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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.

<table><tbody><tr><td>MCF-7 cells</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>T47D cells&amp;nbsp;</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></tr><tr><td>BD BioCoat Fibronectin 60 mm Culture Dishes</td><td>Corning</td><td>354403</td><td></td></tr><tr><td>BD BioCoat Fibronectin 100 mm Culture Dishes</td><td>Corning</td><td>354451</td><td></td></tr><tr><td>BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin</td><td>Corning</td><td>354088</td><td></td></tr><tr><td>6 Well tissue culture plate</td><td>CellTreat</td><td>229106</td><td></td></tr><tr><td>Dulbecco Modified Eagle Medium High Glucose 10X Powder</td><td>Corning Life Sciences</td><td>50-013-PB</td><td></td></tr><tr><td>Heat Inactivated, Fetal Bovine Serum</td><td>Serum Source International</td><td>FB02-500HI</td><td></td></tr><tr><td>0.25% Trypsin/2.21 mM EDTA</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution, 100X</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>L-Glutamine, 100x, Liquid</td><td>Corning</td><td>25-005-CI</td><td></td></tr><tr><td>Recombinant Human FGF&amp;nbsp;basic</td><td>R&amp;amp;D Systems</td><td>234-FSE-025</td><td></td></tr><tr><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></tr><tr><td>LY294002&amp;nbsp;</td><td>CalBiochem</td><td>19-142</td><td>Chenical PI3K inhibitor</td></tr><tr><td>C3 transferase&amp;nbsp;</td><td>Cytoskeleton</td><td>CTO3</td><td>Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1</td></tr><tr><td>Y-27632 dihydrochloride&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>129830-28-2</td><td></td></tr><tr><td>BODIPY FL-Phallacidin (green)&amp;nbsp;</td><td>Molecular Probes</td><td>B607</td><td>Fluorochrome for fibrillar actin staining</td></tr><tr><td>BODIPY FL-Rhodamine phalloidin (red)&amp;nbsp;</td><td>Molecular Probes</td><td>R415</td><td>Fluorochrome for fibrillar actin staining</td></tr><tr><td>Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent&amp;nbsp;</td><td>Molecular Probes</td><td>R37114</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI&amp;nbsp;</td><td>Molecular Probes</td><td>P-36931</td><td></td></tr></tbody></table>

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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Medicine

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

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

وضع نموذج الإكلينيكية البشرية من أوستيوكلاستوجينيسيس من الدم المحيطية وحيدات مثقف يشترك مع خطوط خلايا سرطان الثدي

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Cancer Research

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 ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

نموذج المصفوفة خارج الخلية العظام ثلاثية الأبعاد ل Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

نماذج من الانبثاث العظام

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

زرع العظام لأورام الثدي كنماذج ما قبل السريرية لسرطان الثدي

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

العزل والتقييم الوظيفي للخلايا الجذعية لسرطان الثدي البشري من عينات الخلايا والأنسجة

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

ماماري الظهارية والبطانية الخلايا كرويدات كنموذج وظيفي محتمل في المختبر لأبحاث سرطان الثدي

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

التنميط من MicroRNAs الاستروجين التنظيم في خلايا سرطان الثدي

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

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 ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

طرق التثقيف عظم الفخذ الإنسان إإكسبلنتس الأنسجة لدراسة سرطان الثدي الاستعمار خلية للالمتخصصة الإنتقالية

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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