Name: mirna expression analyses in prostate cancer clinical tissues
Uploaded: 2015-09-08T14:00:00
Description: Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

نشكر الدكتور روجر إريكسون لدعمه والمساعدة في إعداد المخطوط. وأيد هذا العمل من قبل المعهد الوطني للسرطان في المعاهد الوطنية للصحة (منحة رقم RO1CA177984؛ RO1CA138642)، ومشروع برنامج VA على سرطان البروستاتا (BX001604).

تم الحصول عليها الثابتة الفورمالين، جزءا لا يتجزأ من البارافين (FFPE) أو عينات سرطان البروستاتا المثلج من SFVAMC. وكانت عينات من مرضى سرطان البروستاتا الذين خضعوا لاستئصال البروستاتا في SFVAMC. تم الحصول على الموافقة المسبقة الخطية من جميع المرضى وتمت الموافقة على الدراسة م...

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في هذه المقالة، نحن تصف سير العمل مبسط لميرنا التعبير التنميط في FFPE أرشفة أو سرطان البروستاتا المجمدة الأنسجة السريرية جديدة. في سرطان البروستاتا، وتشير العديد من الدراسات دورا هاما من microRNAs في سرطان البروستاتا بدء والتقدم وورم خبيث. ومع ذلك، غالبا ما يتم الحصول على ...

سرطان غدة البروستاتا هي خباثة الذكور تشخيصا التي هي واحدة من الأسباب الرئيسية لوفيات السرطان بين الرجال ذات الصلة. في الولايات المتحدة، وهي 220800 حالة جديدة المقدرة وسيتم إبلاغ 27540 حالة وفاة في عام 2015 1. سرطان البروستاتا هو مرض غير متجانسة مع المرض متغير بدرجة كبيرة طبعا- الأورام يمكن أن يكون المتراخي أو عدوانية جدا. وشكلت تحديا حاسما في سرطان البروستاتا التدبير العلاجي السريري بسبب عدم كفاية الطرق المستخدمة حاليا / المؤشرات الحيوية للكشف عن المرض، والتشخيص، والتشخيص والعلاج 2. وتشمل طرق الفحص الحالية مستضد البروستات محددة (PSA) اختبار وفحص المستقيم الرقمي (DRE)، يليه خزعات البروستاتا 3. مستضد البروستات محددة (PSA) هو الأكثر استخداما على نطاق واسع العلامات البيولوجية سرطان البروستاتا الذي أحدث ثورة بشكل كبير التدبير العلاجي السريري وتحسين معدلات البقاء على قيد الحياة 4. ومع ذلك، ويرجع ذلك إلى القيود المتأصلة في دعم البرامج والإدارة بما في ذلك أمريكا اللاتينية والكاريبيوقد أدى ك التحديد، وفحص القائم على دعم البرامج والإدارة لأكثر من تشخيص وأكثر من العلاج من هذا المرض. في ضوء ذلك، يتم توجيه الجهود المكثفة نحو البحث عن بديل المؤشرات الحيوية سرطان البروستاتا، ولا سيما تلك التي يمكن التنبؤ عدوانية من المرض ومحرك قرارات أفضل علاج 4،5. على مدى السنوات القليلة الماضية، microRNAs (miRNAs) قد برزت بوصفها بديلة المؤشرات الحيوية سرطان البروستاتا واعدة. MicroRNAs (miRNAs) تشكل فئة الحفظ تطويريا من الرنا غير المكودة الصغيرة التي تقمع التعبير الجيني آخر ما بعد transcriptionally عبر التفاعلات تسلسل معين مع 3&#39;- المناطق غير مترجمة (UTRs) الأهداف مرنا وما شابه ذلك. ويقدر أن&gt; 60٪ من من mRNAs يتم حفظها أهداف miRNAs 6. وتقع الجينات ميرنا في المناطق بين الجينات أو ضمن الإنترونات أو الإكسونات من البروتين / غير بروتين الترميز الجينات 7. وكتب هذه الجينات تفضيلي التي كتبها RNA البلمرة II إلى الوجاهةmiRNAs راي (PRI-miRNAs، عدة kilobases طويلة) أن شكل دبوس على شكل حلقة الجذعية الهياكل الثانوية. تتم معالجة هذه miRNAs الحزب الثوري المؤسسي، إلى miRNAs السلائف (ما قبل miRNAs، 60-75 النوكليوتيدات طويلة) التي يتم تصديرها إلى السيتوبلازم ومعالجتها الى مزيد من miRNAs ناضجة (18-25 النوكليوتيدات طويلة) 10/08. miRNAs تنظم العمليات الخلوية الأساسية بما في ذلك انتشار وتطوير والتمايز وموت الخلايا المبرمج 11. وتشير الدراسات إلى التقلبات على نطاق واسع ميرنا ملامح التعبير في مختلف الأورام الخبيثة الإنسان بما في ذلك سرطان البروستاتا 12-15. تم الإبلاغ عن ميرنا ملامح التعبير أن dysregulated على نطاق واسع في سرطان البروستاتا النقيلي الابتدائي و. وقد تم ربط تغير التعبير ميرنا مع تطور سرطان البروستاتا والعدوانية وتكرار تسليط الضوء على إمكانات النذير من miRNAs 12،14،16-19. وهناك مجموعة متزايدة من الأدلة تشير إلى أن miRNAs تلعب أدوارا مهمة في الآلية بدء سرطان البروستاتا، والتنمية والتقدمايون والانبثاث. عموما، miRNAs والمؤشرات الحيوية البديلة واعدة بالنسبة لتشخيص سرطان البروستات والتشخيص يمكن أن تميز بين الأنسجة الطبيعية والسرطان والمساعدة في التقسيم الطبقي للأورام البروستاتا 12 الناشئة. أيضا، miRNAs هي أهداف هامة لتطوير علاجات فعالة ضد سرطان البروستاتا 20. ونظرا لصغر حجمها ومقاومة النشاط ريبونوكلياز الذاتية، miRNAs والمؤشرات الحيوية مستقرة يمكن أن يتم الكشف بسهولة في الأنسجة الثابتة الفورمالين 21 و في خزعات البروستاتا 22. وعلاوة على ذلك، تم مقارنة ملامح التعبير من miRNAs في الأنسجة المجمدة والفورمالين الثابتة وتم العثور على أن تكون بقوة المترابطة 21. ومع ذلك، ميرنا التعبير التنميط في الأنسجة السريرية سرطان البروستاتا غالبا ما تتحدى نظرا لعدم تجانس الورم، وأخطاء أخذ العينات، وتلوث انسجة الخ تطوير miRNAs والمؤشرات الحيوية فعالة لعلاج سرطان البروستاتا ريلى بشدةوفاق على كشف دقيق في الأنسجة السريرية. نحن هنا وصف سير عمل مبسطة المستخدمة في مختبرنا لميرنا التعبير التنميط في FFPE المؤرشفة أو سرطان البروستاتا المجمدة العينات السريرية جديدة. نحن توظيف مجموعة من الكمية في الوقت الحقيقي PCR والتهجين الموضعي لميرنا في تحليل العينات السريرية، مع السابقة العائد على مزيد من المعلومات الكمية وهذه الأخيرة لتصور التعبير التفاضلية من المؤشرات الحيوية ميرنا محتملة في مجموعة من الأنسجة. في هذا العمل، ونحن تحسين المنهجيات القائمة لميرنا الاستخراج من FFPE وأنسجة البروستاتا المجمدة، ويحلل التعبير TAQMAN مسبار استنادا ميرنا RT-PCR وميرنا الموقع تقنية التهجين في استخدام الحمض النووي مؤمن (LNA) المستندة تحقيقات 23. تحقيقات القائم على LNA تقديم المزيد من حساسية وخصوصية مقارنة DNA- أو RNA المرتبط تحقيقات مقرها وتمكن الكشف قوية من جميع متواليات ميرنا، بغض النظر عن محتوى GC وأيضا تسمح discriminaنشوئها الأسر ميرنا. يمكن تطبيقها لدينا الأمثل بروتوكول ميرنا ISH لسرطان البروستاتا شرائح الأنسجة أو ميكروأرس الأنسجة سرطان البروستاتا (TMA)، مع هذا الأخير تقدم القدرة على تسريع ميرنا اكتشاف العلامات البيولوجية.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems&#160;</td>
			<td>RM2255</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus Autopix for LCM</td>
			<td>Arcturus/ Life Technologies</td>
			<td>LCM1621/LCM1110</td>
			<td>Alternatively, Arcutus Xt system from Life Technolgies can be used.&#160;</td>
		</tr>
		<tr>
			<td>CapSure Macro LCM Caps</td>
			<td>Life Technologies</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy FFPE Kit&#160;</td>
			<td>Qiagen</td>
			<td align="right">217504</td>
			<td></td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4351106</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman MicroRNA Reverse Transcription kit&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4366596</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman Fast Universal PCR master mix&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG labeled LNA probe for U6</td>
			<td>Exiqon</td>
			<td>99002-01</td>
			<td></td>
		</tr>
		<tr>
			<td>BM Purple AP substrate</td>
			<td>Roche</td>
			<td align="right">11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-1</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-2</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-8</a></td>
			<td></td>
		</tr>
		<tr>
			<td>AP-conjugated anti-digoxigenin antibody</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-7</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting media&#160;</td>
			<td>Vector Laboratories&#160;</td>
			<td>H-5501&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (guanidine isothiocyanate-phenol reagent)&#160;</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/15596018">15596-018</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Harris hematoxylin</td>
			<td>Statlab</td>
			<td>SL200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin&#160;</td>
			<td>Statlab</td>
			<td>SL201&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

mirna expression analyses in prostate cancer clinical tissues

وشكلت تحديا حاسما في سرطان البروستاتا (PCA) التدبير العلاجي السريري بسبب عدم كفاية المؤشرات الحيوية المستخدمة حاليا لفحص الأمراض والتشخيص والتشخيص والعلاج. في السنوات الأخيرة، microRNAs (miRNAs) ظهرت مؤشرات حيوية بديلة واعدة بالنسبة لتشخيص سرطان البروستاتا والتكهن. ومع ذلك، فإن تطوير miRNAs والمؤشرات الحيوية فعالة لعلاج سرطان البروستاتا يعتمد بشكل كبير على كشف دقيق في الأنسجة السريرية. ميرنا يحلل في سرطان البروستاتا العينات السريرية وغالبا ما يشكل تحديا نظرا لعدم تجانس الورم، وأخطاء أخذ العينات، وتلوث انسجة الخ والهدف من هذه المقالة لوصف سير عمل مبسط لميرنا يحلل في FFPE المؤرشفة أو سرطان البروستاتا المجمدة العينات السريرية جديدة باستخدام مزيج من الكمي في الوقت الحقيقي PCR (RT-PCR) والتهجين في الموقع (ISH). في هذا العمل، ونحن تحسين المنهجيات القائمة لميرنا الاستخراج من FFPE وTISSU البروستاتا المجمدةوفاق والتعبير وتحلل من قبل TAQMAN مسبار استنادا ميرنا RT-PCR. بالإضافة إلى ذلك، نحن تصف طريقة الأمثل لISH يحلل كشف formiRNA في أنسجة البروستاتا باستخدام حمض النووي مؤمن (LNA) - تحقيقات القائمة. يمكن تطبيقها لدينا الأمثل بروتوكول ميرنا ISH لسرطان البروستاتا شرائح الأنسجة أو ميكروأرس الأنسجة سرطان البروستاتا (TMA).

التنميط التعبير مير-203 في LCM سرطان البروستاتا الابتدائية العينات السريرية التي RT-PCR تحليل (الشكل 1) RT-PCR تحليل النسبي التعبير مير-203 في LCM البروستاتا الابتدائية الأنسجة السرطان وأجريت المناطق الطبيعية المجاورة يقابل بها كم...

Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

ميرنا تحليلات التعبير في سرطان البروستاتا الأنسجة السريرية

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

mirna-expression-analyses-in-prostate-cancer-clinical-tissues

Research

JoVE Journal

Medicine

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 ...

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER+ and R26-SmoM2YFP+ mice in this protocol); 2) inducing SmoM2YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.

Carcinogenesis is a process involving interactions between cancer cells and the cancer microenvironment. To dissect the molecular events, one needs to isolate different cell populations at different stages during cancer development. Using a mouse model for basal cell carcinoma, we describe a protocol for cell analyses during carcinogenesis.

تحليلات السكان الخلية خلال الجلد التسرطن

Cancer development is  a multiple-step process involving many cell types including cancer precursor  cells, immune cells, fibroblasts and endothelial ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

MR التصوير الجزيئي للسرطان البروستاتا مع وكيل النقيض المستهدفة الجزيئية الببتيد الصغيرة CLT1

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

تحديد القائمة على ميكروأري من الفردية هيرفي الامكنه التعبير: التطبيق على العلامات البيولوجية ديسكفري في سرطان البروستاتا

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

على الفئران نموذج مثلي من سرطان البروستاتا البشرية الانبثاث

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

عزل الخلايا الجذعية للسرطان من العينات البشرية سرطان البروستاتا

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Mammosphere تشكيل الفحص من الثدي البشري سرطان الأنسجة وخطوط الخلايا

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

جيل من سرطان البروستاتا المريض المشتقة نماذج طعم أجنبي من تعميم الخلايا السرطانية

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

العمل مع الأنسجة البشرية لبالحركة أبحاث السرطان

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients&#039; Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

Exosomal تحليل ميرنا في الخلية غير الصغيرة سرطان الرئة (NSCLC) المرضى البلازما من خلال QPCR: والممكنة السائل أداة خزعة

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...