وأيد هذا العمل في جزء من التمويل المقدم من المجلس الاسكتلندي (عدد المنح HR07005 ؛ <a href="http://www.sfc.ac.uk/">http://www.sfc.ac.uk/</a> ) ، البحوث الطبية اسكتلندا ( <a href="http://www.medicalresearchscotland.org.uk/">http://www.medicalresearchscotland.org.uk/</a> ) ، والسرطان أبحاث السرطان التجريبية في المملكة المتحدة مركز الطب ( <a href="http://www.cancerresearchuk.org/">http://www.cancerresearchuk.org/</a> ) ، واختراق سرطان الثدي ( <a href="http://breakthrough.org.uk/">http://breakthrough.org.uk/</a> ).

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+phosphoprotein+as+a+biological+marker+of+tumor+signaling.">Stability of phosphoprotein as a biological marker of tumor signaling.</a> Clin. Cancer. Res. 11, 4338-4340 (2005).</li><li>Espina, V., Edmiston, K. H., Heiby, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+portrait+of+tissue+phosphoprotein+stability+in+the+clinical+tissue+procurement+process.">A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.</a> Mol. Cell. Proteomics. 7, 1998-2018 (2008).</li><li>Klymkowsky, M. W., Savagner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transition:+a+cancer+researcher's+conceptual+friend+and+foe.">Epithelial-mesenchymal transition: a cancer researcher's conceptual friend and foe.</a> Am. J. Pathol. 174, 1588-1593 (2009).</li><li>Kurrey, N. K., Jalgaonkar, S. P., Joglekar, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snail+and+slug+mediate+radioresistance+and+chemoresistance+by+antagonizing+p53-mediated+apoptosis+and+acquiring+a+stem-like+phenotype+in+ovarian+cancer+cells.">Snail and slug mediate radioresistance and chemoresistance by antagonizing p53-mediated apoptosis and acquiring a stem-like phenotype in ovarian cancer cells.</a> Stem. Cells. 27, 2059-2068 (2009).</li><li>Creighton, C. J., Li, X., Landis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+breast+cancers+after+conventional+therapy+display+mesenchymal+as+well+as+tumor-initiating+features.">Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 13820-13825 (2009).</li><li>Aitken, S. J., Thomas, J. S., Langdon, S. P., Harrison, D. J., Faratian, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+changes+in+ER,+PR+and+HER2+expression+in+primary+breast+cancer+and+paired+nodal+metastases.">Quantitative analysis of changes in ER, PR and HER2 expression in primary breast cancer and paired nodal metastases.</a> Ann. Oncol. 21, 1254-1261 (2010).</li><li>Sayagues, J. M., Abad, M. M., Melchor, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoural+cytogenetic+heterogeneity+of+sporadic+colorectal+carcinomas+suggests+several+pathways+to+liver+metastasis.">Intratumoural cytogenetic heterogeneity of sporadic colorectal carcinomas suggests several pathways to liver metastasis.</a> J. Pathol. 221, 308-319 (2010).</li></ol>

JC ، MG ، CJ ، وخدمات العملاء وموظفي HistoRx ، وشركة

وصف الأسلوب هنا تصاريح الكمي للتغايرية الجزيئية في الفورمالين القياسية الثابتة أقسام البارافين جزءا لا يتجزأ ، من مواد نسيجية الورم. الأسلوب يسمح القيمة التي سيتم تعيينها لدرجة التجانس في قسم الأنسجة ، بحيث يمكن بعد هذا أن تؤخذ بعين الاعتبار كمتغير في تنمية العلامات البيولوجية وتحليلها. بينما في العام فمن الأفضل أن المؤشرات الحيوية الأنسجة متجانسة فيما يتعلق التعبير ، بحيث يكون الفحص أقل عرضة للخطأ المعاينة أو التحيز ، قد يكون في بعض الظروف أن تكون مفيدة لقياس التباين كمعلمة. على سبيل المثال ، كما هو مبين في المثال التوضيحي لائحة وHER2 ، تظهر المؤشرات الحيوية المشتركة تجانس كبير من التعبير و هو الآن غير معروف ما إذا كان هذا يمثل عاملا مستقلا النذير أو التنبؤية فيما يتعلق بنتائج سريرية أو الاستجابة للعلاج ، على التوالي. وبالمثل ، كما يتضح ، ربما يغير العلاج نفسه حيويقد السكان المكونة للخلايا ، وبالتالي قياس تخصيب الهدف مفيدا في توجيه قرارات العلاج. ومع ذلك ، يقتصر هذا الأسلوب من قبل نفس العوامل التي تحد من التحليلات التقليدية النسجية أو العلامات البيولوجية (مثل المناعى). قد تكون النتيجة تعتمد على نوع وحجم ونوعية الأنسجة التي يجري تحليلها ، وبالتالي قسم الأنسجة واحدة لا تكون ممثلة للورم بالكامل. في الواقع ، قد يكون مؤشر سمبسون منحازة على نحو غير ملائم من حيث الحجم من الأنسجة (عدد الإطارات الملتقطة وقياس درجات اكوا). قد استمناع من الحواتم يجري قياسه يخضع لعوامل لا يمكن السيطرة عليها قبل التحليلية التي تغير artifactually التعبير عنها عبر قسم الأنسجة (مثل وقت نقص التروية الباردة ، أو طول التثبيت ، وحافة قطعة أثرية). هذا في حد ذاته مشكلة بالنسبة الحواتم ، الفوسفات ، والتي تشتهر عطوب 14 و 15. ولذلك قد تكون في وضع أفضل تقنيةعلى العينات بدلا من استئصال الخزعات صغيرة وأجريت على مقاطع متعددة بدلا من واحدة فقط. في نهاية المطاف ، قد 3D تقنيات التصوير فرصة لتحديد أفضل هذه المعلمة. ويمكن أيضا هذه التقنية على النحو تكون محدودة بسبب العوامل البيولوجية ، مثل التغير في التعبير عن cytokeratin حاتمة اخفاء. هذا قد يكون مصدر قلق خاص في تلك السرطانات ، بما في ذلك المبيض وسرطان الثدي ، والتي تخضع لالظهارية إلى الوسيطة الانتقالية (EMT) أو غير المخصب لالظهارية المكونات أو الخلايا الجذعية 16 ، كما تم مؤخرا أظهرت أن يحدث في العلاج الكيماوي علاج سرطان المبيض في المختبر 17 عاما ، ولدى مرضى سرطان الثدي في الاستجابة للعلاج 18. ويمكن التغلب على هذا القيد باستخدام الأجسام المضادة اخفاء البديلة (أو خليط من الأضداد) ، مثل cytokeratin vimentin زائد ، وهو upregulated في EMT 16. بالإضافة إلى ذلك ، منذ غيرها من مكونات النسيج (هيئة التصنيع العسكريكما roenvironment) ، مثل الخلايا الليفية أو خلايا بطانة الأوعية الدموية على نحو متزايد هدفا للعلاجات البيولوجية ، يمكن أيضا هذه التقنية يمكن أن تتسع لدرجة أن هذه المكونات ، وذلك باستخدام أقنعة محددة لمقصورات في المصالح ، مثل PECAM / CD31 إلى وصمة عار للخلايا بطانة الأوعية الدموية . على الرغم من استخدام تكييف مؤشر سمبسون كمقياس لعدم تجانس في البروتوكول الحالي نظرا لبساطتها ، يمكن أن تستخدم تدابير أخرى من عدم التجانس ، مثل قياسات بسيطة النزعة المركزية (المتوسط ​​، التباين ، متوسط ​​، الخ). بالإضافة إلى ذلك ، يمكن تعديل حسابات مؤشر سمبسون لتتناسب مع أفضل مجموعة من السكان اختبار معينة. استخدام التحولات - Z درجة تسمح للتسجيل لتكون أكثر قابلية للتطبيق لعلامات متعددة منذ يستند إلى الاختلاف في الانحراف نحو الوسط لمجموعة البيانات. ومع ذلك ، يمكن أن تكون محدودة النطاق الإجمالي للتسجيل في هذا النوع من التحول. قد يكون بعض التصاميم تكون في وضع أفضلبن لمجرد عشرات AQUA الفعلية لجميع العينات من مجموعة علامة خاصة. اختيار عدد من صناديق والانقطاع هو أيضا قيد في مجموعة من القيم التي يمكن أن تؤدي ويمكن أن يكون الأمثل للتصاميم التجريبية البديلة. وقد استخدمنا ER وHER2 في الدراسة الحالية والمؤشرات الحيوية مرشح ، حيث تستخدم فعلا في العيادة ، تساعد الإجابة عن الأسئلة ذات الصلة فيما يتعلق السريرية لفعالية العلاجات المستهدفة ، ومن المعروف أن تظهر التباين الكبير والتغيير في مجال التعليم الابتدائي والبعيد مرض 19. ومع ذلك ، علامة الأمثل للتجانس ما زال يتعين تحديدها. قد تشمل الاحتمالات الأخرى علامات خلوي من قابلية التنسيل ، مثل تلك التي استخدمت سابقا في دراسات الطور البيني السمكية 20. النهج يتطلب التحقق من صحة كبيرة كذلك في الأفواج والسريرية مشروحة بشكل جيد أو تجارب سريرية من أجل إقامة مزيد من أهمية هذه المعلمة في بيولوجيا السرطان.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> ScanScope FL </td><td> Aperio </td><td> ScanScope FL </td><td> كما تستخدم مجهزة من بائع </td></tr><tr><td> طيف </td><td> Aperio </td><td> طيف </td><td> والتكوين والتسويقي الأرقام تختلف حول كيفية إعداد المؤسسات المحلية قاعدة البيانات الخاصة بهم وتكنولوجيا المعلومات الهيكل. </td></tr><tr><td> AQUAnalysis ، بالاشتراك مع AQUAjustment </td><td> HistoRx </td><td> V2.3 </td><td> حزمة AQUAjustment AQUAnalysis يسمح لاستخدام الصور المخزنة في الطيف الترددي. </td></tr><tr><td> HER2 </td><td> داكو </td><td> A0485 </td><td> 1 في 400 تخفيف </td></tr><tr><td> الماوس المضادة للعموم cytokeratin </td><td> داكو </td><td> M3515 </td><td> 1 في 50 تمييع </td></tr><tr><td> الأضداد مخفف </td><td> داكو </TD&gt; <td> S0809 </td><td></td></tr><tr><td> تصور الماعز الأرنب HRP </td><td> داكو </td><td> K4003 </td><td></td></tr><tr><td> البروتين كتلة </td><td> داكو </td><td> X0909 </td><td></td></tr><tr><td> الماعز المضادة للماوس Alexa555 </td><td> Invitrogen </td><td> A21422 </td><td></td></tr><tr><td> دابي تصاعد المتوسطة </td><td> Invitrogen </td><td> P36931 </td><td></td></tr><tr><td> Cy5 Tyramide </td><td> HistoRx </td><td> AQ - EMR1 - 00001 </td><td></td></tr><tr><td> مركز Sequenza Immunostaining </td><td> الحرارية العلمية Shandon </td><td> 73300001 </td><td> تستخدم هنا لتلطيخ المناعي مقعد العلوي </td></tr></tbody></table>

heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Watch this Scientific Journal Video about Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence at JoVE.com

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

نحن هنا وصف طريقة لقياس التباين الجزيئية في المقاطع النسيجية من المواد السرطانية باستخدام كمية المناعي ، وتحليل الصور ، والإحصائي لقياس عدم التجانس. ويهدف الأسلوب لاستخدامها في تطوير العلامات البيولوجية السريرية والتحليل.

تعيين تباين التعبير البروتين في الأورام باستخدام المناعي الكمي

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

heterogeneity-mapping-protein-expression-tumors-using-quantitative

Research

JoVE Journal

Medicine

18.5K Views.  University of Edinburgh. نحن هنا وصف طريقة لقياس التباين الجزيئية في المقاطع النسيجية من المواد السرطانية باستخدام كمية المناعي ، وتحليل الصور ، والإحصائي لقياس عدم التجانس. ويهدف الأسلوب لاستخدامها في تطوير العلامات البيولوجية السريرية والتحليل.

Video: Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

إنشاء والدعوة للأورام الشبكية الإنسان في الفئران أن نقص المناعة

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

تقييم امتصاص الجسيمات النانوية في الأورام في الوقت الحقيقي باستخدام التصوير Intravital

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

الكشف microRNA في أورام البروستات بواسطة PCR الوقت الحقيقي الكمية (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

تحديد<em&gt; الجمال النائم</em&gt; الإدراج ينقول في الأورام الصلبة باستخدام رابط بوساطة PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

في المختبر الثقافة عضوي من أورام القولون ماوس

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

استخدام البروتين عكس صالحة المرحلة (RPPA) لاستكشاف البروتين في التعبير التغير فردية سرطان الخلايا الكلوية

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

في فيفو التصوير الضوئي من أورام الدماغ والتهاب المفاصل عن طريق نيون SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Coordinate Mapping of Hyolaryngeal Mechanics in Swallowing

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement whereas coordinate mapping of hyolaryngeal mechanics using Modified Barium Swallow (MBS) uses one set of coordinates to calculate multiple variables of interest. For demonstration purposes, ten kinematic measurements were generated from one set of coordinates to determine differences in swallowing two different bolus types. Calculations of hyoid excursion against the vertebrae and mandible are correlated to determine the importance of axes of reference.
To demonstrate coordinate mapping methodology, 40 MBS studies were randomly selected from a dataset of healthy normal subjects with no known swallowing impairment. A 5 ml thin-liquid bolus and a 5 ml pudding swallows were measured from each subject. Nine coordinates, mapping the cranial base, mandible, vertebrae and elements of the hyolaryngeal complex, were recorded at the frames of minimum and maximum hyolaryngeal excursion. Coordinates were mathematically converted into ten variables of hyolaryngeal mechanics.
Inter-rater reliability was evaluated by Intraclass correlation coefficients (ICC). Two-tailed t-tests were used to evaluate differences in kinematics by bolus viscosity. Hyoid excursion measurements against different axes of reference were correlated. Inter-rater reliability among six raters for the 18 coordinates ranged from ICC = 0.90 - 0.97. A slate of ten kinematic measurements was compared by subject between the six raters. One outlier was rejected, and the mean of the remaining reliability scores was ICC = 0.91, 0.84 - 0.96, 95% CI. Two-tailed t-tests with Bonferroni corrections comparing ten kinematic variables (5 ml thin-liquid vs. 5 ml pudding swallows) showed statistically significant differences in hyoid excursion, superior laryngeal movement, and pharyngeal shortening (p &#60; 0.005). Pearson correlations of hyoid excursion measurements from two different axes of reference were: r = 0.62, r2 = 0.38, (thin-liquid); r = 0.52, r2 = 0.27, (pudding).
Obtaining landmark coordinates is a reliable method to generate multiple kinematic variables from video fluoroscopic images useful in dysphagia research.

Coordinate mapping is a method of documenting salient features of hyolaryngeal biomechanics in the pharyngeal phase of swallowing. This methodology uses image analysis software to record coordinates of anatomical landmarks. These coordinates are imported into an excel macro and translated into kinematic variables of interest useful in dysphagia research.

تنسيق رسم الخرائط ميكانيكا لامي حنجري في البلع

Characterizing hyolaryngeal movement is important to dysphagia research. Prior methods require multiple measurements to obtain one kinematic measurement ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

الكمي قداس الطيفية من التنميط Proteomes خلايا السرطان المستمدة من السائل والأورام الصلبة

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

الخرائط الكمية للتهوية الخاصة في الرئة البشرية باستخدام التصوير بالرنين المغناطيسي البروتون والأكسجين كعامل تباين

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...