Role of mTOR and VEGFR Inhibition in Prevention of Metastatic Tumor Growth in the Spine

Objective: Spinal metastatic disease remains a major problem of oncological diseases. Patients affectedmay suffer pain, spinal instability, and severe neurological deficits. Today, palliative surgery and radiotherapy are the mainstays of therapy. In contrast, preventive treatment strategies or treatment concepts for an early stage are lacking. Here, we have used a syngeneic, experimental spine metastases model in the mouse to test the efficacy of mTOR inhibition and anti-angiogenesis on the formation and progression of spinal melanoma metastases. Methods: We used our previously established syngeneic spinal metastases mouse model by injecting luciferin-transfected B16 melanoma cells into the common carotid artery. Following injection, mice were treated with everolimus, an inhibitor of the mammalian target of rapamycin (mTOR) complex, axitinib, a tyrosine kinase inhibitor, that blocks vascular endothelial growth factor receptors (VEGFR) 1-3, as well as placebo. Animals were followed-up daily by neurological assessment and by repeat in vivo bioluminescence imaging. With occurrence of neurological deficits, a spinal MRI was performed, and mice were sacrificed. The whole spine was dissected free and analyzed by immunohistochemical techniques. Results: Overall survival was 23 days in the control group, significantly prolonged to 30 days (p = 0.04) in the everolimus group, and to 28 days (p = 0.04) in the axitinib group. While 78% of mice in the placebo group developed symptomatic metastatic epidural spinal cord compression, only 50% did so in the treatment groups. The mean time to manifestation of paralysis was 22 days in the control group, 26 days (p = 0.10) in the everolimus group, and 27 days (p = 0.06) in the axitinib group. Screening for spinal metastases by bioluminescence imaging on two different time points showed a decrease in metastatic tumor formation in the treatment groups compared to the controls. Immunohistochemical analysis confirmed the bioactivity of the two compounds: The Ki67 proliferation labeling index was reduced in the everolimus group and numbers of CD31 positive endothelial cells were reduced in the axitinib group. Conclusion: Both, the mTOR inhibitor everolimus as well as antiangiogenetic effects by the VEGFR inhibitor axitinib showed potential to prevent and retard formation of symptomatic spinal metastases. However, the therapeutic efficacy was only mild in this experimental model

Grundsteine zur Züchtung Marssonina coronaria-robuster Apfelsorte

Seit 2010 fallen in vielen europäischen Apfelanbaugebieten einzelne Bäume oder ganze Anlagen durch einen vorzeitigen Blattfall auf. Biologisch bewirtschaftete Apfelanlagen sind dabei häufiger von dieser Pilzkrankheit betroffen als integrierte. Im Rahmen einer Masterarbeit wurde versucht, dem Pilz auf die Spur zu kommen und die Grundsteine für die Resistenzzüchtung zu legen

Twin-shaped CFRP-sandwich pedestrian bridge

A concept of a nature-inspired pedestrian bridge which tries to merge architectural, structural and manufacturing aspects in an optimized way is presented. The 18.0-m-span bridge is designed as an overhead spatial frame of a complex double-curved shape, which is composed of twelve modules consisting of lightweight carbon fiber-reinforced polymer (CFRP) sandwich construction. The modules are fabricated with only one mould through vacuum assisted resin infusion. The aimed visible texture and black colour of the CFRP fabrics represents an essential architectonical element. The resulting benefits and required compromises of this multi-criteria approach are discussed

CCR2 of Tumor Microenvironmental Cells Is a Relevant Modulator of Glioma Biology.

Glioblastoma multiforme (GBM) shows a high influx of tumor-associated macrophages (TAMs). The CCR2/CCL2 pathway is considered a relevant signal for the recruitment of TAMs and has been suggested as a therapeutic target in malignant gliomas. We found that TAMs of human GBM specimens and of a syngeneic glioma model express CCR2 to varying extents. Using a Ccr2-deficient strain for glioma inoculation revealed a 30% reduction of TAMs intratumorally. This diminished immune cell infiltration occurred with augmented tumor volumes likely based on increased cell proliferation. Remaining TAMs in Ccr2-/- mice showed comparable surface marker expression patterns in comparison to wildtype mice, but expression levels of inflammatory transcription factors (Stat3, Irf7, Cox2) and cytokines (Ifnβ, Il1β, Il12α) were considerably affected. Furthermore, we demonstrated an impact on blood vessel integrity, while vascularization of tumors appeared similar between mouse strains. The higher stability and attenuated leakiness of the tumor vasculature imply improved sustenance of glioma tissue in Ccr2-/- mice. Additionally, despite TAMs residing in the perivascular niche in Ccr2-/- mice, their pro-angiogenic activity was reduced by the downregulation of Vegf. In conclusion, lacking CCR2 solely on tumor microenvironmental cells leads to enhanced tumor progression, whereby high numbers of TAMs infiltrate gliomas independently of the CCR2/CCL2 signal

A novel inhibitor of the PI3K/Akt pathway based on the structure of inositol 1,3,4,5,6-pentakisphosphate

Background: Owing to its role in cancer, the phosphoinositide 3-kinase (PI3K)/Akt pathway is an attractive target for therapeutic intervention. We previously reported that the inhibition of Akt by inositol 1,3,4,5,6- pentakisphosphate (InsP5) results in anti-tumour properties. To further develop this compound we modified its structure to obtain more potent inhibitors of the PI3K/Akt pathway.Methods: Cell proliferation/survival was determined by cell counting, sulphorhodamine or acridine orange/ethidium bromide assay; Akt activation was determined by western blot analysis. In vivo effect of compounds was tested on PC3 xenografts, whereas in vitro activity on kinases was determined by SelectScreen Kinase Profiling Service.Results: The derivative 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5) is active towards cancer types resistant to InsP5 in vitro and in vivo. 2-O-Bn-InsP5 possesses higher pro-apoptotic activity than InsP 5 in sensitive cells and enhances the effect of anti-cancer compounds. 2-O-Bn-InsP5 specifically inhibits 3-phosphoinositide- dependent protein kinase 1 (PDK1) in vitro (IC 50 in the low nanomolar range) and the PDK1-dependent phosphorylation of Akt in cell lines and excised tumours. It is interesting to note that 2-O-Bn-InsP5 also inhibits the mammalian target of rapamycin (mTOR) in vitro.Conclusions: InsP5 and 2-O-Bn-InsP5 may represent lead compounds to develop novel inhibitors of the PI3K/Akt pathway (including potential dual PDK1/mTOR inhibitors) and novel potential anti-cancer drugs

Improved astrophysical rate for the 18O(p,α)15N reaction by underground measurements

The 18O(p,\u3b1)15N reaction affects the synthesis of 15N, 18O and 19F isotopes, whose abundances can be used to probe the nucleosynthesis and mixing processes occurring deep inside asymptotic giant branch (AGB) stars. We performed a low-background direct measurement of the 18O(p,\u3b1)15N reaction cross-section at the Laboratory for Underground Nuclear Astrophysics (LUNA) from center of mass energy Ec.m.=340keV down to Ec.m.=55keV, the lowest energy measured to date corresponding to a cross-section of less than 1 picobarn/sr. The strength of a key resonance at center of mass energy Er=90keV was found to be a factor of 10 higher than previously reported. A multi-channel R-matrix analysis of our and other data available in the literature was performed. Over a wide temperature range, T=0.01\u20131.00GK, our new astrophysical rate is both more accurate and precise than recent evaluations. Stronger constraints can now be placed on the physical processes controlling nucleosynthesis in AGB stars with interesting consequences on the abundance of 18O in these stars and in stardust grains, specifically on the production sites of oxygen-rich Group II grains

Direct measurements of low-energy resonance strengths of the 23Na(p,γ)24Mg reaction for astrophysics

The NeNa and the MgAl cycles play a fundamental role in the nucleosynthesis of asymptotic giant branch stars undergoing hot bottom burning. The 23Na(p, \u3b3)24Mgreaction links these two cycles and a precise determination of its rate is required to correctly estimate the contribution of these stars to the chemical evolution of various isotopes of Na, Mg and Al. At temperatures of 50 <110 MK, narrow resonances at Ep=140and 251 keVare the main contributors to the reaction rate, in addition to the direct capture that dominates in the lower part of the temperature range. We present new measurements of the strengths of these resonances at the Laboratory for Underground Nuclear Astrophysics (LUNA). We have used two complementary detection approaches: high efficiency with a 4\u3c0BGO detector for the 140keV resonance, and high resolution with a HPGe detector for the 251keV resonance. Thanks to the reduced cosmic ray background of LUNA, we were able to determine the resonance strength of the 251keV resonance as \u3c9\u3b3=482(82)\u3bceVand observed new gamma ray transitions for the decay of the corresponding state in 24Mgat Ex=11931 keV. With the highly efficient BGO detector, we observed a signal for the 140keV resonance for the first time in a direct measurement, resulting in a strength of \u3c9\u3b3140=1.46+0.58 120.53neV(68% CL). Our measurement reduces the uncertainty of the 23Na(p, \u3b3)24Mgreaction rate in the temperature range from 0.05 to 0.1GK to at most +50% 1235%at 0.07GK. Accordingly, our results imply a significant reduction of the uncertainties in the nucleosynthesis calculations

Effekte des ephrinB2-EphB4 Signalweges auf spinale Metastasen

Introduction The incidence of spinal metastasis has been increasing constantly over the last 20 years as a consequence of improved diagnostics and oncological therapy. Spinal metastases require extensive surgical intervention in the case of spinal instability or in the presence of a neurological deficit, which impose a significant burden to pa-tients. While surgical procedures are becoming more and more complex for treating spinal metastasis, the molecular principles remain elusive. EphB4 – ephrinB2 has been linked to organ-specific tumor cell dissemination by mediating tumor cell – en-dothelial cell adhesion and repulsion. In this study we investigated the effect of EphB4 – ephrinB2 interaction on B16 tumor cell dissemination and long-term metas- tasis formation. Methods B16-F1 melanoma were lentivirally infected with a firefly luciferase, green fluores-cence protein, puromycin resistance construct (B16-luc). Spinal metastasis of these cells were explanted and recultivated (mB16-luc). EphB4 was retroviraly overex-pressed in B16-luc (B16 -luc-EphB4). The cells were in-vitro characterized for prolif-eration (MTT), migration (wound healing) and EphB4 gene expression (qPCR / Im-munoblot). After retrograde left carotid artery injection, metastasis formation was ob- served by in-vivo bioluminescence imaging. Spinal metastases were identified by T2 small animal magnetic resonance imaging. Number of disseminated cells / metasta-sis was measured after tissue homogenization using ex-vivo bioluminescence. The experiments were performed in wild type and inducible endothelial ephrinB2 knock-out animals (iΔefnb2). Results Endothelial ephrinB2 depletion (1738 ± 806 cells) or EphB4 kinase inhibition (1009 ± 334 cells), led to increased tumor cell dissemination. EphB4 overexpression reduced osseous dissemination of tumor cells (bone: 322 ± 90 cells other: 612 ± 206 cells). In long-term metastasis experiments, the time to neurological deficit was significantly reduced in iΔefnb2 animals (iΔefnb2 median: 21 days, control median: 26 days). Number of spinal metastasis (control: 1.2 ± 0.4472, iΔefnb2: 3.2 ± 1.643) and tumor volume (control: 2.815mm3 ± 2.558mm3 and iΔefnb2 9.999mm3 ± 5.596mm3) was significantly increased in iΔefnb2 mice. In contrast, EphB4 overexpression signifi-cantly reduced spinal metastasis number (B16-luc- pLXSN: 1.4 ± 0.5477, B16-luc-EphB4: 0.4 ± 0.5477). Secondary metastatic tumor cells (mB16-luc) lead to in-creased, organ unspecific cell dissemination. Long-term metastasis showed faster neurological deficits (B16-luc median: 26 days, mB16-luc median: 22 days), higher number of spinal metastasis (B16-luc 1.2 ± 0.4472, mB16-luc 3.8 ± 1.643) and in-creased tumor volume in the spine (B16-luc 2.815mm3 ± 2.558mm3 and mB16-luc 9.209mm3 ± 5.561mm3). Conclusion We found generally increased aggressiveness of sequential metastatic cells in- vivo. Endothelial ephrinB2 depletion mediates metastasis formation to the spine and may predispose to increased metastasis formation in osseous organs. EphB4 overex-pression on tumor cells decreased spinal metastasis. This research helps facilitate the development of a priory-targeted therapy for spinal metastasis.Einführung Die Entstehung klinisch relevanter spinaler Metastasen ist in den vergangenen 20 Jahren stetig gestiegen. Diese Prognose limitierende onkologische Manifestation kann momentan nicht suffizient therapiert werden. Die molekularen Mechanismen, welche dieser Krankheit zu Grunde liegen, sind weitestgehend unbekannt. Tumorzell – Endothelzell-Interaktionen vermitteln die Extravasation von Tumorzellen und nehmen damit bei der Entstehung von Metastasen eine entscheidende Rolle ein. Der EphrinB2-ephB4 Signalweg vermittelt in diesem Zusammenhang Tumorzell – Endothelzell-Interaktionen und könnte damit eine zentrale Rolle in der Pathophysiologie dieser Erkrankung einnehmen. In dieser Studie wurde der Effekt des EphrinB2-ephB4-Systems auf die Dissemination von B16 Melanomzellen und die Manifestation von spinalen Metastasen untersucht. Methodik Luziferase infizierte B16-F1 Melanomzellen (B16-luc) sowie EphB4 überexprimieren-de Tumorzellen (B16-luc-EphB4) wurden retrograd über die Arteria carotis interna in die Aorta descendens injiziert. Spinale Metastasen dieser Zellen wurden explantiert und rekultiviert (mB16-luc). Alle Zellen wurden in-vitro auf Proliferation (MTT), Migra-tion (Wundheilungs assay) und Genexpression von EphB4 (qPCR / Immunoblot) charakterisiert. Die Metastasenbildung wurde mittels in-vivo Biolumineszenzbildge-bung überwacht. Bei Nachweis spinaler Metastasen wurden die Größe und Anzahl mittels T2 Kleintier-Magnetresonanztomographie charakterisiert. Anschließend wurden die Organe extrahiert. Mittels Gewebehomogenisation sowie anschließender Lumineszenzanalyse wurde die Anzahl disseminierter Tumorzellen bzw. metastati-scher Tumorzellen pro Organ bestimmt. Die Experimente wurden in Kontrollen und induzierbaren endothelspezifischen ephrinB2 knockout Tieren (iΔefnb2) durchge-führt. Ergebnisse Die Depletion von ephrinB2 auf dem Endothel (1738 ± 806 Zellen) oder die Inhibition der EphB4 Kinase (1009 ± 334 Zellen) führten zu einer verstärkten Tumorzelldisse-mination, während im Gegensatz dazu eine EphB4 Überexpression zu einer reduzierten ossären Dissemination führte (Knochen: 322 ± 90 Zellen, Weichteilorgane: 612 ± 206 Zellen). Dementsprechend zeigten iΔefnb2 Tiere eine signifikant erhöhte Metastasierung (Kontrolle: 2.815mm3 ± 2.558mm3, iΔefnb2: 9.999mm3± 5.596mm3) mit multiplen spinalen Metastasen (Kontrolle: 1.2 ± 0.4472, iΔefnb2: 3.2 ± 1.643) so-wie ein korrespondierend früher einsetzendes neurologisches Defizit als Kontrolltiere (iΔefnb2 median: 21 Tage, Kontrolle: median: 26 Tage). Unter Überexpression von EphB4 wurde eine signifikante Reduktion spinaler Metastasen beobachtet (B16-luc-pLXSN:1.4 ± 0.5477, B16-luc-EphB4: 0.4 ± 0.5477) mit einem signifikant später ein- setzenden neurologischen Defizit. Die Reinjektion von spinal metastasierten Tumor-zellen (mB16-luc) hatte eine organunspezifische Dissemination zur Folge mit einer unspezifischen Erhöhung spinaler Metastasen (B16-luc: 1.2 ± 0.4472, mB16-luc: 3.8 ± 1.643) und einem früher einsetzenden neurologischen Defizit (B16-luc median: 26 Tage, mB16-luc median: 22 Tage) im Vergleich zur Kontrollgruppe (B16-luc median: 26 Tage, mB16-luc median: 22 Tage). Schlussfolgerung EphrinB2–EphB4-Interaktionen spielen eine zentrale Rolle bei der organspezifi-schen Metastasierung mittels Steuerung der Tumorzelldissemination. Insbesondere bei der Entstehung experimenteller spinaler Metastasen stellt dieser Signalweg damit ein vielversprechendes therapeutisches Ziel dar