The integrin inhibitor cilengitide enhances the anti-glioma efficacy of vasculostatin-expressing oncolytic virus

Oncolytic viral (OV) therapy has been considered as a promising treatment modality for brain tumors. Vasculostatin, the fragment of brain-specific angiogenesis inhibitor-1, shows anti-angiogenic activity against malignant gliomas. Previously, a vasculostatin-expressing oncolytic herpes simplex virus-1, Rapid Antiangiogenesis Mediated By Oncolytic virus (RAMBO), was reported to have a potent antitumor effect. Here, we investigated the therapeutic efficacy of RAMBO and cilengitide, an integrin inhibitor, combination therapy for malignant glioma. In vitro, tube formation was significantly decreased in RAMBO and cilengitide combination treatment compared with RAMBO or cilengitide monotherapy. Moreover, combination treatment induced a synergistic suppressive effect on endothelial cell migration compared with the control virus. RAMBO, combined with cilengitide, induced synergistic cytotoxicity on glioma cells. In the caspase-8 and -9 assays, the relative absorption of U87 Delta EGFR cell clusters treated with cilengitide and with RAMBO was significantly higher than that of those treated with control. In addition, the activity of caspase 3/7 was significantly increased with combination therapy. In vivo, there was a significant increase in the survival of mice treated with combination therapy compared with RAMBO or cilengitide monotherapy. These results indicate that cilengitide enhanced vasculostatin-expressing OV therapy for malignant glioma and provide a rationale for designing future clinical trials combining these two agents

Myxovirus resistance 1 (MX1) is an independent predictor of poor outcome in invasive breast cancer

Background: Breast cancer (BC) is a disease with variable morphology, clinical behaviour and response to therapy. Identifying factors associated with the progression of early stage BC can help understand the risk of metastasis and guide treatment decisions. Myxovirus resistance 1 (MX1), which is involved in the cellular antiviral mechanism, plays a role in some solid tumours; however, its role in invasive BC remains unknown. In this study, we aimed to explore the clinicopathological and prognostic significance of MX1 in BC.Methods: MX1 was assessed at the protein level using tissue microarrays from a large well-annotated BC cohort (n=845). The expression of MX1 mRNA was assessed at the transcriptomic level using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n=1980) and validated using three publicly available cohorts on Breast Cancer Gene-Expression Miner (bc-GenExMiner version 4.4). The associations between MX1 expression and clinicopathological factors, and outcome were evaluated.Results: High MX1 protein expression was associated with features of aggressiveness, including large tumour size, high tumour grade, high Nottingham prognostic index scores, hormone receptor negativity and high Ki67 expression. High MX1 expression showed an association with poor patient outcome and it was an independent predictor of short BC- specific survival (p=0.028; HR=1.5; 95%CI=1.0–2.2). Consistent with the protein results, high MX1 mRNA levels showed an association with features of aggressive behaviour and with shorter survival.Conclusion: This study identified MX1 as an independent predictor of poor outcome in patients with BC. Further functional studies are needed to investigate the biological role of MX1 in BC and its potential value as a therapeutic target

Therapeutic effect of suicide gene-transferred mesenchymal stem cells in a rat model of glioma

We evaluated a new therapeutic strategy for malignant glioma, which combines intratumoral inoculation of mesenchymal stem cells (MSCs) expressing cytosine deaminase gene with 5-fluorocytosine (5-FC) administration. For in vitro and in vivo experiments, MSCs were transfected with adenovirus carrying either enhanced green fluorescent protein gene (AdexCAEGFP) or cytosine deaminase gene (AdexCACD), to establish MSC-expressing EGFP (MSC-EGFP) or CD (MSC-CD). Co-culture of 9L glioma cells with MSC-CD in a medium containing 5-FC resulted in a remarkable reduction in 9L cell viability. The migratory ability of MSC-EGFP toward 9L cells was demonstrated by double-chamber assay. For the in vivo study, rats harboring 9L brain tumors were inoculated with MSC-EGFP or MSC-CD. Immunohistochemistry of rat brain tumors inoculated with MSC-EGFP showed intratumoral distribution of MSC-EGFP. Survival analysis of rats bearing 9L gliomas treated with intratumoral MSC-CD and intraperitoneal 5-FC resulted in significant prolongation of survival compared with control animals. In conclusion, molecular therapy combining suicide gene therapy and MSCs as a targeting vehicle represents a potential new therapeutic approach for malignant glioma, both with respect to the antitumor potential of this system and its neuroprotective effect on normal brain tissue

Targetable ERBB2 mutation status is an independent marker of adverse prognosis in estrogen receptor positive, ERBB2 non-amplified primary lobular breast carcinoma: a retrospective in silico analysis of public datasets

© 2020 The Author(s). Background: Invasive lobular carcinoma (ILC) accounts for 10-15% of primary breast cancers and is typically estrogen receptor alpha positive (ER+) and ERBB2 non-amplified. Somatic mutations in ERBB2/3 are emerging as a tractable mechanism underlying enhanced human epidermal growth factor 2 (HER2) activity. We tested the hypothesis that therapeutically targetable ERBB2/3 mutations in primary ILC of the breast associate with poor survival outcome in large public datasets. Methods: We performed in silico comparison of ERBB2 non-amplified cases of ER+ stage I-III primary ILC (N = 279) and invasive ductal carcinoma (IDC, N = 1301) using METABRIC, TCGA, and MSK-IMPACT information. Activating mutations amenable to HER2-directed therapy with neratinib were identified using existing functional data from in vitro cell line and xenograft experiments. Multivariate analysis of 10-year overall survival (OS) with tumor size, grade, and lymph node status was performed using a Cox regression model. Differential gene expression analyses by ERBB2 mutation and amplification status was performed using weighted average differences and an in silico model of response to neratinib derived from breast cancer cell lines. Results: ILC tumors comprised 17.7% of all cases in the dataset but accounted for 47.1% of ERBB2-mutated cases. Mutations in ERBB2 were enriched in ILC vs. IDC cases (5.7%, N = 16 vs. 1.4%, N = 18, p < 0.0001) and clustered in the tyrosine kinase domain of HER2. ERBB3 mutations were not enriched in ILC (1.1%, N = 3 vs. 1.8%, N = 23; p = 0.604). Median OS for patients with ERBB2-mutant ILC tumors was 66 months vs. 211 months for ERBB2 wild-type (p = 0.0001), and 159 vs. 166 months (p = 0.733) for IDC tumors. Targetable ERBB2 mutational status was an independent prognostic marker of 10-year OS - but only in ILC (hazard ratio, HR = 3.7, 95% CI 1.2-11.0; p = 0.021). Findings were validated using a novel ERBB2 mutation gene enrichment score (HR for 10-year OS in ILC = 2.3, 95% CI 1.04-5.05; p = 0.040). Conclusions: Targetable ERBB2 mutations are enriched in primary ILC and their detection represents an actionable strategy with the potential to improve patient outcomes. Biomarker-led clinical trials of adjuvant HER-targeted therapy are warranted for patients with ERBB2-mutated primary ILC

The fate of Böhler's angle in conservatively-treated displaced intra-articular calcaneal fractures

Purpose: Although the predictive value of Böhler's angle on outcome remains subject of debate, the initial angle at the time of trauma still guides treatment. Changes in Böhler's angle during follow-up are frequently reported following surgical treatment of displaced intra-articular calcaneal fractures (DIACF). The aim of the present study was to determine the changes in Böhler's angle as a measure of secondary fracture displacement following conservative management of DIACF. Methods: Thirty-eight patients with a total of 44 displaced intra-articular calcaneal fractures treated conservatively with a minimum of two lateral radiographs during follow-up were analysed. Böhler's angle at different follow-up times was measured by three observers. The change in angle was compared with the angle at trauma, and influence of trauma mechanism and common calcaneal fracture classifications were determined. Results: The results showed a significant decline over time of the Böhler's angle in conservatively-treated patients of more than 11° on average at a mean follow-up of 29.2 weeks. This decrease was not related to gender, the initial angle, or the Essex-Lopresti or Sanders classification. A statistically significantly higher decrease was detected in high energetic trauma compared with low energetic trauma. Conclusion: The conservative treatment of displaced intra-articular calcaneal fractures is still a viable option, yet a significant secondary displacement in time should be taken into account, as reflected in a decrease of Böhler's angle of 11° up to one year following trauma

Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection

<p>Abstract</p> <p>Background</p> <p>In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV).</p> <p>Methods</p> <p>Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII⁺-cell depletion) in nude mice.</p> <p>Results</p> <p>Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII⁺/CD31⁺vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII⁺-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis.</p> <p>Conclusions</p> <p>Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.</p

Stem Cell Mediation of Functional Recovery after Stroke in the Rat

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Regenerative strategies of stem cell grafting have been demonstrated to be effective in animal models of stroke. In those studies, the effectiveness of stem cells promoting functional recovery was assessed by behavioral testing. These behavioral studies do, however, not provide access to the understanding of the mechanisms underlying the observed functional outcome improvement. [Methodology/Principal Findings]: In order to address the underlying mechanisms of stem cell mediated functional improvement, this functional improvement after stroke in the rat was investigated for six months after stroke by use of fMRI, somatosensory evoked potentials by electrophysiology, and sensorimotor behavior testing. Stem cells were grafted ipsilateral to the ischemic lesion. Rigorous exclusion of spontaneous recovery as confounding factor permitted to observe graft-related functional improvement beginning after 7 weeks and continuously increasing during the 6-month observation period. The major findings were i) functional improvement causally related to the stem cells grafting; ii) tissue replacement can be excluded as dominant factor for stem cell mediated functional improvement; iii) functional improvement occurs by exclusive restitution of the function in the original representation field, without clear contributions from reorganization processes, and iv) stem cells were not detectable any longer after six months. [Conclusions/Significance]: A delayed functional improvement due to stem cell implantation has been documented by electrophysiology, fMRI and behavioral testing. This functional improvement occurred without cells acting as a tissue replacement for the necrotic tissue after the ischemic event. Combination of disappearance of grafted cells after six months on histological sections with persistent functional recovery was interpreted as paracrine effects by the grafted stem cells being the dominant mechanism of cell activity underlying the observed functional restitution of the original activation sites. Future studies will have to investigate whether the stem cell mediated improvement reactivates the original representation target field by using original connectivity pathways or by generating/activating new ones for the stimulus.Financial support from the Hertie Foundation (Germany), and EU grants of the FP-6: DiMI (LSHB-CT-2005-512146), EMIL (LSHC-CT-2004-503569) and Stem Stroke (LSHB-CT-2006-037526) are gratefully acknowledged.Peer Reviewe

Elevated MMP9 expression in breast cancer is a predictor of shorter patient survival

Purpose: MMP9 is a matricellular protein associated with extracellular matrix (ECM) remodelling, that promotes tumour progression, and modulates the activity of cell adhesion molecules and cytokines. This study aims to assess the prognostic value of MMP9 and its association with cytoskeletal modulators in early-stage invasive breast cancer (BC). Methods: MMP9 expression was evaluated by immunohistochemistry using a well-characterised series of primary BC patients with long-term clinical follow-up. Association with clinicopathological factors, patient outcome and ECM remodelling BC-biomarkers were investigated. METABRIC dataset, BC-GenExMiner v4.0 and TCGA were used for the external validation of MMP9 expression. GSEA gene enrichment analyses were used to evaluate MMP9 associated pathways.Results: MMP9 immunopositivity was observed in the stroma and cytoplasm of BC cells. Elevated MMP9 protein levels was associated with high tumour grade, high Nottingham Prognostic Index, and hormonal receptor negativity. Elevated MMP9 protein expression correlated significantly with cytokeratin 17 (Ck17), Epidermal Growth Factor Receptor (EGFR), proliferation (Ki67) biomarkers, cell surface adhesion receptor (CD44) and cell division control protein 42 (CDC42). Cytoplasmic MMP9 expression was an independent prognostic factor associated with shorter BC-specific survival. In the external validation cohorts, MMP9 expression was also associated with poor patients’ outcome. Transcriptomic analysis confirmed a positive association between MMP9 and ECM remodelling biomarkers. GSEA analysis supports MMP9 association with ECM and cytoskeletal pathways. Conclusion: This study provides evidence for the prognostic value of MMP9 in BC. Further functional studies to decipher the role of MMP9 and its association with cytoskeletal modulators in BC progression are warranted

Lung fibroblasts from patients with emphysema show markers of senescence in vitro

BACKGROUND: The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD. METHODS: Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema (E) and 15 controls (C) undergoing surgery for lung tumor resection or volume reduction (n = 2). Fibroblasts (8E/9C) were stained for senescence-associated β-galactosidase (SA-β-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant. RESULTS: The median (quartiles) percentage of fibroblasts positive for SA-β-Gal was 4.4 (3.2;4.7) % in controls and 16.0 (10.0;24.8) % in emphysema (p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array (factor ≥ 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-rP1 (p = 0.029, p = 0.0002), while expression of IGFBP-5, -rP2 (CTGF), -rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 (p = 0.006) in emphysema. CONCLUSION: These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence-associated IGFBP-3 and -rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate

Clinicopathological significance of lipocalin 2 nuclear expression in invasive breast cancer

PURPOSE: The epithelial–mesenchymal transition (EMT) plays a key role in breast cancer progression and metastasis. Lipocalin 2 (LCN2) is involved in the regulation of EMT. The aim of this study was to investigate the clinicopathological significance of LCN2 expression in breast cancer.METHODS: The expression of LCN2 protein was immunohistochemically assessed in two well-characterised annotated cohorts of breast cancer (discovery cohort, n = 612; validation cohort, n = 1,363). The relationship of LCN2 expression and subcellular location with the clinicopathological factors and outcomes of patients was analysed.RESULTS: Absent or reduced nuclear LCN2 expression was associated with features of aggressive behaviour, including high histological grade, high Nottingham Prognostic Index, high Ki67 labelling index, hormone receptor negativity and human epidermal growth factor receptor 2 positivity. The high cytoplasmic expression of LCN2 was correlated with lymph node positivity. The nuclear downregulation of LCN2 was correlated with the overexpression of EMT associated proteins (N-cadherin and Twist-related protein 2) and basal biomarkers (cytokeratin 5/6 and epidermal growth factor receptor). Unlike the cytoplasmic expression of LCN2, the loss of nuclear expression was a significant predictor of poor outcome. The combinatorial expression tumours with high cytoplasmic and low nuclear expression were associated with the worst prognosis.CONCLUSIONS: Tumour cell expression of LCN2 plays a role in breast cancer progression with loss of its nuclear expression is associated with aggressive features and poor outcome. Further functional analysis is warranted to confirm the relationship between the subcellular localisation LCN2 and behaviour of breast cancer