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MULTIMODAL MOLECULAR IMAGING FOR HIF-1ALPHA NON-INVASIVE ASSESSMENT IN A MURINE GLIOMA MODEL
No full textIntroduction: Molecular imaging is an emerging discipline in biomedical research that allows the visual representation, the early characterization and quantification of biological processes at the cellular and subcellular level directly in vivo. Imaging techniques are of great importance since they allow the translation of the data obtained in preclinical models directly to the clinic. In this research, molecular imaging has been applied to the study of a preclinical glioma model, to assess intratumoural hypoxia, as a negative prognostic factor, since it has been correlated to chemo and radio-resistance, tumour progression, increase of invasiveness and to a poor prognosis. For these reasons, hypoxia represents a promising therapeutic target, both in GBM but also in other types of cancer, and an interesting marker able to estimate the prognosis and to predict the therapeutic outcome. In this perspective, the molecular study of hypoxia presented herein was focused mainly on the evaluation of the transcriptional activity of HIF-1α , which is a key factor of the hypoxia process.
Objectives: The main purpose of this project was to study, in a non invasive manner, HIF-1α transcriptional activity as a result of the development and progression of neoplastic disease and after treatment with selective inhibitors and drugs already used in the clinical setting. The first objectives were the characterization and validation of the proposed cell and animal models and the evaluation of HIF-1α activity after treatments of various inhibitors and drugs, to understand its potential role as a prognostic biomarker. The results obtained could be used in the evaluation of the possibility of performing personalized treatments as a result of stratification of individuals depending on HIF-1α activity.
Materials and Methods: The human U251 glioma cell line was used after its appropriatey engineering in order to obtain three models: in the first, U251-HRE, the luciferase reporter gene is under the control of the HRE sequences; in the second, U251-pGL3, the luciferase gene is expressed constitutively, whereas in the third, U251-HRE-mCherry, the mCherry reporter gene is constitutively expressed while the luciferase reporter gene is again under the control of HRE sequences. These lines were first used in vitro for the study of different agents modulating the transcriptional activity of HIF- 1α, whose action has been evaluated primarily by biochemical luciferase assay. Subsequently, the same cells were inoculated orthotopically in nude mice and tumor growth-related events, transcriptional activity of HIF-α, intratumoural hypoxia and therapeutic response, were analyzed using different imaging techniques (BLI, FLI, PET and MRI). The data obtained in vitro and in vivo were also validated ex vivo by histological and immunohistochemical staining.
Results: The in vitro studies have shown that in U251-HRE model,the luciferase activity can be modulated through hypoxia mimetic drugs, such DFX, and it was related to HIF-1α nuclear accumulation In addition, in vitro experiments have allowed to evaluate the contribution of different molecular pathways (involving PI3K/Akt and Ras/MEK/ERK) on HIF-1α activity. As expected, in U251-pGL3 model, the luciferase activity was not modulated. In vivo studies, performed after cell injection in murine models receiving U251-pGL3 cells, showed a linear increase of luciferase activity, proportionally to tumour growth. In contrast, in U251-HRE models a bimodal trend was observed, since it was dependent on hypoxia establishment HIF-1α mediated. These results suggested that this model can be used both in vitro and in vivo for the study of the modulation of HIF-1α activity. Although optical imaging cannot be used in humans, cross- validation of the results obtained with techniques routinely used in the clinic, such as PET and MRI (which gave results comparable and complementary to those provided by optical imaging), allowed to enhance the value of the results obtained in bioluminescence and fluorescence studies due to the possibility to translate these procedures to the clinical setting. The preliminary study conducted with the TMZ has shown, both in vitro and in vivo, that previously to the onset of the cytotoxic effect of the treatment, an early reduction of HIF-1α activity was detectable. The mechanism of action and the significance of this finding, however, have to be explored by further studies.
Conclusions: The U251-HRE model, recapitulating GMB features, could be a reliable model for the study of HIF-1α role in tumour progression. In this context, the optical imaging could be considered a sensitive and versatile technique for the study of the processes related to tumour progression especially those mediated by HIF-1α, but it is limited since it is impossible to translate these data into the clinics. For these reasons, it is extremely important the cross-validation with imaging techniques routinely used in the clinical practice, such as PET and MRI. Moreover, HIF-1α activity could be considered as an early efficacy biomarker after treatment with TMZ, although this crucial process has to be studied in depth
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Multimodal hypoxia imaging in a preclinical glioma model
Get PDFIntroduction: Molecular imaging techniques allow early characterization of tumors and quantification of biological processes in vivo. The aim of our study is to analyze the relationship between tumor growth and tumor hypoxia in an orthotopic glioma murine model by using a multimodal procedure, and to compare the features of the different imaging techniques in revealing hypoxia induction. Materials and methods: Engineered U251 cells were gently provided by Dr. Giovanni Melillo, National Cancer Institute, Frederick (MD). These cells express the luciferase reporter gene under control of a constitutive promoter (U251-LUC) or under control of three copies of a Hypoxic Responsive Element (U251-HRE). Cells has been analyzed by means of three different approaches: 1) In vitro evaluation of transcriptional activation of HIF-1α at different times after deferoxamine (DFX) treatment by immunocytochemistry (ICC). 2) In vivo analysis of tumoral progression in orthotopic murine models obtained by stereotaxic injection of 105 glioma cells; animals were monitored weekly with BLI (CCD camera), PET ([18F]FDG,[18F]FAZA,[18F]FLT) and MRI to evaluate tumoral progression and hypoxia activation. 3) Ex vivo analysis by H&E staining and hypoxia markers (HIF-1α and CAIX) were performed. Results: In vitro studies showed no differences among the two cell lines as regards to HIF-1α activation kinetic with a peak of activation between 3 and 6h and a decrease at 20h. In vivo, U251-HRE model showed a detectable and progressive luciferase activity induction starting at 18 days from injection demonstrating that luminescence expression is dependent on HRE-mediated activation. On the other hand, in U251-LUC model luciferase activity was detectable immediately after injection and remained proportional to tumor growth. Lesions were highly proliferative ( [18F]FLT) but hypo-metabolic ([18F]FDG). In partial agreement with the results of U251-HRE, a hypoxia dependent [18F]FAZA uptake was observed at later times (30 days). MRI provided morphological characterization and diffusion studies showed hypoxic necrotic areas only at later days. Ex vivo H&E staining demonstrates that tumors were characterized by nuclear atypia, brisk mitotic activity, microvascular proliferation and necrosis. HIF-1α and CAIX were clearly expressed in hypoxic areas, especially in the inner part of the tumor. Conclusions: This study demonstrates that the U251-HRE orthotopic murine model may be proposed as a predictive and reliable tool to evaluate hypoxia dependent processes of human glioma in preclinical studies by BLI. Differences among the three imaging techniques may be related to methods sensitivity. Further ex vivo post mortem measurements of HIF-1α and CAIX at earlier times will be performed. Additional studies will be conducted to understand the clinical meaning of early or delayed hypoxia identification in terms of response to treatment. References: SEMENZA GL. Curr Opin Cell Biol 13(2):167-71, 2001. Review. RAPISARDA A. Cancer Research 62, 4316-4324, 200
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Validation of an engineered cell model for in vitro and in vivo HIF-1α evaluation by different imaging modalities
No full textPURPOSE:
The aim of this study was to characterize a cell-based model for the molecular study of hypoxia-inducible factor (HIF)-1α activity, in the context of hypoxia, by means of different imaging techniques.
PROCEDURES:
Engineered U251-HRE glioma cells were used to analyze the molecular mechanisms underlying HIF-1α activity in vitro in relation to luciferase expression. The same cells were orthotopically implanted in mice to evaluate tumor progression and hypoxia induction by bioluminescence imaging, fluorescence imaging, positron emission tomography (PET), and magnetic resonance imaging (MRI).
RESULTS:
In vitro analyses highlighted the relationship between HIF-1α and luciferase activity in hypoxic conditions and after pharmacological treatments in U251-HRE cells. Through in vivo studies, it was possible to assess hypoxia establishment in relation to tumor growth by optical imaging, PET and MRI.
CONCLUSIONS:
The findings of this study indicate that the U251-HRE orthotopic murine model can be used to reliably evaluate processes modulating HIF-1α activity, using both molecular and preclinical non-invasive imaging techniques
PET and MRI studies applied on characterization of Fisher/F98 rat glioma model
No full textIntroduction: Preclinical brain tumor models
have provided a wealth of information on the
biology, imaging and experimental therapeuticsof brain tumors. The aim of our study is characterized Fisher/F98 rat glioma model using Positron Emission Tomography (PET) and Magnetic Resonance (MR) analysis to set up an experimental model useful to study the efficacy of new colloidal vectors for chemotherapy.
Methods: Syngenic rat brain-glioma models
(Fisher/F98) was obtained by stereotactic (x=2; y=5; z=3) implantation of different cell concentrations (102, 103, 104 and 105). To monitor tumor growth progression, rats underwent once a week Gadolinium enhanced T1-MRI studies followed by [18F]FDG PET studies, starting from 7 days after surgery. A group of animals performed also [18F]FAZA PET studies to evaluate regional tissue
hypoxia. To improve quantification, PET and
MRI images were fused using PMOD 2.7 software.
Max radiotracers uptake was calculated for tumor,frontal cortex, cerebellum and background using region of interest (ROI) analysis. Radioactivity concentration values expressed in MBq/g were then transformed into percentage of injected dose per gram of tissue (%ID/g). Moreover, histological
analysis of proliferation, apoptosis, differentiation, neoangiogenesis and hypoxia markers were performed.
Results: Mean survival time of rats injected with 104 and 105 cells was nine days. One week after surgery, MRI revealed a rapid growth that reached 0.11 cm3 mean tumour volume. Animals injected with 103 and 102 cells showed a mean survival time of 18 and 24 days respectively. In rats injected with
103 cells, tumor was revealed 14 days after surgery at MRI and [18F]FDG PET and successively tumors rapidly increased. Disease course in 102 cells injected rats was slower. Tumors were characterized
by high [18F]FDG uptake and hypoxic subareas
which only partially overlapped. Hypoxic areas were mainly localized in correspondence to Gd-enhanced regions whereas hyper glucose metabolic areas were localized in the outer part of the tumors. At histological analysis tumoral masses showed an infiltrative pattern of growth
and moderate neoangiogenesis. Tumors obtained
at animals death showed diffused necrotic areas.
HIF1 was clearly expressed by glial and neuronal cells in oedematous and hypoxic areas.
Conclusions: Our study indicates that Fisher/
F98 rat glioma model reproduces the characteristic of aggressiveness of human glioblastoma.
Tumor was characterized by high glucose
metabolism and by hypoxic sub-areas. The concentration of 102 cells permits to better monitor disease onset and progression and to plan experiments to test new anti-neoplastic therapies efficacy
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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