CD44 expressed by myeloid cells promotes glioma invasion.

Glioblastoma multiforme (GBM) is one of the most common and malignant brain tumors in adulthood with a median survival of only 15 months. This poor prognosis is related to GBM's ability to extensively infiltrate the surrounding brain parenchyma resulting in diffuse spread of neoplastic cells in the brain, responsible for high rate of recurrence. CD44 (Cluster of Differentiation 44) is a transmembrane protein, overexpressed in multiple cancer types, including gliomas, and implicated in cell motility, proliferation and angiogenesis. Multiple studies have investigated the role of CD44 in GBM cells and have highlighted a link between tumor malignancy and CD44 expression. However up to date, little is known of the role of CD44 on cells from the tumor microenvironment (TME). Here, we have investigated a potential role of CD44 in the TME in regards to GBM invasiveness. Using an ex-vivo organotypic brain slice invasion assay, we show that absence of CD44 from the TME impairs the ability of glioma cells to invade the surrounding brain parenchyma. By deleting CD44 in the astrocytic, endothelial and myeloid compartments, we show that it is specifically CD44 expression in myeloid cells that is responsible for the observed phenotype. Combining in vivo studies in cell-specific knock-out mice and in vitro analyses on primary microglia we demonstrate that myeloid CD44 is implicated in Toll Like Receptor 2 signaling and is a major regulator of Matrix metalloproteinase 9 expression.

Nutritional Calcium Supply Dependent Calcium Balance, Bone Calcification and Calcium Isotope Ratios in Rats.

Serum calcium isotopes (δ44/42Ca) have been suggested as a non-invasive and sensitive Ca balance marker. Quantitative δ44/42Ca changes associated with Ca flux across body compartment barriers relative to the dietary Ca and the correlation of δ44/42CaSerum with bone histology are unknown. We analyzed Ca and δ44/42Ca by mass-spectrometry in rats after two weeks of standard-Ca-diet (0.5%) and after four subsequent weeks of standard- and of low-Ca-diet (0.25%). In animals on a low-Ca-diet net Ca gain was 61 ± 3% and femur Ca content 68 ± 41% of standard-Ca-diet, bone mineralized area per section area was 68 ± 15% compared to standard-Ca-diet. δ44/42Ca was similar in the diets, and decreased in feces and urine and increased in serum in animals on low-Ca-diet. δ44/42CaBone was higher in animals on low-Ca-diet, lower in the diaphysis than the metaphysis and epiphysis, and unaffected by gender. Independent of diet, δ44/42CaBone was similar in the femora and ribs. At the time of sacrifice, δ44/42CaSerum inversely correlated with intestinal Ca uptake and histological bone mineralization markers, but not with Ca content and bone mineral density by µCT. In conclusion, δ44/42CaBone was bone site specific, but mechanical stress and gender independent. Low-Ca-diet induced marked changes in feces, serum and urine δ44/42Ca in growing rats. δ44/42CaSerum inversely correlated with markers of bone mineralization.

In Vitro Characterization of a Novel Type of Radiopaque Doxorubicin-Loaded Microsphere.

PURPOSE: To evaluate and compare the material characteristics of a novel type of radiopaque doxorubicin-loaded microsphere (V-100) with radiopaque and non-radiopaque doxorubicin-loaded microspheres. MATERIALS AND METHODS: The prototype V-100 featuring inherent radiopacity and three available commercial controls (DC-Bead-LUMI™-70-150, Embozene-Tandem™-100 and DC-Bead™-M1) were analyzed before and after doxorubicin loading (37.5 mg doxorubicin/1 ml microspheres) in suspension with aqua and/or aqua/iodixanol-320. Study goals included inherent radiopacity [e.g., using conventional computed tomography (CT)], doxorubicin loading efficacy, morphology using light and fluorescence microscopy, size distribution using laser diffraction/light scattering, time-in-suspension, rheological properties using rheometer analysis, and microsphere stability observed over a period of 5 days after doxorubicin loading. RESULTS: V-100 showed good inherent radiopacity without adverse imaging artifacts. Under conventional CT, the quantitative radiopacity was as follows: 480.4 ± 2.9HU for V-100, 2432.7 ± 3.2HU for DC-Bead-LUMI™-70-150, 118.1 ± 3.0HU for Embozene-Tandem™-100, and 19.8 ± 1.5HU for DC-Bead™-M1. All of the types of microspheres showed a similar loading efficiency (> 98%) after 24 h; however, there were slower doxorubicin loading velocities for the radiopaque microspheres. The doxorubicin-loaded V-100 and Embozene-Tandem™-100 showed typical narrow-sized distributions. In aqua/iodixanol-320 suspension, doxorubicin-loaded V-100 showed the best suspension features and ideal deformability and elasticity characteristics. Similar to other microspheres, doxorubicin-loaded V-100 was very stable and storable for at least 5 days. CONCLUSION: V-100 is a promising novel type of radiopaque doxorubicin-loaded microsphere. Compared with the controls, V-100 shows good inherent radiopacity without adverse imaging artifacts and with comparable doxorubicin loading efficacy. Further advantages of V-100 include narrow-sized distribution and excellent suspension, rheology, and stability features.

The Novel X-Ray Visible Zein-Based Non-adhesive Precipitating Liquid Embolic HEIE1_2017: An Exploratory Study.

PURPOSE: To investigate the novel zein-based non-adhesive precipitating liquid embolic HEIE1_2017. MATERIALS AND METHODS: Zein-based liquid embolics are an own class of embolization material. In this study, HEIE1_2017, a novel zein-based liquid embolic, was investigated. Visibility was assessed in vitro in CT and MRI phantoms, embolization characteristics were assessed in vivo in the kidneys of 12 pigs. Components of HEIE1_2017 were zein as occlusion material, ethanol as solvent, and iodized oil as radiopaque material. HEIE1_2017 was used in pure (HEI-PURE) and manually modified (HEI-MOD) form and compared with 6% ethylene vinyl alcohol copolymer (EVOH). Different radiological methods (CT, MRI, DSA, cone-beam CT, and micro-CT) and histopathologic analyses were applied to compare visibility and vascular occlusion patterns. RESULTS: In CT phantoms, all embolics were definitely visible as hyperdense materials. In MRI phantoms, signal-to-noise ratio was highest for HEI-PURE, followed by HEI-MOD and EVOH. In all kidneys, embolization procedures were technically successful and without complications. In DSA, all embolics were definitely visible during and after embolization. Only EVOH caused substantial artifacts in cone-beam CT and CT. In micro-CT and histopathology, HEI-PURE showed a homogeneous occlusion from segmental arteries to glomerular capillaries. HEI-MOD demonstrated the deepest vascular penetration (up to the level of peritubular capillaries), but with an inhomogeneous distribution. For EVOH, there was inhomogeneous vascular occlusion from segmental arteries to glomerular capillaries. CONCLUSION: HEIE1_2017 is a promising novel zein-based liquid embolic. Further preclinical and clinical studies with higher case numbers and long-term follow-up are needed to further assess the value of this embolic material.

Pan-mutant IDH1 inhibitor BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo.

Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.

Endosialin Promotes Atherosclerosis Through Phenotypic Remodeling of Vascular Smooth Muscle Cells.

OBJECTIVE: Vascular smooth muscle cells (VSMC) play a key role in the pathogenesis of atherosclerosis, the globally leading cause of death. The transmembrane orphan receptor endosialin (CD248) has been characterized as an activation marker of cells of the mesenchymal lineage including tumor-associated pericytes, stromal myofibroblasts, and activated VSMC. We, therefore, hypothesized that VSMC-expressed endosialin may display functional involvement in the pathogenesis of atherosclerosis. APPROACH AND RESULTS: Expression of endosialin was upregulated during atherosclerosis in apolipoprotein E (ApoE)-null mice and human atherosclerotic samples analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry. Atherosclerosis, assessed by Oil Red O staining of the descending aorta, was significantly reduced in ApoE/endosialin-deficient mice on Western-type diet. Marker analysis of VSMC in lesions induced by shear stress-modifying cast implantation around the right carotid artery identified a more pronounced contractile VSMC phenotype in the absence of endosialin. Moreover, in addition to contributing to neointima formation, endosialin also potentially regulated the proinflammatory phenotype of VSMC as evidenced in surrogate cornea pocket assay experiments in vivo and corresponding flow cytometry and ELISA analyses in vitro. CONCLUSIONS: The experiments identify endosialin as a potential regulator of phenotypic remodeling of VSMC contributing to atherosclerosis. The association of endosialin with atherosclerosis and its absent expression in nonatherosclerotic samples warrant further consideration of endosialin as a therapeutic target and biomarker.

Increased x-ray attenuation in malignant vs. benign mediastinal nodes in an orthotopic model of lung cancer.

PURPOSE: Staging of lung cancer is typically performed with fluorodeoxyglucose-positron emission tomography-computed tomography (FDG-PET/CT); however, false positive PET scans can occur due to inflammatory disease. The CT scan is used for anatomic registration and attenuation correction. Herein, we evaluated x-ray attenuation (XRA) within nodes on CT and correlated this with the presence of malignancy in an orthotopic lung cancer model in rats. METHODS: 1×106 NCI-H460 cells were injected transthoracically in six National Institutes of Health nude rats and six animals served as controls. After two weeks, animals were sacrificed; lymph nodes were extracted and scanned with a micro-CT to determine their XRA prior to histologic analysis. RESULTS: Median CT density in malignant lymph nodes (n=20) was significantly higher than benign lymph nodes (n=12; P = 0.018). Short-axis diameter of metastatic lymph nodes was significantly different than benign nodes (3.4 mm vs. 2.4 mm; P = 0.025). Area under the curve for malignancy was higher for density-based lymph node analysis compared with size measurements (0.87 vs. 0.7). CONCLUSION: XRA of metastatic mediastinal lymph nodes is significantly higher than benign nodes in this lung cancer model. This suggests that information on nodal density may be useful when used in combination with the results of FDG-PET in determining the likelihood of malignant adenopathy.

A Comparison of microCT and microPET for Evaluating Lymph Node Metastasis in a Rat Model.

PURPOSE: The demand to optimize multidisciplinary treatment strategies in patients with benign and malignant diseases of the lung and other organs has led to the increased need of mechanistic proof-of-concept studies in preclinical small animal models using new non-invasive imaging methods. Therefore, we evaluated the role of microPET and microCT for mediastinal lymph node staging in an orthotopic lung cancer model in rats. PROCEDURES: Human lung cancer cells (NCI-H460) were injected transthoracically in nude rats (NIH-RNU). After 2 weeks of tumour growth, animals underwent multiphase contrast-enhanced microCT using ExiTron nano 12000 as a contrast agent and dynamic microPET using the tracer 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG). Thereafter, animals were sacrificed for histological analysis. RESULTS: Late phase micro X-ray computed tomography (microCT) revealed the best delineation of lymph node metastases, as compared to earlier scans. In terms of an increased [(18)F]FDG uptake over time, dynamic micro positron emission tomography (microPET) delineated lymph node metastases and enabled metabolic examinations of the induced lung cancer metastases. CONCLUSION: The combination of contrast-enhanced microCT and dynamic microPET is feasible in rats for the visualization of mediastinal lymph node metastases.

Establishment of a patient-derived orthotopic osteosarcoma mouse model.

BACKGROUND: Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. METHODS: Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. RESULTS: A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. CONCLUSION: We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.

mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy.

A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O(6)-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.