Jisook Lee

Magnetic targeting of nanoparticles across the intact blood–brain barrier

Delivery of therapeutic or diagnostic agents across an intact blood-brain barrier (BBB) remains a major challenge. Here we demonstrate in a mouse model that magnetic nanoparticles (MNPs) can cross the normal BBB when subjected to an external magnetic field. Following a systemic administration, an applied external magnetic field mediates the ability of MNPs to permeate the BBB and accumulate in a perivascular zone of the brain parenchyma. Direct tracking and localization inside endothelial cells and in the perivascular extracellular matrix in vivo was established using fluorescent MNPs. These MNPs were inert and associated with low toxicity, using a non-invasive reporter for astrogliosis, biochemical and histological studies. Atomic force microscopy demonstrated that MNPs were internalized by endothelial cells, suggesting that trans-cellular trafficking may be a mechanism for the MNP crossing of the BBB observed. The silica-coated magnetic nanocapsules (SiMNCs) allow on-demand drug release via remote radio frequency (RF) magnetic field. Together, these results establish an effective strategy for regulating the biodistribution of MNPs in the brain through the application of an external magnetic field.

Pharmacologic Blockade of FAK Autophosphorylation Decreases Human Glioblastoma Tumor Growth and Synergizes with Temozolomide

Malignant gliomas are characterized by aggressive tumor growth with a mean survival of 15 to 18 months and frequently developed resistance to temozolomide. Therefore, strategies that sensitize glioma cells to temozolomide have a high translational impact. We have studied focal adhesion kinase (FAK), a tyrosine kinase and emerging therapeutic target that is known to be highly expressed and activated in glioma. In this report, we tested the FAK autophosphorylation inhibitor, Y15, in DBTRG and U87 glioblastoma cells. Y15 significantly decreased viability and clonogenicity in a dose-dependent manner, increased detachment in a dose- and time-dependent manner, caused apoptosis, and inhibited cell invasion in both cell lines. In addition, Y15 treatment decreased autophosphorylation of FAK in a dose-dependent manner and changed cell morphology by causing cell rounding in DBTRG and U87 cells. Administration of Y15 significantly decreased subcutaneous DBTRG tumor growth with decreased Y397-FAK autophosphorylation, activated caspase-3 and PARP. Y15 was administered in an orthotopic glioma model, leading to an increase in mouse survival. The combination of Y15 with temozolomide was more effective than either agent alone in decreasing viability and activating caspase-8 in DBTRG and U87 cells in vitro. In addition, the combination of Y15 and temozolomide synergistically blocked U87 brain tumor growth in vivo. Thus, pharmacologic blockade of FAK autophosphorylation with the oral administration of a small-molecule inhibitor Y15 has a potential to be an effective therapy approach for glioblastoma either alone or in combination with chemotherapy agents such as temozolomide. Mol Cancer Ther; 12(2); 162–72. ©2012 AACR.

Glioma-induced remodeling of the neurovascular unit

The normal BBB (blood-brain barrier) consists of a series of structures collectively known as neurovascular units, or NVU, that are composed of endothelial cells and astrocyte endfeet separated by a basal lamina at their interface. The integrity of the BBB and specifically endothelial tight junctions is maintained by interactions between these different components and the local microenvironment of the NVU. Central nervous system cancers such as gliomas disrupt the integrity of the BBB and this compromise is associated with increased tumor growth and invasion of the surrounding brain parenchyma. Because the relationship between glioma-induced BBB breakdown and glioma invasion remains poorly understood, and the host microenvironment can influence tumor cell migration, we used immunohistochemical techniques to characterize tumor associated BBB remodeling. Using an orthotopic xenograft model of glioma, we demonstrate that tumor cells induce specific changes in the composition of the basal lamina and in astrocytic components of the NVU. We suggest that these changes may be essential to understand the capacity of gliomas to regulate BBB integrity and as such, glioma invasion into brain parenchyma.

Enhanced room temperature ferromagnetism in Co- and Mn-ion-implanted silicon

The authors report on ferromagnetism at room temperature in cluster-free, cobalt- and manganese-ion-implanted crystalline silicon. Through magnetic and structural analysis it is shown that the ion-implanted Si consists of two layers of Co- and Mn-containing silicon: (1) an amorphous Si layer on the surface and (2) single crystalline Si beneath. The amorphous layer shows very little magnetism by itself but seems to be responsible for partially canceling out or masking the ferromagnetism in the crystalline Si. The authors also observe that etching of the amorphous Si layer dramatically enhances the measured magnetism by as much as 400%.

Non-invasive quantification of brain tumor-induced astrogliosis

BackgroundCNS injury including stroke, infection, and tumor growth lead to astrogliosis, a process that involves upregulation of glial fibrillary acidic protein (GFAP) in astrocytes. However, the kinetics of astrogliosis that is related to these insults (i.e. tumor) is largely unknown.ResultsUsing transgenic mice expressing firefly luciferase under the regulation of the GFAP promoter (GFAP-luc), we developed a model system to monitor astrogliosis upon tumor growth in a rapid, non-invasive manner. A biphasic induction of astrogliosis was observed in our xenograft model in which an early phase of activation of GFAP was associated with inflammatory response followed by a secondary, long-term upregulation of GFAP. These animals reveal GFAP activation with kinetics that is in parallel with tumor growth. Furthermore, a strong correlation between astrogliosis and tumor size was observed.ConclusionsOur results suggest that non-invasive, quantitative bioluminescent imaging using GFAP-luc reporter animal is a useful tool to monitor temporal-spatial kinetics of host-mediated astrogliosis that is associated with glioma and metastatic brain tumor growth.

Acute Lower Gi Bleeding for the Acute Care Surgeon: Current Diagnosis and Management

Lower gastrointestinal bleeding is a common cause for hospital admission that results in significant morbidity and mortality. After initial resuscitation of the patient, the diagnosis and treatment of lower gastrointestinal bleeding remains a challenge for acute care surgeons. Identifying the source of bleeding can be difficult since many patients bleed intermittently or stop bleeding spontaneously. It is therefore important for the acute care surgeon to be familiar with the different diagnostic and therapeutic modalities and their advantages and disadvantages in order to guide the management of the acutely bleeding patient. This review summarizes the current methods available for the diagnosis and treatment of acute lower gastrointestinal bleeding and proposes an algorithm for the management of these patients.

Cell surface localization and release of the candidate tumor suppressor Ecrg4 from polymorphonuclear cells and monocytes activate macrophages

We identified fresh human leukocytes as an abundant source of the candidate epithelial tumor suppressor gene, Ecrg4, an epigenetically regulated gene, which unlike other tumor suppressor genes, encodes an orphan‐secreted, ligand‐like protein. In human cell lines, Ecrg4 gene expression was low, Ecrg4 protein undetectable, and Ecrg4 promoter hypermethylation high (45–90%) and reversible by the methylation inhibitor 5‐AzaC. In contrast, Ecrg4 gene expression in fresh, normal human PBMCs and PMNs was 600–800 times higher than in cultured cell lines, methylation of the Ecrg4 promoter was low (<3%), and protein levels were readily detectable in lysates and on the cell surface. Flow cytometry, immunofluorescent staining, and cell surface biotinylation established that full‐length, 14‐kDa Ecrg4 was localized on PMN and monocyte cell surfaces, establishing that Ecrg4 is a membrane‐anchored protein. LPS treatment induced processing and release of Ecrg4, as detected by flow and immunoblotting, whereas an effect of fMLF treatment on Ecrg4 on the PMN cell surface was detected on the polarized R2 subpopulation of cells. This loss of cell surface Ecrg4 was associated with the detection of intact and processed Ecrg4 in the conditioned media of fresh leukocytes and was shown to be associated with the inflammatory response that follows severe, cutaneous burn injury. Furthermore, incubation of macrophages with a soluble Ecrg4‐derived peptide increased the P‐p65, suggesting that processing of an intact sentinel Ecrg4 on quiescent circulating leukocytes leads to processing from the cell surface following injury and macrophage activation.

Thrombin-processed Ecrg4 recruits myeloid cells and induces antitumorigenic inflammation

BACKGROUND Extensive infiltration of brain tumors by microglia and macrophages is a hallmark of tumor progression, and yet the overall tumor microenvironment is characterized by an immunosuppressive phenotype. Here we identify esophageal cancer-related gene 4 (Ecrg4) as a novel thrombin-processed monocyte chemoattractant that recruits myeloid cells, promotes their activation, and leads to a blockade of tumor progression. METHODS Both xenograft glioma and syngeneic glioma models were used to measure orthotopic tumor progression and overall survival. Flow cytometry and immunohistochemical analyses were performed to assess myeloid cell localization, recruitment, and activation. RESULTS Ecrg4 promotes monocyte recruitment and activation of microglia in a T-/B-cell-independent mechanism, which leads to a reduction in glioma tumor burden and increased survival. Mutational analysis reveals that the biological activity of Ecrg4 is dependent on a thrombin-processing site at the C-terminus, inducing monocyte invasion in vivo and in vitro. Furthermore, tumor-induced myeloid cell recruitment is impaired in Ecrg4 knockout mice, leading to increased tumor burden and decreased survival. CONCLUSIONS Together, these results identify Ecrg4 as a paracrine factor that activates microglia and is chemotactic for monocytes, with potential as an antitumor therapeutic.

Esophageal cancer-related gene 4 at the interface of injury, inflammation, infection, and malignancy.

In humans, esophageal cancer-related gene 4 (ECRG4) is encoded by four exons in the c2orf40 locus of chromosome 2. Translation of ECRG4 messenger ribonucleic acid produces a 148 amino acid-secreted 17 KDa protein that is then processed to 14, ten, eight, six, four, and two KDa peptides, depending on the cell in which the gene is expressed. As hypermethylation at the c2orf40 locus inhibits ECRG4 gene expression in many epithelial cancers, several investigators have speculated that ECRG4 is a candidate tumor suppressor. Indeed, overexpression of ECRG4 inhibits cell proliferation in vitro, but it also has a wide range of effects in vivo beyond its antitumor activity. ECRG4 overexpression affects apoptosis, senescence, cell migration, inflammation, injury, and infection responsiveness. ECRG4 activities also depend on its cellular localization, secretion, and post-translational processing. These cytokine/chemokine-like characteristics argue that ECRG4 is not a traditional candidate tumor suppressor gene, as originally predicted by its downregulation in cancer. We review how insights into the regulation of ECRG4 gene expression, knowledge of its primary structure, and the study of its emerging physiological functions come together to support a much more complex role for ECRG4 at the interface of inflammation, infection, and malignancy.

In vivo measurement of glioma-induced vascular permeability.

The normal blood-brain barrier (BBB) consists of tight interendothelial cell junctions and adjacent astrocyte end feet separated by a basal lamina surrounding the endothelium. The interactions between the different cell types of BBB are disrupted in distinct patterns in the microenvironment of glioma. Malignant gliomas infiltrate the surrounding normal brain parenchyma; a process associated with vascular permeability (VP) and breakdown of the BBB. Herein, we describe methods to quantitatively measure glioma-induced vascular permeability, utilizing an orthotopic xenograft model of glioma.