A web-based system for tissue microarray data management

BACKGROUND: Tissue Microarray is a novel technique for analysing large amounts of immunohistochemically stained specimens. However, those large amounts make it difficult to design, prepare and analyze a tissue microarray, so that software support is almost inevitable. METHODS: We designed a tissue microarray data management system starting from specifications obtained by pathologists, and arranged for a preliminary validation in thyroid pathology. RESULTS: A web-based system has been developed, basing on open-source software and principles, that was well accepted by pathologists and allowed to carry out a study on 52 thyroid pathology cases. CONCLUSION: Though limited in functionalities, the developed system is effective and can be downloaded at the address

Identification of a Prognostic Microenvironment-Related Gene Signature in Glioblastoma Patients Treated with Carmustine Wafers

SIMPLE SUMMARY: Carmustine wafer (CW) implantation into the resection cavity of patients operated for glioblastoma (GBM) was approved as an adjuvant treatment before the Stupp Protocol. Although contrasting clinical results limited its use, our retrospective study on 116 GBM treated with CW showed a significant benefit in terms of OS in a subgroup of patients. Since GBM growth, progression, and drug resistance are supported by the surrounding environment, and since the tumor microenvironment (TME) is the source of druggable targets, we hypothesized that the TME of patients who benefited from CW could have different characteristics compared to patients who did not show any advantage. Exploiting a human in vitro model of glioma microenvironment and a transcriptomic approach, we found a different gene signature suggesting the importance of developing in vitro models that mimic the properties of human cancers and that can help to study individual patient characteristics at the cellular and molecular level. ABSTRACT: Despite the state-of-the-art treatment, patients diagnosed with glioblastoma (GBM) have a median overall survival (OS) of 14 months. The insertion of carmustine wafers (CWs) into the resection cavity as adjuvant treatment represents a promising option, although its use has been limited due to contrasting clinical results. Our retrospective evaluation of CW efficacy showed a significant improvement in terms of OS in a subgroup of patients. Given the crucial role of the tumor microenvironment (TME) in GBM progression and response to therapy, we hypothesized that the TME of patients who benefited from CW could have different properties compared to that of patients who did not show any advantage. Using an in vitro model of the glioma microenvironment, represented by glioma-associated-stem cells (GASC), we performed a transcriptomic analysis of GASC isolated from tumors of patients responsive and not responsive to CW to identify differentially expressed genes. We found different transcriptomic profiles, and we identified four genes, specifically down-regulated in GASC isolated from long-term survivors, correlated with clinical data deposited in the TCGA–GBM dataset. Our results highlight that studying the in vitro properties of patient-specific glioma microenvironments can help to identify molecular determinants potentially prognostic for patients treated with CW

Cardiac Cell Senescence and Redox Signaling

Aging is characterized by a progressive loss of the ability of the organism to cope with stressors and to repair tissue damage. As a result, chronic diseases, including cardiovascular disease, increase their prevalence with aging, underlining the existence of common mechanisms that lead to frailty and age-related diseases. In this frame, the progressive decline of the homeostatic and reparative function of primitive cells has been hypothesized to play a major role in the evolution of cardiac pathology to heart failure. Although initially it was believed that reactive oxygen species (ROS) were produced in an unregulated manner as a byproduct of cellular metabolism, causing macromolecular damage and aging, accumulating evidence indicate the major role played by redox signaling in physiology. Aim of this review is to critically revise evidence linking ROS to cell senescence and aging and to provide evidence of the primary role played by redox signaling, with a particular emphasis on the multifunctional protein APE1/Ref in stem cell biology. Finally, we will discuss evidence supporting the role of redox signaling in cardiovascular cells

Immunohistochemical evaluation of vitamin D receptor (VDR) expression in cutaneous melanoma tissues and four VDR gene polymorphisms

ObjectiveVitamin D receptor (VDR) mediates vitamin D activity. We examined whether VDR expression in excised melanoma tissues is associated with VDR gene (VDR) polymorphisms. MethodsWe evaluated VDR protein expression (by monoclonal antibody immunostaining), melanoma characteristics, and carriage of VDR-FokI-rs2228570 (C>T), VDR-BsmI-rs1544410 (G>A), VDR-ApaI-rs7975232 (T>G), and VDR-TaqI-rs731236 (T>C) polymorphisms (by restriction fragment length polymorphism). Absence or presence of restriction site was denoted by a capital or lower letter, respectively: \u201cF\u201d and \u201cf\u201d for FokI, \u201cB\u201d and \u201cb\u201d for BsmI, \u201cA\u201d and \u201ca\u201d for ApaI, and \u201cT\u201d and \u201ct\u201d for TaqI endonuclease. Seventy-four Italian cutaneous primary melanomas (52.1\ub112.7 years old) were studied; 51.4% were Stage I, 21.6% Stage II, 13.5% Stage III, and 13.5% Stage IV melanomas. VDR expression was categorized as follows: 100% positive vs. <100%; over the median 20% (high VDR expression) vs. 6420% (low VDR expression); absence versus presence of VDR-expressing cells. ResultsStage I melanomas, Breslow thickness of <1.00 mm, level II Clark invasion, Aa heterozygous genotype, and AaTT combined genotype were more frequent in melanomas with high versus low VDR expression. Combined genotypes BbAA, bbAa, AATt, BbAATt, and bbAaTT were more frequent in 100% versus <100% VDR-expressing cells. Combined genotype AATT was more frequent in melanomas lacking VDR expression (odds ratio=14.5; P=0.025). VDR expression was not associated with metastasis, ulceration, mitosis >1, regression, tumor-infiltrating lymphocytes, tumoral infiltration of vascular tissues, additional skin and non-skin cancers, and melanoma familiarity. ConclusionsWe highlighted that VDR polymorphisms can affect VDR expression in excised melanoma cells. Low VDR expression in AATT carriers is a new finding that merits further study. VDR expression possibly poses implications for vitamin D supplementation against melanoma. VDR expression and VDR genotype may become precise medicinal tools for melanoma in the future

Role of microenvironment in glioma invasion: What we learned from in vitro models

The invasion properties of glioblastoma hamper a radical surgery and are responsible for its recurrence. Understanding the invasion mechanisms is thus critical to devise new therapeutic strategies. Therefore, the creation of in vitro models that enable these mechanisms to be studied represents a crucial step. Since in vitro models represent an over-simplification of the in vivo system, in these years it has been attempted to increase the level of complexity of in vitro assays to create models that could better mimic the behaviour of the cells in vivo. These levels of complexity involved: 1. The dimension of the system, moving from two-dimensional to three-dimensional models; 2. The use of microfluidic systems; 3. The use of mixed cultures of tumour cells and cells of the tumour micro-environment in order to mimic the complex cross-talk between tumour cells and their micro-environment; 4. And the source of cells used in an attempt to move from commercial lines to patient-based models. In this review, we will summarize the evidence obtained exploring these different levels of complexity and highlighting advantages and limitations of each system used

Dissecting the heterogeneity of circulating tumor cells in metastatic breast cancer: Going far beyond the needle in the Haystack

6noAlthough the enumeration of circulating tumor cells (CTC) defined as expressing both epithelial cell adhesion molecule and cytokeratins (EpCAM+/CK+) can predict prognosis and response to therapy in metastatic breast, colon and prostate cancer, its clinical utility (i.e., the ability to improve patient outcome by guiding therapy) has not yet been proven in clinical trials. Therefore, scientists are now focusing on the molecular characterization of CTC as a way to explore its possible use as a “surrogate” of tumor tissues to non-invasively assess the genomic landscape of the cancer and its evolution during treatment. Additionally, evidences confirm the existence of CTC in epithelial-to-mesenchymal transition (EMT) characterized by a variable loss of epithelial markers. Since the EMT process can originate cells with enhanced invasiveness, stemness and drug-resistance, the enumeration and characterization of this population, perhaps the one truly responsible of tumor recurrence and progression, could be more clinically useful. For these reasons, several devices able to capture CTC independently from the expression of epithelial markers have been developed. In this review, we will describe the types of heterogeneity so far identified and the key role played by the epithelial-to-mesenchymal transition in driving CTC heterogeneity. The clinical relevance of detecting CTC-heterogeneity will be discussed as well.openopenBulfoni, Michela; Turetta, Matteo; Del Ben, Fabio; Di Loreto, Carla; Beltrami, Antonio Paolo; Cesselli, DanielaBulfoni, Michela; Turetta, Matteo; Del Ben, Fabio; DI LORETO, Carla; Beltrami, Antonio Paolo; Cesselli, Daniel

The Effects of Exercise and Kinesio Tape on Physical Limitations in Patients with Knee Osteoarthritis

(2) exercise KT with tension application (stabilizing effect) grou

Identification of tumorigenesis-related mRNAs associated with RNA-binding protein HuR in thyroid cancer cells

RNA binding proteins (RBPs) play a central role in cell physiology and pathology. Among them, HuR is a nuclear RBP, which shuttles to the cytoplasm to allow its RNA targets processing. HuR over-expression and delocalization are often associated to cell transformation. Numerous cancers display increased HuR protein levels and its high cytoplasmic levels has been associated with a worse prognosis.In our study, we first evaluated HuR expression in normal and cancer thyroid tissues and then evaluated its function in thyroid cell lines. HuR is over-expressed in all thyroid tumor tissues; high cytoplasmic levels are detected in all thyroid carcinomas. HuR silencing decreased cell viability and determined apoptotic cell death, in a non-tumorigenic (Nthy-ori-3.1) and a tumorigenic (BCPAP) thyroid cell line. Global transcriptome analysis indicated that HuR silencing, though having similar biological effects, induces distinct gene expression modifications in the two cell lines. By using the RIP-seq approach, the HuR-bound RNA profiles of different thyroid cell lines were evaluated. We show that in distinct cell lines HuR-bound RNA profiles are different. A set of 114 HuR-bound RNAs distinguishing tumorigenic cell lines from the non-tumorigenic one was identified.Altogether, our data indicate that HuR plays a role in thyroid tumorigenesis. Moreover, our findings are a proof of concept that RBP targets differ between cells with the same origin but with distinct biological behavior

MCM5 as a target of BET inhibitors in thyroid cancer cells

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer subtype, refractory to the current medical treatment. Among various epigenetic anticancer drugs, bromodomain and extra-terminal inhibitors (BETis) are considered to be an appealing novel class of compounds. BETi target the bromodomain and extra-terminal of BET proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlies the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated the effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETis, JQ1 and I-BET762, decreased cell viability, reduced cell cycle S-phase, and determined cell death. In order to find BETi effectors, FRO and SW1736 were subjected to a global transcriptome analysis after JQ1 treatment. A significant portion of deregulated genes belongs to cell cycle regulators. Among them, MCM5 was decreased at both mRNA and protein levels in both tested cell lines. Chromatin immunoprecipitation (ChIP) experiments indicate that MCM5 is directly bound by the BET protein BRD4. MCM5 silencing reduced cell proliferation, thus underlining its involvement in the block of proliferation induced by BETis. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its overexpression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also overexpressed in a murine model of ATC, and JQ1 treatment reduced Mcm5 mRNA expression in two murine ATC cell lines. Thus, MCM5 could represent a new target in the therapeutic approach against ATC

Assessment of the mutational status of NSCLC using hypermetabolic circulating tumor cells

Molecular characterization is currently a key step in NSCLC therapy selection. Circulating tumor cells (CTC) are excellent candidates for downstream analysis, but technology is still lagging behind. In this work, we show that the mutational status of NSCLC can be assessed on hypermetabolic CTC, detected by their increased glucose uptake. We validated the method in 30 Stage IV NSCLC patients: peripheral blood samples were incubated with a fluorescent glucose analog (2-NBDG) and analyzed by flow cytometry. Cells with the highest glucose uptake were sorted out. EGFR and KRAS mutations were detected by ddPCR. In sorted cells, mutated DNA was found in 85% of patients, finding an exact match with primary tumor in 70% of cases. Interestingly, in two patients multiple KRAS mutations were detected. Two patients displayed different mutations with respect to the primary tumor, and in two out of the four patients with a wild type primary tumor, new mutations were highlighted: EGFR p.746_750del and KRAS p.G12V. Hypermetabolic CTC can be enriched without the need of dedicated equipment and their mutational status can successfully be assessed by ddPCR. Finally, the finding of new mutations supports the possibility of probing tumor heterogeneity