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بدعم من إدارة المنح الدفاع DAMD17-01-C-0343 وDAMD17-03-1-0524، لجنة ولاية نيو جيرسي في أبحاث السرطان 02-1140-CCR-E0 وروث إيسترن غولدبرغ نصب تذكاري لأبحاث السرطان (RW)

يتكون لدينا نموذج من عدة عناصر أساسية من السكون في نخاع العظام. وهو يتألف من الاستروجين الخلايا الحساسة، والتي هي نوع من المرجح أن تظل كامنة في نخاع لفترات طويلة 10، وتتكون من فبرونيكتين، وهو العنصر الهيكلي الرئيسي للنخاع، FGF-2، وهو عامل نمو توليفها بوفرة من قبل ?...

خلايا سرطان الثدي metastasize إلى نخاع العظم قبل المرض قابلا للاكتشاف 1، في أقرب وقت تتطور الاورام الصغيرة الأوعية الدموية 2،3. عملية النقيلي سريعا ولكن غير فعالة. الخلايا تدخل الأوعية الدموية الجديدة بسرعة، في ملايين في اليوم الواحد 4 ولكن قلة البقاء على قيد الحياة في رحلة إلى أعضاء بعيدة 5. ومع ذلك، فإن بعض micrometastases البقاء على قيد الحياة في العظام ويمكن العثور عليها كما الخلايا واحد أو كتل الخلايا الصغيرة في شفاطات نخاع العظام من المرضى الذين شخصت حديثا 1. هذه الخلايا تقاوم العلاج الكيميائي المساعد، والتي تدار لغرض جدا القضاء عليها 6. وهبت هذه المقاومة، إلى حد كبير، من خلال بقاء الإشارات التي بدأها التفاعل مع نخاع العظام المكروية 7،8. Micrometastases يمكن العثور عليها في حوالي ثلث النساء المصابات بسرطان الثدي الموضعي وتمثل مؤشرا مستقلا من البقاء على قيد الحياة عند تحليلها عن طريق التحليل وحيد المتغير 9. بعض micrometastases ل بنمو بدأت، ولكن تعتمد أنماط تكرار على نوع من الخلايا. المرضى الذين يعانون من سرطان الثدي ثلاثي السلبية تميل إلى تتكرر بين 1-4 سنوات، مما يشير إلى ضعف السيطرة على حالة سبات. أنواع الخلايا الأخرى، بما في ذلك ER / PR + الخلايا، يمكن أن تظل كامنة لمدة تصل إلى 20 عاما، مع مطرد وبمعدل المستمر من تكرار 10. في حين الاختلافات في السكون التوقيعات التعبير الجيني بين ER + وER- خطوط خلايا الثدي والأورام تعكس إمكانات السكون مختلفة 11، والتفاعلات مع نخاع العظم سدى المرجح تمثل مساهمة كبيرة في السكون. دراسة السكون في الجسم الحي هي صعبة للغاية بسبب micrometastases نادرة وفاق عدد من الخلايا المكونة للدم بأكثر من 10 6 أضعاف. وبالتالي، لا بد من إنشاء النماذج ذات الصلة التي تقدم في بيانات المختبر التي يمكن اقتراح آليات وتوليد فرضيات قابلة للاختبار في الجسم الحي. وهناك عدد من النماذج السكون، بما في ذلك ماثينماذج matical 12،13، في المختبر نماذج 7،8،14،15، في النماذج الحية طعم أجنبي 16، مزيج من في المختبر ونماذج طعم أجنبي 17،18 والورم والانبثاث نماذج عفوية 19، وقد أسفرت بعض التبصر الخلايا السرطانية السكون 20 . كل هذه النماذج لدينا القيود الخاصة بها وهي في حد ذاتها مفيدة في المقام الأول لتوليد الفرضيات بشأن الإشارات الجزيئية والتفاعلات التي تحكم السكون لفحصها في أكثر النماذج ذات الصلة بيولوجيا. مع الهدف العام المتمثل في تحديد الآليات الجزيئية من السكون، والتفاعل مع المكروية التي تؤدي إلى اعتقال دورة، عودة التمايز والمقاومة والآليات العلاجية التي تؤدي إلى تكرار في ER + الخلايا، وضعنا نموذجا في المختبر أن يوفر اختيار العناصر ذات الصلة من انسجة المكروية 7. هذا النموذج، في حين متفرق نسبيا في كومبو لnents، هو قوي بما فيه الكفاية للسماح للمحققين لاستخلاص الآليات الجزيئية المحددة التي تؤثر على وظائف كبيرة من السكون. هذه التجارب تولد الفرضيات التي يمكن اختبارها بشكل مباشر في الجسم الحي. ويعتمد هذا النموذج على عدد قليل من العناصر الرئيسية التي أثبتنا أن تكون ذات صلة في السكون. وهي تشمل استخدام خلايا سرطان الثدي تعتمد على هرمون الاستروجين والثقافة من الخلايا في مناطق ذات كثافة مولد الرمع حيث تفاعلها هو في المقام الأول مع التحتية ومكونات قابلة للذوبان من المتوسط، وهو التحتية فبرونيكتين وجود عامل نمو الخلايا الليفية الأساسية (FGF-2) في المتوسط. نحن تتميز الآليات التي تحكم النظام في المختبر، بما في ذلك تحريض اعتقال دورة الخلية التي كتبها FGF-2 21، بوساطة من خلال TGFβ 22 والبقاء يشير من خلال PI3 كيناز 7،8 و 8 ERK والتمايز morphogenic إلى النمط الظاهري الظهارية، التي تعتمد على RhoA تعطيل، إنتغرين45؛ 5β1 upregulation وربط فبرونيكتين انسجة من أجل البقاء 7،15 (الشكل 1). الآثار في المختبر دورة الخلية من FGF-2 على MCF-7 الخلايا تبدأ في تركيزات سجل واحد على الأقل أقل من 10 نانوغرام / مل 21،23. واستند المبرر على السيطرة الزمنية للFGF-2 التعبير التي تحكم الثديية التشكل الأقنية، والتوسع دوري والركود في عدد من النظم الثدييات 24-27. أثبتنا أن FGF-2 يدفع التمايز، بما في ذلك التشكل الأقنية في الثقافة 3D 28، والتي تضيع عادة مع التحول السرطاني للأورام الإنسان 29 FGF-2 التعبير. بقي التعبير عن FGR1 سليمة في سرطان الثدي التي شملتها الدراسة 29 و MCF-7 الخلايا الاستمرار في التعبير عن كل مستقبلات FGF 4 30. في سياق السكون، ويتم تصدير FGF-2 التي وأودعت بشكل كبير على نخاع العظم سدى 31،32 حيث أنه يعمل في الحفاظ على الخلايا الجذعية المكونة للدم 33. نحن ماركاonstrated أن FGF-2 يؤدي الى حالة سبات في ER + سرطان الثدي الخلايا المستزرعة على الطبقات التحتية فبرونيكتين، وفيرة أيضا في نخاع، حيث يدفع التمايز morphogenic 7. في هذا النموذج، خلايا سرطان الثدي ل بنمو مستعمل، تعطيل رو A من خلال RhoGap غراف، redifferentiate إلى النمط الظاهري الظهارية وإعادة اكسبريس integrins α5β1 lost مع تطور خبيث. أنها تربط الفيبرونكتين من خلال إنتغرين α5β1 وتفعيل البقاء على قيد الحياة مما يشير إلى أن تجعلها مقاومة للعلاج السامة للخلايا 7،8،15 (الشكل 1). وقد تجلى تثبيط GTPases الدرجة رو سابقا للحث على النمط الظاهري نائمة 34. ونحن هنا سوف الخطوط العريضة لإجراءات محددة من شأنها أن تسمح للمحققين لإنشاء نموذج ودراسة الآليات الجزيئية والخلوية المحددة التي تحكم السكون من ER + سرطان الثدي الخلايا. في التجارب المقدمة هنا لتوضيح استخدام نموذج، نحن استهدف مسار PI3K (الشكل 1B) مع مثبط AKT ومثبط PI3K وجميع أفراد الأسرة رو (الشكل 1B) مع مثبط لعموم رو رو وكيناز (ROCK) المانع.

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			<td>MCF-7 cells</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
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		<tr>
			<td>T47D cells&#160;</td>
			<td>ATCC</td>
			<td>HTB-123</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 24 Well Clear Flat Bottom TC-Treated Multiwell Plate</td>
			<td>Corning</td>
			<td>354411</td>
			<td></td>
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		<tr>
			<td>BD BioCoat Fibronectin 60 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354403</td>
			<td></td>
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		<tr>
			<td>BD BioCoat Fibronectin 100 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354451</td>
			<td></td>
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			<td>BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin</td>
			<td>Corning</td>
			<td>354088</td>
			<td></td>
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		<tr>
			<td>6 Well tissue culture plate</td>
			<td>CellTreat</td>
			<td>229106</td>
			<td></td>
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			<td>Dulbecco Modified Eagle Medium High Glucose 10X Powder</td>
			<td>Corning Life Sciences</td>
			<td>50-013-PB</td>
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			<td>Heat Inactivated, Fetal Bovine Serum</td>
			<td>Serum Source International</td>
			<td>FB02-500HI</td>
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			<td>0.25% Trypsin/2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
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			<td>Penicillin-Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-CI</td>
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			<td>L-Glutamine, 100x, Liquid</td>
			<td>Corning</td>
			<td>25-005-CI</td>
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			<td>Recombinant Human FGF&#160;basic</td>
			<td>R&#38;D Systems</td>
			<td>234-FSE-025</td>
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			<td>Akt Inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate)</td>
			<td>CalBiochem</td>
			<td>124005</td>
			<td></td>
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			<td>LY294002&#160;</td>
			<td>CalBiochem</td>
			<td>19-142</td>
			<td>Chenical PI3K inhibitor</td>
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		<tr>
			<td>C3 transferase&#160;</td>
			<td>Cytoskeleton</td>
			<td>CTO3</td>
			<td>Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1</td>
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			<td>Y-27632 dihydrochloride&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>129830-28-2</td>
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			<td>BODIPY FL-Phallacidin (green)&#160;</td>
			<td>Molecular Probes</td>
			<td>B607</td>
			<td>Fluorochrome for fibrillar actin staining</td>
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			<td>BODIPY FL-Rhodamine phalloidin (red)&#160;</td>
			<td>Molecular Probes</td>
			<td>R415</td>
			<td>Fluorochrome for fibrillar actin staining</td>
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			<td>Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent&#160;</td>
			<td>Molecular Probes</td>
			<td>R37114</td>
			<td></td>
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		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI&#160;</td>
			<td>Molecular Probes</td>
			<td>P-36931</td>
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an in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: a tool for molecular mechanism studies and hypothesis generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

<html> وأجريت التجارب أن ألخص الفحص. يظهر مدار الساعة من التجربة في الشكل 2A. يتم تحضين الخلايا في كثافة مولد الرمع يوم -1، يضاف FGF-2 في متوسطة جديدة في اليوم 0 ويتم استزراع خلايا حتى يوم 6 عندما الملون أنها وتحسب المستعمرات. تدار أي اضطرابات للنظام في يوم 3 في 100 مجلدا...

Watch this Scientific Journal Video about An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation at JoVE.co

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

ل<em&gt; في المختبر</em&gt; الخمول نموذج من الاستروجين تراعي سرطان الثدي في نخاع العظام: أداة للدراسات الآلية الجزيئية والجيل الفرضية

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

8.2K Views.  Rutgers New Jersey Medical School. We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo. 

Video: An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

و في المختبر النظام الدراسي لاستئصال ورم من الخمول وتبديل للنمو النقيلي

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

متعدد الفوتون التصوير من غزو خلايا الورم في الفأر نموذج مثلي الفم سرطان الخلايا الحرشفية

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

ومتعددة الوسائط التصوير من الأوعية الدموية في نموذج الفئران عاري من سرطان الثدي الإنبثاث العظام باستخدام التصوير بالرنين المغناطيسي، الحجمي التصوير المقطعي والموجات فوق الصوتية

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

الكشف عن سرطان الجلد باستخدام المعدلات اسماك الزرد الموديل ورم الأصليين

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

باستخدام الكمي في الوقت الحقيقي PCR لتحديد الجهات المانحة Engraftment خلية في التنافسية نموذج الفئران زرع نخاع العظم

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

excitotoxic المواد تحفيز الدماغ باعتبارها Microslices<em&gt; في المختبر</em&gt; نموذج من السكتة الدماغية

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

الفخذ نخاع العظم الطموح في الفئران لايف

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

ل<em&gt; السابقين فيفو</em&gt; نموذج لدراسة هرمون العمل في الثدي البشري

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

في فيفو وفيفو السابقين المداخل لدراسة سرطان المبيض النقيلي الاستعمار من الهياكل التبانة بقعة في البريتوني الدهنية

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

الاستفادة من الفحص لينة آجار مستعمرة تشكيل لالتعرف مثبطات Tumorigenicity في خلايا سرطان الثدي

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...