Browsing by Subject "Xenograft"
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Item Open Access Analysis of the Wnt/B-catenin/TCF4 pathway using SAGE, genome-wide microarray and promoter analysis: identification of BRI3 and HSF2 as novel targets(Elsevier, 2010) Kavak, E.; Najafov, A.; Ozturk, N.; Seker, T.; Cavusoglu, K.; Aslan, T.; Duru, A. D.; Saygili, T.; Hoxhaj, G.; Hiz, M. C.; Unal, D. O.; Birgül-Iyison, N.; Ozturk, M.; Koman, A.The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. β-catenin, a cytoplasmic component, plays a major role in the transduction of canonical Wnt signaling. The aim of this study was to identify novel genes that are regulated by active β-catenin/TCF signaling in hepatocellular carcinoma-derived Huh7 cells with high (transfected) and low β-catenin/TCF activities. High TCF activity Huh7 cells led to earlier and larger tumor formation when xenografted into nude mice. SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis were performed in parallel, to compare gene expression between low and high β-catenin/TCF activity clones, and also those that had been rescued from the xenograft tumors. SAGE and genome-wide microarray data were compared and contrasted. BRI3 and HSF2 were identified as novel targets of Wnt/β-catenin signaling after combined analysis and confirming experiments including qRT-PCR, ChIP, luciferase assay and lithium treatment. © 2010 Elsevier Inc.Item Open Access Development of a specialized zebrafish xenotransplantation database and establishment of ALU-based tumor DNA quantification method in zebrafish: focus on models of overexpression and microenvironment(2020-09) Targen, SeniyeSuccessful xenotransplantation of human cancer cells into zebrafish host marked a new era in cancer research enabling high throughput in vivo screens. Zebrafish xenotransplantation literature continues to rapidly accumulate, and this necessitates the development of an interactive database for accommodating the collective data for fined-tune search, visualization and statistical representation purposes. Herein, I have introduced an interactive database, ZenoFishDb v1.1 (https://konulab.shinyapps.io/zenofishdb), housing manually curated details on molecularly-modified cell transplantations, PDXs, stem cell and cancer stem cell transplantation studies as well as transplantation studies bearing modified host details. The database projects collected data in a table format via various attributes and provides graphical representation of the curated details as well as statistical analyses yielding information on incorporated numbers and frequencies of selected attributes; hence can be used for reviews and designing new experiments. Zebrafish PDX studies are separately conceptualized and displayed through ZenoFishDb v1.1. Development of the ZenoFishDb v1.1 leads to a better understanding of tumor analysis methods such as assessment of proliferation and/or tumor growth in xenotransplantation studies and further marks the need for development of novel methods for precise quantification of tumor size. In the light of these findings, I have helped establish a novel qRT-PCRbased proliferation assessment method for xenografts in zebrafish, adapted from previous mouse xenotransplantation studies. Herein, the use and precision of ALU repeat-based quantification of transplanted liver cancer cells in genotyped zebrafish ache mutants and wildtype siblings was shown exemplifying microenvironment as an important factor for tumor growth. I further demonstrated the power of ALU repeatbased quantification in Mineralocorticoid Receptor (MR) overexpressing breast cancer cells (MCF7) injected to the transparent casper zebrafish as a case study. First, I demonstrated that MR expression and signaling was important in breast cancer biology and prognosis based on in silico TCGA and custom RNA sequencing as well as other in vitro and ex vivo assays. I further showed that results obtained from ALU repeatbased quantification of tumor growth in MR-overexpressing MCF7 cells paralleled fluorescent image-based intensity measurements while the former being relatively less time-consuming and more high-throughput. In this study, accurate quantification of MR overexpression in xenografts was also successfully performed by a cDNA-specific primer pair; and the rate of tumor growth based on image analysis, did not correlate with the amount of MR DNA in casper fish xenografts. However, MCF7 cells overexpressing MR exhibited lower cell viability in vitro although some of these effects were due to empty vector (EV) integration. Accordingly, tumor size in xenografts of naïve, EV- and MR-transfected MCF7 cells injected into pigmented AB larvae were quantified for ALU-repeats yet no significant difference was observed due to high within-group variability in vivo. Future studies are needed to assess the role of varying the volume and placement of injected cells along with the amount of MR gene transfected on tumor growth in vivo.Item Open Access Zebrafish as an in vivo model of drug screens for liver cancer: the role of phenothiazines(2021-08) Korkmaz, BüşraHepatocellular carcinoma (HCC) is one of the most dangerous cancer types and sorafenib (SFB) is a commonly used drug against HCC, being the only FDA-approved medication until 2018. However, its limited efficacy and severe side effects emphasize the importance of developing alternative approaches. Regulation of cholesterol has been implicated in the progression of various cancers including HCC. Phenothiazines, which are in use as antipsychotic drugs and with effects on cholesterol biosynthesis, have drawn recent attention as anti-cancer drugs against different cancers. The elucidation of how phenothiazines exert their effects at cellular level might pave the way for proposing better strategies for HCC treatment. Zebrafish serves as a good cancer model for in vivo validations, since it enables efficient tumor formation and tracking. Its conserved genes and similar organ system to human make it even better of a model to reveal both molecular and morphological alterations caused by drugs. Based on previous cell viability studies from our lab, the combination of trifluoperazine (TFP) with SFB was found to exhibit synergism in Hep3B cells, while it was antagonistic in SkHep1 cells. Moreover, RNA-seq analysis on Hep3B cells has demonstrated that steroid biosynthesis and cholesterol metabolism was among the modulated pathways. Therefore, in the present thesis, the regulation of cholesterol levels in response to phenothiazine derivatives was investigated in HCC cell lines SkHep1 and Hep3B. The combinatorial approach was examined on Huh7 cells in terms of the expression of cholesterol biosynthesis genes based on RT-qPCR. After showing the ability of phenothiazine derivatives and TFP-SFB combinations to modulate cellular cholesterol, zebrafish drug screens were performed to determine applicable derivative and combination doses. Assessment of safe doses for TFP, SFB and their combination directed us for xenograft studies, which resulted in enhanced Hep3B cell survival by the combination of low dose (6 μM) TFP and 1 μM of SFB. This finding was further validated based on Alu-based DNA quantifiation, a powerful method we have established. With the help of RNA-seq analysis results, orthologous zebrafish genes, involved in cholesterol biosynthesis were identified and used in cross-species testing. The impact of TFP on liver vasculature was evaluated using transgenic (Fli1a:EGFP) zebrafish and found insignificant. Overall, the findings presented in this thesis highlight that TFP alone or in combination was able to change cellular cholesterol levels and tumor growth.Item Open Access Zebrafish glioma xenograft models: in vıvo investıgatıon of injection methods and development of a streamlit application(2023-10-20) Tombuloğlu, RüyaGlioblastoma (GBM) is one of the most aggressive and lethal forms of primary brain cancer, posing significant challenges to effective treatment and patient outcomes. Despite extensive research efforts, our understanding of GBM biology and the development of novel therapeutic strategies remains limited. Zebrafish xenograft models have emerged as a promising tool in cancer research, offering unique advantages in studying GBM. This thesis explores the utility of zebrafish xenograft models in advancing our understanding of GBM. Due to their genetic and physiological similarities to humans, zebrafish provide an excellent platform for studying GBM pathogenesis, tumor progression, and drug screening. Their transparency during early development allows for real-time visualization of tumor growth, invasion, and response to treatments. Moreover, zebrafish models enable rapid and cost-effective high-throughput drug screening, accelerating the identification of potential GBM therapeutics. In this thesis, I focused on creating an application named ZenofishDb Glioma, which is a more evolved and focused version of our previous database called ZenofishDb. ZenofishDb Glioma has been created using Python Streamlit, and comprises only the glioma studies and uses Natural Language Processing (NLP) to classify better, and effectively find and summarize information about zebrafish glioma xenograft models. In addition, after searching ZenofishDb Glioma, I decided to investigate an experimental protocol for injection of glioblastoma cells in the zebrafish model to test effects of injected cell numbers. Using MGG-119-GFP cells and Casper zebrafish, I injected different numbers of cells at different locations and stages, i.e., blastula and 2 days post fertilization, and observed there were significant differences between groups at 5 dpf using multiple quantification strategies. In conclusion, ZenofishDb Glioma can help design effective xenotransplantation strategies and make comparisons to understand how different experimental parameters affect the outcome of zebrafish glioma xenograft models.Item Open Access Zebrafish xenotransplantation models for studying gene function and drug treatment in hepatocellular carcinoma(2022-01-15) Konu, Ozlen; Targen, SeniyeIntroduction Zebrafish is a promising model organism for human disease including hepatocellular cancer (HCC). Recently, zebrafish has emerged also as a host for xenograft studies of liver cancer cell lines and patient derived tumors of HCC. Zebrafish embryos enable drug screening and gene function studies of xenografted cells via ease of microinjection and visualization of tumor growth and metastasis. Objectives In this review, we aimed to overview zebrafish HCC and liver cancer xenotransplantation studies focusing on ‘gene functional analysis’ and ‘drug/chemical screening’. Methods Herein, a comprehensive literature search was performed for liver and HCC xenografts in zebrafish on PubMed using different key words and filters for molecular modifications or drug exposure. Results Our literature search revealed around 250 studies which were filtered and summarized in a table (Table 1) revealing comprehensive collection of experimental and technical details on microinjection, injected cell lines, molecular modifications of injected cells, types and doses of drug treatments as well as biological assessments. Conclusion This review provides a platform for HCC and liver xenografts and highlights studies performed to understand gene functionality and drug efficacy in vivo in zebrafish.Item Open Access ZenoFishDb v1.1: a database for xenotransplantation studies in zebrafish(Mary Ann Liebert, 2020) Targen, Seniye; Kaya, Tuğberk; Avcı, M. E.; Güneş, Damla; Keşküş, Ayşe Gökçe; Konu, ÖzlenRapidly accumulating literature has proven feasibility of the zebrafish xenograft models in cancer research. Nevertheless, online databases for searching the current zebrafish xenograft literature are in great demand. Herein, we have developed a manually curated database, called ZenoFishDb v1.1 (https://konulab.shinyapps.io/zenofishdb), based on R Shiny platform aiming to provide searchable information on ever increasing collection of zebrafish studies for cancer cell line transplantation and patient-derived xenografts (PDXs). ZenoFishDb v1.1 user interface contains four modules: DataTable, Visualization, PDX Details, and PDX Charts. The DataTable and Visualization pages represent xenograft study details, including injected cell lines, PDX injections, molecular modifications of cell lines, zebrafish strains, as well as technical aspects of the xenotransplantation procedures in table, bar, and/or pie chart formats. The PDX Details module provides comprehensive information on the patient details in table format and can be searched and visualized. Overall, ZenoFishDb v1.1 enables researchers to effectively search, list, and visualize different technical and biological attributes of zebrafish xenotransplantation studies particularly focusing on the new trends that make use of reporters, RNA interference, overexpression, or mutant gene constructs of transplanted cancer cells, stem cells, and PDXs, as well as distinguished host modifications.