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

Impact of pre-hospital handling and initial time to cranial computed tomography on outcome in aneurysmal subarachnoid hemorrhage patients with out-of-hospital sudden cardiac arrest—a retrospective bi-centric study

BackgroundAneurysmal subarachnoid hemorrhage (SAH) presents occasionally with cardiac arrest (CA). The impact of pre-hospital and emergency room (ER) treatment on outcome remains unclear. Therefore, we investigated the impact of pre-hospital treatment, focusing on lay cardiopulmonary resuscitation (CPR), and ER handling on the outcome of SAH patients with out-of-hospital CA (OHCA).MethodsIn this bi-centric retrospective analysis, we reviewed SAH databases for OHCA and CPR from January 2011 to June 2021. Patients were analyzed for general clinical and epidemiological parameters. CPR data were obtained from ambulance reports and information on ER handling from the medical records. Data were correlated with patient survival at hospital discharge as a predefined outcome parameter.ResultsOf 1,120 patients with SAH, 45 (4.0%) were identified with OHCA and CPR, 38 of whom provided all required information and were included in this study. Time to resuscitation was significantly shorter with lay resuscitation (5.3 ± 5.2 min vs. 0.3 ± 1.2 min, p = 0.003). Nineteen patients were not initially scheduled for cranial computed tomography (CCT), resulting in a significantly longer time interval to first CCT (mean ± SD: 154 ± 217 min vs. 40 ± 23 min; p < 0.001). Overall survival to discharge was 31.6%. Pre-hospital lay CPR was not associated with higher survival (p = 0.632). However, we observed a shorter time to first CCT in surviving patients (p = 0.065)ConclusionsOHCA in SAH patients is not uncommon. Besides high-quality CPR, time to diagnosis of SAH appears to play an important role. We therefore recommend considering CCT diagnostics as part of the diagnostic algorithm in patients with OHCA

Der Ephrin-B2-EphB4-Signalweg als therapeutisches Ziel in der Entstehung spinaler Metastasen

Introduction The development of spinal metastases is a common and complex problem in the management of cancer patients, with the spine being one of the main sites to where metastases will spread. If untreated, spinal metastases rapidly lead to progressive myelopathy with extensive effects on ambulatory status, quality of life and survival rates of affected patients. Although major developments in the field of surgical and oncological procedures have led to improved therapeutic options, the molecular steps of spinal metastasis formation still remain inadequately understood. EphB4 and Ephrin-B2 have repeatedly been identified as key regulators in metastatic cell dissemination, tumor cell – endothelial cell interaction and tumor growth. We investigated the effects of a therapeutic alteration of this molecular interaction on long- term metastasis formation. Methods Bioluminescent B16 melanoma cells were injected retrogradely into the carotid artery of Ephrin-B2-knockout (efnb2iΔEC) and control (efnb2lox/lox) mice in a pre- and post- metastatic setting (“pre-“ and “post-tumor”). Potential therapeutic effects were evaluated after applying soluble Ephrin-B2-Fc, as well as NVP-BHG 712, a small molecular inhibitor of the EphB4 receptor tyrosine kinase. Tumor growth and dissemination were surveyed utilizing in-vivo bioluminescent imaging procedures and compressive spinal loci were identified using magnetic resonance imaging. Immunohistochemical evaluation of tumor cell proliferation and vasculature were performed and the organ specific tumor load was examined using a luciferase detection assay. Results In pre-tumor treated efnb2lox/lox mice the application of Ephrin-B2-Fc (median: 20.5 days, n=7, p=0.0048) and NVP-BHG 712 (median: 21 days, n=14, p=0.0002) induced significantly earlier neurologic deficits when compared to the placebo-treated group (median: 24.5 days, n=11), by increasing the number and volume of spinal metastases. In post-tumor treated efnb2lox/lox mice there was no significant difference in survival times or number of spinal metastases in the MRI. The earlier appearance of neurological deficits in efnb2iΔEC-knockout mice (median efnb2i∆EC placebo: 18 days, n=7, p=<0.0001) could be significantly delayed by pre-tumor treatment with Ephrin-B2- Fc. (median: 23 days, n=8). Conclusion The molecular interaction between EphB4 and Ephrin-B2 significantly affected the formation of spinal metastases. The physiological EphrinB2-EphB4 interaction in efnb2lox/lox control mice showed tumor suppressive effects by increasing tumor cell repulsion from the endothelium. The therapeutic disruption of these repulsive effects decreased neurological survival through an increased number and volume of metastases. Under the effects of the prometastatic endothelial Ephrin-B2 knockout (efnb2i∆EC), these physiologically inert repulsive functions could be partially reestablished through Ephrin-B2-Fc.Einführung Die Entstehung spinaler Metastasen ist eine häufige und schwerwiegende Komplikation in der Behandlung von Krebspatienten. Die Wirbelsäule stellt hierbei eine der häufigsten Lokalisationen ossärer Metastasierung dar. Ohne adäquate Therapie führen spinale Metastasen binnen kürzester Zeit zu einer zunehmenden Myelopathie und Destruktion der neuralen Elemente, mit weitreichenden Auswirkungen auf das Überleben und die Lebensqualität der betroffenen Patienten. Trotz stetiger Entwicklungen in der onkologischen und neurochirurgischen Therapie dieses komplexen Krankheitsbildes bleiben die grundlegenden Entstehungsmechanismen weiterhin weitestgehend unverstanden. EphB4 und Ephrin-B2 wurden wiederholt als wichtige Regulatoren in der Metastasenentstehung, sowie in der Interaktion zwischen Tumor- und Endothelzellen identifiziert. Wir haben die Auswirkungen dieser molekularen Interaktion, sowie mögliche therapeutische Beeinflussungen der Entstehung spinaler Metastasen untersucht. Methodik BIolumineszierende B16-Melanomzellen wurden jeweils in einem prä- und postmetastatischen Ansatz retrograd in die A. carotis communis von Ephrin-B2- Knockout- (efnb2iΔEC) und Kontrollmäusen (efnb2lox/lox) injiziert. Potenzielle therapeutische Effekte des löslichen Antikörpers Ephrin-B2-Fc, sowie des spezifischen EphB4 Tyrosinkinaseinhibitors NVP-BHG 712 wurden evaluiert. Tumorwachstum und Verteilungsmuster wurden durch Biolumineszenz-Bildgebungsverfahren in vivo überwacht und Tumorwachstum und Myelonkompression mittels spinalem MRT dargestellt. Immunhistochemische Färbungen wurden hinsichtlich Proliferation und Angiogenese ausgewertet, sowie die Anzahl der metastatischen Tumorzellen pro Organ mittels eines Luziferase-Assays. Ergebnisse In prä-tumor vorbehandelten efnb2lox/lox Mäusen zeigten sowohl die mittels Ephrin-B2- Fc (Mittelwert: 20.5 Tage, n=7, p=0.0048) als auch die mit NVP-BHG 712 (Mittelwert: 21 Tage, n=14, p=0.0002) therapierte Gruppe signifikant frühere neurologische Defizite durch vermehrte spinale Metastasen als die Placebo-behandelten Tiere (Mittelwert: 24,5 Tage, n=11). In den post-tumor behandelten efnb2lox/lox Tieren zeigte sich kein Unterschied im Überleben oder der spinalen Tumorlast. Das signifikant frühere Auftreten neurologischer Defizite in Ephrin-B2-knockout (efnb2iΔEC ) Mäusen (Mittelwert efnb2i∆EC Placebo: 18 Tage, n=7, p=<0.0001) konnte durch die prämetastatische Gabe von Ephrin-B2-Fc deutlich verzögert werden (Mittelwert: 23 Tage, n=8). Schlussfolgerung Die molekulare Interaktion zwischen EphB4 und Ephrin-B2 beeinflusst die Entstehung spinaler Metastasen. Der physiologische Zustand inhibiert das Tumorwachstum durch vermehrte Abstoßung der zirkulierenden Tumorzellen. Die therapeutische Unterbrechung dieses protektiven Effektes führte zu einem signifikant früheren neurologischen Defizit durch vermehrtes Wachstum spinaler Metastasen. Unter dem prometastatischen endothelialen Ephrin-B2-knockout konnte durch prämetastatische Applikation des Ephrin-B2-Fc Antikörpers die physiologisch bestehende Abstoßungsreaktion partiell wiederhergestellt werden, sodass eine signifikante Verlängerung des neurologischen Überlebens erreicht wurde

ICAM1 depletion reduces spinal metastasis formation in vivo and improves neurological outcome

INTRODUCTION Clinical treatment of spinal metastasis is gaining in complexity while the underlying biology remains unknown. Insufficient biological understanding is due to a lack of suitable experimental animal models. Intercellular adhesion molecule-1 (ICAM1) has been implicated in metastasis formation. Its role in spinal metastasis remains unclear. It was the aim to generate a reliable spinal metastasis model in mice and to investigate metastasis formation under ICAM1 depletion. MATERIAL AND METHODS B16 melanoma cells were infected with a lentivirus containing firefly luciferase (B16-luc). Stable cell clones (B16-luc) were injected retrogradely into the distal aortic arch. Spinal metastasis formation was monitored using in vivo bioluminescence imaging/MRI. Neurological deficits were monitored daily. In vivo selected, metastasized tumor cells were isolated (mB16-luc) and reinjected intraarterially. mB16-luc cells were injected intraarterially in ICAM1 KO mice. Metastasis distribution was analyzed using organ-specific fluorescence analysis. RESULTS Intraarterial injection of B16-luc and metastatic mB16-luc reliably induced spinal metastasis formation with neurological deficits (B16-luc:26.5, mB16-luc:21 days, p<0.05). In vivo selection increased the metastatic aggressiveness and led to a bone specific homing phenotype. Thus, mB16-luc cells demonstrated higher number (B16-luc: 1.2±0.447, mB16-luc:3.2±1.643) and increased total metastasis volume (B16-luc:2.87±2.453 mm3, mB16-luc:11.19±3.898 mm3, p<0.05) in the spine. ICAM1 depletion leads to a significantly reduced number of spinal metastasis (mB16-luc:1.2±0.84) with improved neurological outcome (29 days). General metastatic burden was significantly reduced under ICAM1 depletion (control: 3.47×10(7)±1.66×10(7); ICAM-1-/-: 5.20×10(4)±4.44×10(4), p<0.05 vs. control) CONCLUSION Applying a reliable animal model for spinal metastasis, ICAM1 depletion reduces spinal metastasis formation due to an organ-unspecific reduction of metastasis development

Comparison of B16-luc, LLC1-luc and TRAMP-C2-luc tumor cell dissemination versus microsphere dissemination (7 and 16 μm diameter) in osseous organs.

<p>(A) Only ~5% of tumor cells and microbeads disseminated to the spine. No significant difference was found between the groups. (B) Long bones revealed dissemination fractions between ~10–25%. TRAMP-C2-luc disseminated significantly higher to long bones compared to LLC1-luc cells (LLC1-luc vs. TRAMP-C2-luc: p = 0.0406). No other significant dissemination differences were found. (C) Low tumor cell and microbeads dissemination was found in the thorax (~5–10%). No significant difference was found between the groups. (D) Dissemination to the cranium was ~2–6%. No significant dissemination differences were found. Mean ± SD, (B16-luc n = 4, LLC1-luc n = 5, TRAMP-C2-luc n = 5, 7 μm beads n = 5, 16 μm beads n = 5) for all experiments shown, one way ANOVA with Bonferroni post hoc analysis.</p

B16-luc, LLC1-luc, TRAMP-C2-luc and microsphere dissemination (7 and 16 μm diameter) in soft tissues.

<p>(A) Individual lung dissemination of tumor cells varied between ~30–40%. In contrast, very few microbeads (independent of their size) were found in the lung. All cell types disseminated significantly higher to the lung compared to microbeads (B16-luc vs. 7 μm beads: p < 0.0001, B16-luc vs. 16 μm beads: p < 0.0001, LLC1-luc vs. 7 μm beads: p = 0.0025, LLC1-luc vs. 16 μm beads: p = 0.0039, TRAMP-C2-luc vs. 7 μm beads: p = 0.0024, TRAMP-C2-luc vs. 16 μm beads: p = 0.0037). (B) Tumor cell and microbeads dissemination to the brain was relatively low (~5%). LLC1-luc cells disseminated significantly higher to the brain compared to B16-luc. Cell sized (16 μm) microbeads also disseminated significantly higher to the brain compared to B16-luc and even TRAMP-C2-luc (B16-luc vs. LLC1-luc: p = 0.0288, B16-luc vs. 7 μm beads: p = 0.0386, B16-luc vs. 16 μm beads: p = 0.0012, TRAMP-C2-luc vs. 16 μm beads: p = 0.0019). (C) The kidneys’ dissemination fraction ranged from ~10 to 25%. Small (7 μm) microbeads disseminated significantly higher to the kidneys compared to B16-luc and LLC1-luc tumor cells (LLC1-luc vs. 7μm beads: p = 0.0327). (D) Microbeads dissemination was high in the liver (~20%), whereas cell dissemination varied by cell type. LLC1-luc cells disseminated significantly stronger to the liver compared to the other cells. Comparably high amounts of microbeads were found in the liver, 7 μm beads disseminated significantly higher to the liver compared to B16-luc and TRAMP-C2, 16 μm beads showed significantly higher dissemination compared to TRAMP-C2 (B16-luc vs. LLC1-luc: p = 0.0029, B16-luc vs. 7μm beads: p = 0.0116, B16-luc vs. 16μm beads: p = 0.0338, LLC1-luc vs. TRAMP-C2-luc: p < 0.0001, TRAMP-C2-luc vs. 7μm beads: p = 0.0003, TRAMP-C2-luc vs. 16μm beads: p = 0.0003). (E) The skin showed high dissemination of microbeads and TRAMP-C2-luc cells, with a significant effect compared to B16-luc cells (B16-luc vs. TRAMP-C2-luc: p = 0.0366, B16-luc vs. 7μm beads: p = 0.0082, B16-luc vs. 16μm beads: p = 0.0084). Mean ± SD, (B16-luc n = 4, LLC1-luc n = 5, TRAMP-C2-luc n = 5, 7 μm beads n = 5, 16 μm beads n = 5) for all experiments shown, one way ANOVA with Bonferroni post hoc analysis.</p

Characterization of firefly luciferase—eGFP infected B16-F1 melanoma, LLC1 lung carcinoma and TRAMP-C2 prostate carcinoma cells.

<p>(A) Cells were lentivirally transduced to express firefly luciferase, enhanced GFP and puromycin. Representative phase contrast and green fluorescence image of B16-luc, LLC1-luc and TRAMP-C2-luc cells (bar represents 100 μm). (B) Analysis of luciferase activity by spectrometer demonstrates relative light units (RLU) emission by lysed cells. Bars represent mean ± SD, Student’s two tailed t-test (B16-luc vs. B16: p = < 0.0001, n = 7, LLC1-luc vs. LLC1: p = < 0.0001, n = 6, TRAMP-C2-luc vs. TRAMP-C2: p = < 0.0001, n = 6). (C) Viability was measured by absorbance of MTT. The absorbance was significantly higher in B16-luc cells seeded at 10’000 cells per well compared to control B16 cells (p < 0.0001). There was no viability difference in LLC1-luc and TRAMP-C2-luc cells compared to LLC1 and TRMAP-C2 cells respectively. Mean ± SD, n = 5 for all experiments shown, two way ANOVA with Bonferroni post hoc analysis. (D) Wound healing 2D migration analysis shows no difference in B16-luc, LLC1-luc andTRAMP-C2-luc cell migration 24 hours post scratch. Mean ± SD, n = 3 for all experiments shown, two way ANOVA with Bonferroni post hoc analysis.</p

Initial pupil status is a strong predictor for in-hospital mortality after aneurysmal subarachnoid hemorrhage

Prognosis of patients with high-grade aneurysmal subarachnoid hemorrhage (aSAH) is only insufficiently displayed by current standard prognostic scores. This study aims to evaluate the role of pupil status for mortality prediction and provide improved prognostic models. Anonymized data of 477 aSAH patients admitted to our medical center from November 2010 to August 2018 were retrospectively analyzed. Identification of variables independently predicting in-hospital mortality was performed by multivariable logistic regression analysis. Final regression models included Hunt & Hess scale (H&H), pupil status and age or in a simplified variation only H&H and pupil status, leading to the design of novel H&H-Pupil-Age score (HHPA) and simplified H&H-Pupil score (sHHP), respectively. In an external validation cohort of 402 patients, areas under the receiver operating characteristic curves (AUROC) of HHPA (0.841) and sHHP (0.821) were significantly higher than areas of H&H (0.794; p<0.001) or World Federation of Neurosurgical Societies (WFNS) scale (0.775; p<0.01). Accordingly, including information about pupil status improves the predictive performance of prognostic scores for in-hospital mortality in patients with aSAH. HHPA and sHHP allow simple, early and detailed prognosis assessment while predictive performance remained strong in an external validation cohort suggesting adequate generalizability and low interrater variability

Genome-wide DNA methylation profiles distinguish silent from non-silent ACTH adenomas

Early active multiple sclerosis (MS) lesions can be classified histologically into three main immunopathological patterns of demyelination (patterns I-III), which suggest pathogenic heterogeneity and may predict therapy response. Patterns I and II show signs of immune-mediated demyelination, but only pattern II is associated with antibody/complement deposition. In pattern III lesions, which include Baló's concentric sclerosis, primary oligodendrocyte damage was proposed. Serum antibody reactivities could reflect disease pathogenesis and thus distinguish histopathologically defined MS patterns. We established a customized microarray with more than 700 peptides that represent human and viral antigens potentially relevant for inflammatory demyelinating CNS diseases, and tested sera from 66 patients (pattern I n = 12; II n = 29; III n = 25, including 8 with Baló's), healthy controls, patients with Sjögren's syndrome and stroke patients. Cell-based assays were performed for aquaporin 1 (AQP1) and AQP4 antibody detection. No single peptide showed differential binding among study cohorts. Because antibodies can react with different peptides from one protein, we also analyzed groups of peptides. Patients with pattern II showed significantly higher reactivities to Nogo-A peptides as compared to patterns I (p = 0.02) and III (p = 0.02). Pattern III patients showed higher reactivities to AQP1 (compared to pattern I p = 0.002, pattern II p = 0.001) and varicella zoster virus (VZV, compared to pattern II p = 0.05). In patients with Baló's, AQP1 reactivity was also significantly higher compared to patients without Baló's (p = 0.04), and the former revealed distinct antibody signatures. Histologically, Baló's patients showed loss of AQP1 and AQP4 in demyelinating lesions, but no antibodies binding conformational AQP1 or AQP4 were detected. In summary, higher reactivities to Nogo-A peptides in pattern II patients could be relevant for enhanced axonal repair and remyelination. Higher reactivities to AQP1 peptides in pattern III patients and its subgroup of Baló's patients possibly reflect astrocytic damage. Finally, latent VZV infection may cause peripheral immune activation

CD74 and CD44 Expression on CTCs in Cancer Patients with Brain Metastasis

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies