support a common downstream cytoskeletal alteration in diabetic CD34+ cells that is independent of growth factor receptor activation and is correctable with exogenous NO. This inability of diabetic EPCs to respond to SDF-1 may contribute to aberrant tissue vascularization and endothelial repair in diabetic patients.