Single cell molecular recognition of migrating and invading tumor cells using a targeted fluorescent probe to receptor PTPmu.

Detection of an extracellular cleaved fragment of a cell-cell adhesion molecule represents a new paradigm in molecular recognition and imaging of tumors. We previously demonstrated that probes that recognize the cleaved extracellular domain of receptor protein tyrosine phosphatase mu (PTPmu) label human glioblastoma brain tumor sections and the main tumor mass of intracranial xenograft gliomas. In this article, we examine whether one of these probes, SBK2, can label dispersed glioma cells that are no longer connected to the main tumor mass. Live mice with highly dispersive glioma tumors were injected intravenously with the fluorescent PTPmu probe to test the ability of the probe to label the dispersive glioma cells in vivo. Analysis was performed using a unique three-dimensional (3D) cryo-imaging technique to reveal highly migratory and invasive glioma cell dispersal within the brain and the extent of colabeling by the PTPmu probe. The PTPmu probe labeled the main tumor site and dispersed cells up to 3.5 mm away. The cryo-images of tumors labeled with the PTPmu probe provide a novel, high-resolution view of molecular tumor recognition, with excellent 3D detail regarding the pathways of tumor cell migration. Our data demonstrate that the PTPmu probe recognizes distant tumor cells even in parts of the brain where the blood-brain barrier is likely intact. The PTPmu probe has potential translational significance for recognizing tumor cells to facilitate molecular imaging, a more complete tumor resection and to serve as a molecular targeting agent to deliver chemotherapeutics to the main tumor mass and distant dispersive tumor cells.

Synthesis and evaluation of a peptide targeted small molecular Gd-DOTA monoamide conjugate for MR molecular imaging of prostate cancer.

Tumor extracellular matrix has an abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. Innovative design and development of safe and effective targeted contrast agents to these biomarkers would allow effective MR cancer molecular imaging with high spatial resolution. In this study, we synthesized a low molecular weight CLT1 peptide targeted Gd(III) chelate CLT1-dL-(Gd-DOTA)(4) specific to clotted plasma proteins in tumor stroma for cancer MR molecular imaging. CLT1-dL-(Gd-DOTA)(4) was synthesized by conjugating four Gd-DOTA monoamide chelates to a CLT1 peptide via generation 1 lysine dendrimer. The T(1) relaxivity of CLT1-dL-(Gd-DOTA)(4) was 40.4 mM(-1) s(-1) per molecule (10.1 mM(-1) s(-1) per Gd) at 37 °C and 1.5 T. Fluorescence imaging showed high binding specificity of CLT1 to orthotopic PC3 prostate tumor in mice. The contrast agent resulted in improved tumor contrast enhancement in male athymic nude mice bearing orthotopic PC3 prostate tumor xenograft at a dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for high-resolution MR molecular imaging of prostate tumor.

MR molecular imaging of prostate cancer with a peptide-targeted contrast agent in a mouse orthotopic prostate cancer model.

PURPOSE: To study the effectiveness of a peptide targeted nanoglobular Gd-DOTA complexes for MR molecular imaging of prostate cancer in a mouse orthotopic PC-3 prostate cancer model. METHODS: A CLT1 (CGLIIQKNEC) peptide-targeted generation 2 nanoglobular Gd-DOTA monoamide conjugate [CLT1-G2-(Gd-DOTA)] was used for imaging fibrin-fibronectin complexes in prostate tumor using a non-specific peptide KAREC modified conjugate, KAREC-G2-(Gd-DOTA) as a control. Cy5 conjugates of CLT1 and KAREC were synthesized for binding studies. Orthotopic PC-3 prostate tumors were established in the prostate of athymic male nude mice. MRI study was performed on a Bruker 7T small animal MRI system. RESULTS: CLT1 peptide showed specific binding in the prostate tumor with no binding in normal tissues. The control peptide had little binding in normal and tumor tissues. CLT1-G2-(Gd-DOTA) resulted in stronger contrast enhancement in tumor tissue than KAREC-G2-(Gd-DOTA). CLT1-G2-(Gd-DOTA) generated ~100% increase in contrast-to-noise ratio (CNR) in the tumor compared to precontrast CNR at 1 min post-injection, while KAREC-G2-(Gd-DOTA) resulted in 8% increase. CONCLUSION: CLT1-G2-(Gd-DOTA) is a promising molecular MRI contrast agent for fibrin-fibronectin complexes in tumor stroma. It has potential for diagnosis and assessing prognosis of malignant tumors with MRI.

Identification of phospholipase C gamma1 as a protein tyrosine phosphatase mu substrate that regulates cell migration.

The receptor protein tyrosine phosphatase PTPµ has a cell-adhesion molecule-like extracellular segment and a catalytically active intracellular segment. This structure gives PTPµ the ability to transduce signals in response to cell-cell adhesion. Full-length PTPµ is down-regulated in glioma cells by proteolysis which is linked to increased migration of these cells in the brain. To gain insight into the substrates PTPµ may be dephosphorylating to suppress glioma cell migration, we used a substrate trapping method to identify PTPµ substrates in tumor cell lines. We identified both PKCδ and PLCγ1 as PTPµ substrates. As PLCγ1 activation is linked to increased invasion of cancer cells, we set out to determine whether PTPµ may be upstream of PLCγ1 in regulating glioma cell migration. We conducted brain slice assays using U87-MG human glioma cells in which PTPµ expression was reduced by shRNA to induce migration. Treatment of the same cells with PTPµ shRNA and a PLCγ1 inhibitor prevented migration of the cells within the brain slice. These data suggest that PLCγ1 is downstream of PTPµ and that dephosphorylation of PLCγ1 is likely to be a major pathway through which PTPµ suppresses glioma cell migration.

Protein tyrosine phosphatase-mu differentially regulates neurite outgrowth of nasal and temporal neurons in the retina.

Cell adhesion molecules play an important role in the development of the visual system. The receptor-type protein tyrosine phosphatase, PTPmu is a cell adhesion molecule that mediates cell aggregation and may signal in response to adhesion. PTPmu is expressed in the chick retina during development and promotes neurite outgrowth from retinal ganglion cell (RGC) axons in vitro (Burden-Gulley and Brady-Kalnay, 1999). The axons of RGC neurons form the optic nerve, which is the sole output from the retina to the optic tectum in the chick. In this study, we observed that PTPmu expression in RGC axons occurs as a step gradient, with temporal axons expressing the highest level of PTPmu. PTPmu expression in the optic tectum occurred as a smooth descending gradient from anterior to posterior regions during development. Because temporal RGC axons innervate anterior tectal regions, PTPmu may regulate the formation of topographic projections to the tectum. In agreement with this hypothesis, a differential response of RGC neurites to a PTPmu substrate was also observed: RGCs of temporal retina were unable to extend neurites on PTPmu compared with neurites of nasal retina. When given a choice between PTPmu and a second substrate, the growth cones of temporal neurites clustered at the PTPmu border and stalled, thus avoiding additional growth on the PTPmu substrate. In contrast, PTPmu was permissive for growth of nasal neurites. Finally, application of soluble PTPmu to retinal cultures resulted in the collapse of temporal but not nasal growth cones. Therefore, PTPmu may specifically signal to temporal RGC axons to cease their forward growth after reaching the anterior tectum, thus allowing for subsequent innervation of deeper tectal layers.

Molecular Magnetic Resonance Imaging of Tumors with a PTPµ Targeted Contrast Agent.

Molecular magnetic resonance imaging (MRI) of tumors improves the specificity of MRI by using targeted probes conjugated to contrast-generating metals. The limitation of this approach is in the identification of a target molecule present in sufficient concentration for visualization and the development of a labeling reagent that can penetrate tumor tissue with the fast kinetics required for use in a clinical setting. The receptor protein tyrosine phosphatase PTPµ is a transmembrane protein that is continuously proteolyzed in the tumor microenvironment to generate a high concentration of extracellular fragment that can be recognized by the SBK2 probe. We conjugated the SBK2 peptide to a gadolinium chelate [SBK2-Tris-(Gd-DOTA)3] to test whether the SBK2 probe could be developed as an MR molecular imaging probe. When intravenously injected into mice bearing flank tumors of human glioma cells, SBK2-Tris-(Gd-DOTA)3 labeled the tumors within 5 minutes with a high level of contrast for up to 2 hours post-injection. The contrast enhancement of SBK2-Tris-(Gd-DOTA)3 was significantly higher than that observed with a current MRI macrocyclic gadolinium chelate (Gadoteridol, ProHance) alone or a scrambled control. These results demonstrate that SBK2-Tris-(Gd-DOTA)3 labeling of the PTPµ extracellular fragment is a more specific MR molecular imaging probe than ProHance or a scrambled control. Consequently, the SBK2 probe may be more useful than the current gold standard reagent for MRI to identify tumors and to co-register tumor borders during surgical resection.

Protein tyrosine phosphatase mu regulates glioblastoma cell growth and survival in vivo.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor. Extensive proliferation and dispersal of GBM tumor cells within the brain limits patient survival to approximately 1 year. Hence, there is a great need for the development of better means to treat GBM. Receptor protein tyrosine phosphatase (PTP)µ is proteolytically cleaved in GBM to yield fragments that promote dispersal of GBM cells. While normal brain tissue retains expression of full-length PTPµ, low-grade human astrocytoma samples have varying amounts of full-length PTPµ and cleaved PTPµ. In the highest-grade astrocytomas (i.e., GBM), PTPµ is completely proteolyzed into fragments. We demonstrate that short hairpin RNA mediated knockdown of full-length PTPµ and PTPµ fragments reduces glioma cell growth and survival in vitro. The reduction in growth and survival following PTPµ knockdown is enhanced when cells are grown in the absence of serum, suggesting that PTPµ may regulate autocrine signaling. Furthermore, we show for the first time that reduction of PTPµ protein expression decreases the growth and survival of glioma cells in vivo using mouse xenograft flank and i.c. tumor models. Inhibitors of PTPµ could be used to reduce the growth and survival of GBM cells in the brain, representing a promising therapeutic target for GBM.

Cryo-image analysis of tumor cell migration, invasion, and dispersal in a mouse xenograft model of human glioblastoma multiforme.

PURPOSE: The goals of this study were to create cryo-imaging methods to quantify characteristics (size, dispersal, and blood vessel density) of mouse orthotopic models of glioblastoma multiforme (GBM) and to enable studies of tumor biology, targeted imaging agents, and theranostic nanoparticles. PROCEDURES: Green fluorescent protein-labeled, human glioma LN-229 cells were implanted into mouse brain. At 20-38 days, cryo-imaging gave whole brain, 4-GB, 3D microscopic images of bright field anatomy, including vasculature, and fluorescent tumor. Image analysis/visualization methods were developed. RESULTS: Vessel visualization and segmentation methods successfully enabled analyses. The main tumor mass volume, the number of dispersed clusters, the number of cells/cluster, and the percent dispersed volume all increase with age of the tumor. Histograms of dispersal distance give a mean and median of 63 and 56 µm, respectively, averaged over all brains. Dispersal distance tends to increase with age of the tumors. Dispersal tends to occur along blood vessels. Blood vessel density did not appear to increase in and around the tumor with this cell line. CONCLUSION: Cryo-imaging and software allow, for the first time, 3D, whole brain, microscopic characterization of a tumor from a particular cell line. LN-229 exhibits considerable dispersal along blood vessels, a characteristic of human tumors that limits treatment success.

Cadherin-11, a marker of the mesenchymal phenotype, regulates glioblastoma cell migration and survival in vivo.

Glioblastoma multiforme (GBM) is the most malignant and lethal form of astrocytoma. The GBM patient survival time of approximately 1 year necessitates the identification of novel molecular targets and more effective therapeutics. Cadherin-11, a calcium-dependent cell-cell adhesion molecule and mesenchymal marker, plays a role in both normal tissue development and in cancer cell migration. The functional significance of cadherin-11 in GBM has not been investigated. Here, we show that cadherin-11 is expressed in human GBM tumors and human glioma stem-like cells by immunohistochemical labeling. In addition, we show that cadherin-11 is expressed in human glioma cell lines by immunoblotting. Short hairpin RNA-mediated knockdown of cadherin-11 expression in human glioma cell lines results in decreased migration and growth factor-independent cell survival in vitro. More importantly, knockdown of cadherin-11 inhibits glioma cell survival in heterotopic and orthotopic mouse xenograft models. Together, our results show the functional significance of cadherin-11 expression in GBM and provide evidence for a novel role of cadherin-11 in promoting glioma cell survival in an in vivo environment. Thus, our studies suggest cadherin-11 is a viable molecular target for therapeutic intervention in GBM.

Novel cryo-imaging of the glioma tumor microenvironment reveals migration and dispersal pathways in vivid three-dimensional detail.

Traditional methods of imaging cell migration in the tumor microenvironment include serial sections of xenografts and standard histologic stains. Current molecular imaging techniques suffer from low resolution and difficulty in imaging through the skull. Here we show how computer algorithms can be used to reconstruct images from tissue sections obtained from mouse xenograft models of human glioma and can be rendered into three-dimensional images offering exquisite anatomic detail of tumor cell dispersal. Our findings identify human LN-229 and rodent CNS-1 glioma cells as valid systems to study the highly dispersive nature of glioma tumor cells along blood vessels and white matter tracts in vivo. This novel cryo-imaging technique provides a valuable tool to evaluate therapeutic interventions targeted at limiting tumor cell invasion and dispersal.

Stimulation of N-cadherin-dependent neurite outgrowth by small molecule peptide mimetic agonists of the N-cadherin HAV motif.

N-cadherin is a cell adhesion molecule that promotes axon outgrowth and synapse formation during the development of the central nervous system. In addition, N-cadherin promotes glial cell adhesion and myelination of axons. Therefore, stimulating N-cadherin function with N-cadherin agonists could be used therapeutically to promote regeneration of the nervous system and remyelination after injury or disease. In the extracellular domain of N-cadherin, the amino acid sequence HAV is required for N-cadherin-mediated adhesion and neurite outgrowth. The ADH-1 cyclic peptide, derived from the N-cadherin HAV site, is an effective antagonist of N-cadherin-mediated neurite outgrowth and is currently being tested in clinical trials for cancer chemotherapy. Of interest, a dimeric version of this cyclic peptide, N-Ac-CHAVDINGHAVDIC-NH(2), functions as an N-cadherin agonist. This dimeric peptide agonist and the peptide antagonist ADH-1 both have limitations as drugs due to their metabolic instability and lack of oral delivery. To address this issue Adherex Technologies Inc. generated a small molecule library of peptidomimetics to the HAV region of N-cadherin, which would be more amenable to therapeutic use. We screened the Adherex library for compounds that altered neurite outgrowth and identified eight N-cadherin agonists that stimulated N-cadherin-dependent neurite outgrowth. Five of these agonists also stimulated retinal cell migration. These small molecule agonists may be effective reagents for promoting axon growth and remyelination after injury or disease.

A novel molecular diagnostic of glioblastomas: detection of an extracellular fragment of protein tyrosine phosphatase mu.

We recently found that normal human brain and low-grade astrocytomas express the receptor protein tyrosine phosphatase mu (PTPmu) and that the more invasive astrocytomas, glioblastoma multiforme (GBM), downregulate full-length PTPmu expression. Loss of PTPmu expression in GBMs is due to proteolytic cleavage that generates an intracellular and potentially a cleaved and released extracellular fragment of PTPmicro. Here, we identify that a cleaved extracellular fragment containing the domains required for PTPmicro-mediated adhesion remains associated with GBM tumor tissue. We hypothesized that detection of this fragment would make an excellent diagnostic tool for the localization of tumor tissue within the brain. To this end, we generated a series of fluorescently tagged peptide probes that bind the PTPmu fragment. The peptide probes specifically recognize GBM cells in tissue sections of surgically resected human tumors. To test whether the peptide probes are able to detect GBM tumors in vivo, the PTPmu peptide probes were tested in both mouse flank and intracranial xenograft human glioblastoma tumor model systems. The glial tumors were molecularly labeled with the PTPmu peptide probes within minutes of tail vein injection using the Maestro FLEX In Vivo Imaging System. The label was stable for at least 3 hours. Together, these results indicate that peptide recognition of the PTPmu extracellular fragment provides a novel molecular diagnostic tool for detection of human glioblastomas. Such a tool has clear translational applications and may lead to improved surgical resections and prognosis for patients with this devastating disease.

Novel peptide mimetic small molecules of the HAV motif in N-cadherin inhibit N-cadherin-mediated neurite outgrowth and cell adhesion.

The cell adhesion molecule, N-cadherin, stabilizes cell-cell junctions and promotes cellular migration during tissue morphogenesis in development. N-cadherin is also implicated in mediating tumor progression and metastasis in cancer. Therefore, developing antagonists of N-cadherin adhesion may be of therapeutic value in cancer treatment. The amino acid sequence HAV in the extracellular domain of N-cadherin is required for N-cadherin-mediated adhesion and migration. A cyclic peptide, ADH-1, derived from the N-cadherin HAV site is an effective antagonist of N-cadherin-mediated processes and is now in clinical trials for cancer chemotherapy. Because it is a peptide, ADH-1 has certain limitations as a drug, namely its metabolic instability and lack of oral delivery. Adherex set out to identify small molecule antagonists of N-cadherin, which would be more amenable to therapeutic use. Using three-dimensional computational screening, Adherex identified a set of small molecules as potential antagonists with sufficient structural similarity to the HAV region of N-cadherin. We tested the ability of these small molecules to interfere with two N-cadherin-dependent processes: neurite outgrowth (axonal migration) and N-cadherin-dependent cell adhesion. We identified 21 N-cadherin antagonists of varying potency. More importantly, our studies demonstrate that these compounds are significantly more potent than ADH-1 at perturbing N-cadherin-mediated processes. The IC(50) of ADH-1 is 2.33 mM while the IC(50) of the small molecules ranges from 4.5 to 30 microM. Given the efficacy of ADH-1 for treating cancer, these small molecule antagonists will be highly effective in treatment of cancer metastasis and conditions of aberrant neurite outgrowth, such as neuropathic pain.

Proteolytic cleavage of protein tyrosine phosphatase mu regulates glioblastoma cell migration.

Glioblastoma multiforme (GBM), the most common malignant primary brain tumor, represents a significant disease burden. GBM tumor cells disperse extensively throughout the brain parenchyma, and the need for tumor-specific drug targets and pharmacologic agents to inhibit cell migration and dispersal is great. The receptor protein tyrosine phosphatase mu (PTPmu) is a homophilic cell adhesion molecule. The full-length form of PTPmu is down-regulated in human glioblastoma. In this article, overexpression of full-length PTPmu is shown to suppress migration and survival of glioblastoma cells. Additionally, proteolytic cleavage is shown to be the mechanism of PTPmu down-regulation in glioblastoma cells. Proteolysis of PTPmu generates a series of proteolytic fragments, including a soluble catalytic intracellular domain fragment that translocates to the nucleus. Only proteolyzed PTPmu fragments are detected in human glioblastomas. Short hairpin RNA-mediated down-regulation of PTPmu fragments decreases glioblastoma cell migration and survival. A peptide inhibitor of PTPmu function blocks fragment-induced glioblastoma cell migration, which may prove to be of therapeutic value in GBM treatment. These data suggest that loss of cell surface PTPmu by proteolysis generates catalytically active PTPmu fragments that contribute to migration and survival of glioblastoma cells.

PTPmu suppresses glioma cell migration and dispersal.

The cell-surface receptor protein tyrosine phosphatase mu (PTPmu) is a homophilic cell adhesion molecule expressed in CNS neurons and glia. Glioblastomas (GBMs) are the highest grade of primary brain tumors with astrocytic similarity and are characterized by marked dispersal of tumor cells. PTPmu expression was examined in human GBM, low-grade astrocytoma, and normal brain tissue. These studies revealed a striking loss of PTPmu protein expression in highly dispersive GBMs compared to less dispersive low-grade astrocytomas and normal brain. We hypothesized that PTPmu contributes to contact inhibition of glial cell migration by transducing signals in response to cell adhesion. Therefore, loss of PTPmu may contribute to the extensive dispersal of GBMs. The migration of brain tumor cells was assessed in vitro using a scratch wound assay. Parental U-87 MG cells express PTPmu and exhibited limited migration. However, short-hairpin RNA (shRNA)-mediated knockdown of PTPmu induced a morphological change and increased migration. Next, a brain slice assay replicating the three-dimensional environment of the brain was used. To assess migration, labeled U-87 MG glioma cells were injected into adult rat brain slices, and their movement was followed over time. Parental U-87 MG cells demonstrated limited dispersal in this assay. However, PTPmu shRNA induced migration and dispersal of U-87 MG cells in the brain slice. Finally, in a mouse xenograft model of intracranially injected U-87 MG cells, PTPmu shRNA induced morphological heterogeneity in these xenografts. Together, these data suggest that loss of PTPmu in human GBMs contributes to tumor cell migration and dispersal, implicating loss of PTPmu in glioma progression.

Expression of the receptor protein-tyrosine phosphatase, PTPmu, restores E-cadherin-dependent adhesion in human prostate carcinoma cells.

Normal prostate expresses the receptor protein-tyrosine phosphatase, PTPmu, whereas LNCaP prostate carcinoma cells do not. PTPmu has been shown previously to interact with the E-cadherin complex. LNCaP cells express normal levels of E-cadherin and catenins but do not mediate either PTPmu- or E-cadherin-dependent adhesion. Re-expression of PTPmu restored cell adhesion to PTPmu and to E-cadherin. A mutant form of PTPmu that is catalytically inactive was re-expressed, and it also restored adhesion to PTPmu and to E-cadherin. Expression of PTPmu-extra (which lacks most of the cytoplasmic domain) induced adhesion to PTPmu but not to E-cadherin, demonstrating a requirement for the presence of the intracellular domains of PTPmu to restore E-cadherin-mediated adhesion. We previously observed a direct interaction between the intracellular domain of PTPmu and RACK1, a receptor for activated protein kinase C (PKC). We demonstrate that RACK1 binds to both the catalytically active and inactive mutant form of PTPmu. In addition, we determined that RACK1 binds to the PKCdelta isoform in LNCaP cells. We tested whether PKC could be playing a role in the ability of PTPmu to restore E-cadherin-dependent adhesion. Activation of PKC reversed the adhesion of PTPmuWT-expressing cells to E-cadherin, whereas treatment of parental LNCaP cells with a PKCdelta-specific inhibitor induced adhesion to E-cadherin. Together, these studies suggest that PTPmu regulates the PKC pathway to restore E-cadherin-dependent adhesion via its interaction with RACK1.