Michael J. Ting

EphA3 Maintains Tumorigenicity and Is a Therapeutic Target in Glioblastoma Multiforme

Significant endeavor has been applied to identify functional therapeutic targets in glioblastoma (GBM) to halt the growth of this aggressive cancer. We show that the receptor tyrosine kinase EphA3 is frequently overexpressed in GBM and, in particular, in the most aggressive mesenchymal subtype. Importantly, EphA3 is highly expressed on the tumor-initiating cell population in glioma and appears critically involved in maintaining tumor cells in a less differentiated state by modulating mitogen-activated protein kinase signaling. EphA3 knockdown or depletion of EphA3-positive tumor cells reduced tumorigenic potential to a degree comparable to treatment with a therapeutic radiolabelled EphA3-specific monoclonal antibody. These results identify EphA3 as a functional, targetable receptor in GBM.

Activation of ephrin A proteins influences hematopoietic stem cell adhesion and trafficking patterns

OBJECTIVE To determine if Eph receptors and ephrins can modulate the homing of hematopoietic cells in a murine bone marrow transplantation model. MATERIALS AND METHODS EphA and ephrin A gene expression by mouse hematopoietic stem cells and the progenitor cell line FDCP-1 was determined by real-time reverse transcription polymerase chain reaction and flow cytometry. The effect of ephrin A activation on adhesion of hematopoietic progenitors was determined by in vitro adhesion assays in which cells were exposed to fibronectin or vascular cell adhesion molecule-1 (VCAM-1) and an increasing gradient of immobilized EphA3-Fc. Adhesion to fibronectin and VCAM-1 was further investigated using soluble preclustered EphA3-Fc. We used soluble unclustered EphA3-Fc as an antagonist to block endogenous EphA-ephrin A interactions in vivo. The effect of injecting soluble EphA3-Fc on the mobilization of hematopoietic progenitor cells was examined. We determined the effect on short-term homing by pretreating bone marrow cells with EphA3-Fc or the control IgG before infusion into lethally irradiated mice. RESULTS Preclustered and immobilized EphA3-Fc increased adhesion of progenitor cells and FDCP-1 to fibronectin and VCAM-1 (1.6- to 2-fold higher adhesion; p < 0.05) relative to control (0 μ/cm(2) EphA3-Fc extracellular molecule alone). Injection of the antagonist soluble EphA3-Fc increased progenitor cell and colony-forming unit-spleen cells in the peripheral blood (42% greater colony-forming unit in culture; p < 0.05, 3.8-fold higher colony-forming unit-spleen) relative to control. CONCLUSION Treating bone marrow cells with EphA3-Fc resulted in a reduction by 31% in donor stem cells homing to the bone marrow and accumulation of donor cells in recipient spleens (50% greater than control) and greater recovery of donor stem cells from the peripheral blood.

Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells

BackgroundFetal mesenchymal stem/stromal cells (MSC) represent a developmentally-advantageous cell type with translational potential.To enhance adult MSC migration, studies have focussed on the role of the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12), but more recent work implicates an intricate system of CXCR4 receptor dimerization, intracellular localization, multiple ligands, splice variants and nuclear accumulation. We investigated the intracellular localization of CXCR4 in fetal bone marrow-derived MSC and role of intracellular trafficking in CXCR4 surface expression and function.ResultsWe found that up to 4% of human fetal MSC have detectable surface-localized CXCR4. In the majority of cells, CXCR4 is located not at the cell surface, as would be required for ‘sensing’ migratory cues, but intracellularly. CXCR4 was identified in early endosomes, recycling endosomes, and lysosomes, indicating only a small percentage of CXCR4 travelling to the plasma membrane. Notably CXCR4 was also found in and around the nucleus, as detected with an anti-CXCR4 antibody directed specifically against CXCR4 isoform 2 differing only in N-terminal sequence. After demonstrating that endocytosis of CXCR4 is largely independent of endogenously-produced SDF-1, we next applied the cytoskeletal inhibitors blebbistatin and dynasore to inhibit endocytotic recycling. These increased the number of cells expressing surface CXCR4 by 10 and 5 fold respectively, and enhanced the number of cells migrating to SDF1 in vitro (up to 2.6 fold). These molecules had a transient effect on cell morphology and adhesion, which abated after the removal of the inhibitors, and did not alter functional stem cell properties.ConclusionsWe conclude that constitutive endocytosis is implicated in the regulation of CXCR4 membrane expression, and suggest a novel pharmacological strategy to enhance migration of systemically-transplanted cells.

EPHA4-FC treatment reduces ischemia/reperfusion-induced intestinal injury by inhibiting vascular permeability

ABSTRACT The inflammatory response is characterized by increased endothelial permeability, which permits the passage of fluid and inflammatory cells into interstitial spaces. The Eph/ephrin receptor ligand system plays a role in inflammation through a signaling cascade, which modifies Rho-GTPase activity. We hypothesized that blocking Eph/ephrin signaling using an EphA4-Fc would result in decreased inflammation and tissue injury in a model of ischemia/reperfusion (I/R) injury. Mice undergoing intestinal I/R pretreated with the EphA4-Fc had significantly reduced intestinal injury compared to mice injected with the control Fc. This reduction in I/R injury was accompanied by significantly reduced neutrophil infiltration, but did not affect intestinal inflammatory cytokine generation. Using microdialysis, we identified that intestinal I/R induced a marked increase in systemic vascular leakage, which was completely abrogated in EphA4-Fc-treated mice. Finally, we confirmed the direct role of Eph/ephrin signaling in endothelial leakage by demonstrating that EphA4-Fc inhibited tumor necrosis factor-&agr;–induced vascular permeability in human umbilical vein endothelial cells. This study identifies that Eph/ephrin interaction induces proinflammatory signaling in vivo by inducing vascular leak and neutrophil infiltration, which results in tissue injury in intestinal I/R. Therefore, therapeutic targeting of Eph/ephrin interaction using inhibitors, such as EphA4-Fc, may be a novel method to prevent tissue injury in acute inflammation by influencing endothelial integrity and by controlling vascular leak.

The role of Eph receptor tyrosine kinases and ephrin ligands in hematopoietic cell development and function

The Eph receptor tyrosine kinases form the largest sub-family of the Receptor tyrosine kinases. Eph receptors interact with membrane bound ligands, termed ephrins. Eph-ephrin interactions have significant roles during mammalian development where they mediate such processes as cell adhesion and migration. Although Eph/ephrin expression is low in adult tissues evidence is accumulating which indicates that Eph and ephrins continue to play significant roles in postembryonic tissues. In this review we describe the current understanding of the function of Eph-ephrin signaling in human and mouse hematopoiesis. Ephs and ephrins have been shown to regulate the differentiation of hematopoietic cells, to direct lymphoid development as well as immune function. Whilst much still remains to be learned about the role of Ephephrin signaling in hematopoieis it is already clear that this system exerts important regulatory effects on differentiation, cell migration and effector function. The possibility that these molecules may be therapeutic targets is also discussed.

EphA2KO mice survive longer in a mouse model of sepsis

CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at sites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. In this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion led to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. In order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs were too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. Collectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.Changes in microbiome, mucosal immunity and intestinal integrity have been associated with the onset of Type 1 Diabetes (T1D) in children. Toll-like Receptors (TLR) have been associated all three factors. The role of TLR and their effects on microbiome in autoimmunity were studied by crossing TLR1,2,4,6,9 and MyD88 targeted deficiency mutations to the type 1 diabetes (T1D)-prone NOD mouse strain. While NOD.Tlr9-/- and NOD.Tlr6-/- mice were unaffected, T1D in NOD.Tlr4-/- and NOD.Tlr1-/- mice was exacerbated and that in NOD.Myd88-/- and NOD.Tlr2-/- mice ameliorated. Physical parameters of the intestines were compared; ileal weight was reduced in NOD.Tlr1-/-mice. Similarly, by histology, these mice had reduced villus length and width. The intestinal microbiomes of NOD wild-type (WT), NOD.Tlr1-/-, NOD.Tlr2-/- and NOD.Tlr4-/- mice were compared by high throughput sequencing of 16S ribosomal DNA (rDNA), in two cohorts, 18 months apart. Analysis of caecal 16S sequences clearly resolved the mouse lines and there were significant differences in beta diversity between the strains, with individual bacterial species contributing greatly to the differences in the microbiota of individual TLR-deficient strains. To test the relationship between microbiome and T1D, all strains were re-derived onto the parental NOD/Lt line and the incidence of T1D re-assessed within two generations. All rederived lines expressed an incidence of disease similar to that of the parental line. TLR deficiencies are associated with changes in microbiome; changes of microbiome are associated with T1D; the effects of TLR deficiencies on T1D appear to be mediated by their effects on gut flora.Intestinal TCRb+CD4-CD8b-CD8a+ (CD8aa) IELs alleviate T cell induced colitis and have been suggested to play a role in virus infection and cancer. Their thymic development has been elucidated to some extent, as IEL precursors (IELp) are known to be CD4-CD8-CD5+TCRb+, but is not yet fully understood. Within the thymus, mature IELp were identified based on their expression of CD122 and MHC class I. Two major phenotypic subsets exist within this mature thymic IELp population: a PD1+Tbet- population that preferentially expresses a4b7, and a PD1-Tbet+ population with preferential CD103 expression. These two populations were also distinct in their Valpha repertoire. The PD1+a4b7+ population contains clones that are strongly self-reactive as judged by Nur77GFP and their dramatic increase in Bim deficient mice, while the PD1-Tbet+ population did not show these characteristics. Both gave rise to CD8aa IELs upon adoptive transfer into RAG-/- recipients. However intrathymic labeling revealed that PD1+a4b7+ IELp were the major thymic emigrating population, and emigration was S1P1-dependent. In contrast, PD1-Tbet+ IELp expressed CXCR3, were retained, and accumulated in the thymus with age. Preliminary immunofluorescence data furthermore indicate differential thymic cortico-medullary localization of the IELp subtypes. These experiments more precisely define the behavior of IEL precursors.

Abstract 1197: EphA3 kinase ablation induces glioblastoma differentiation and prevents tumor progression

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Eph receptors constitute the largest sub-family of receptor tyrosine kinases and interact with membrane-bound ligands termed ephrins. Eph and ephrins have many vital functions including cell adhesion, migration and axon guidance. Eph and ephrins have been found to be aberrantly expressed in many malignancies including brain tumor. The purpose of this study was to investigate EphA3 receptor function in the most common and aggressive form of brain tumor, Glioblastoma (GBM). Methods: Gene expression was investigated by Q-PCR, IHC and flow cytometry in high grade glioma (HGG) surgical specimens and primary derived serum free cell cultures. Targeted reduction of Eph expression was performed using both a constitutive and inducible shRNA system. Murine in-vivo studies were performed using both subcutaneous and orthotopic ‘intracranial’ xenografts in immuno-compromised animals. Signaling pathways were assessed by western blotting. Results: To establish whether the receptor tyrosine kinase EphA3 was over expressed in HGG we assessed 12 normal human brain specimens, 56 HGG specimens and 26 HGG primary cultures. EphA3 mRNA expression was negligible in normal brain while 30% of clinical specimens and 46% of primary cultured tumor cells expressed EphA3. EphA3 protein was also detected in HGG clinical specimens using IHC. To further investigate EphA3 function the receptor was down regulated using shRNA in an EphA3+ GBM neurosphere cell line. Constitutive and inducible down regulation of the EphA3 receptor resulted in initiation of neuronal and glial cell differentiation following activation of the ERK/MAPK pathway. A reduction in stem/progenitor cell proliferation was also observed following EphA3 knockdown by shRNA (46%) or by alternately inhibiting EphA3 function using soluble ephrin A5-Fc (33%). CFSE division tracking identified slower cell division in populations in which EphA3 signaling was attenuated. In-vivo studies were performed using a NOD/SCID mouse subcutaneous and intracranial xenograft model. Results highlighted a marked reduction in tumor formation in the EphA3 knockdown as opposed to control tumors. Subcutaneous control tumors formed with a median survival of 66 days while EphA3 knockdown animals survived beyond 100 days (p<0.05). Similar to the subcutaneous xenograft model a marked lack of intracranial tumor formation was observed when EphA3 was neutralized. Control mice formed large well vascularized invasive tumors with a median survival of 62 days. All EphA3 knockdown animals were free of tumor following autopsy at 145 days when the experiment was terminated (p<0.05). Importantly a mutant EphA3 rescue of the knockdown culture returned the tumorigenic potential of these cells. Conclusions: We propose EphA3, in part, regulates cancer stem cell self renewal and cell division rate in GBM and could prove a potential therapeutic target and marker of brain tumor initiating stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1197. doi:10.1158/1538-7445.AM2011-1197

Fetal stem cell therapy

Introduction. Recent advances in molecular diagnostics and imaging technology now provide an unprecedented capacity for prenatal identification of a wide range of serious genetic and chromosomal disorders. Early diagnosis has far exceeded our ability to correct debilitating disorders, especially those for which there is no satisfactory postnatal treatment, and this has been the impetus for research into prenatal therapy. The rationale is that early detection allows early treatment, thus potentially curing a uniformly fatal disorder or preventing irreversible postnatal sequelae, especially those affecting the central nervous system. Stem cells hold far-reaching possibilities for the treatment of both acquired and congenital diseases. They can be used therapeutically to replace dysfunctional cells and tissues, or via ex vivo genetic manipulation to reconstitute a missing gene product (stem cell-based gene therapy). Stem cell transplantation in utero offers the exciting prospect of effectively treating inherited haematological, metabolic and other early-onset genetic diseases. This chapter addresses the current status of fetal stem cell therapy, its limitations and its future development. Sources of stem cells. Stem cells are rare primitive cells that share two distinct properties regardless of their source: the capacity for self-renewal. multi-lineage potential. Embryonic stem (ES) cells from the inner cell mass have the advantage of pluripotency or even totipotency but their clinical use is hindered by the real possibility of teratoma formation in vivo, which largely limits clinical application to tissue engineering rather than cell therapy.