המחברים מבקשים להודות לBofan וו והנוצרי Raithel לקבלת סיוע טכני ומיכאל ריגר להקדמתו להדמיה חייה תאים. נתמך על ידי NCI, NIH RO1CA112176 (PAF), NCI, NIH. R01CA89041-10S1 (PAF), דויטשר Akademischer Austaush Dienst eV A/09/72227 Ref. 316 (REN), משרד ההגנה W81XWH-11-1-0074 (REN), תכנית WCU (World Class אוניברסיטה) באמצעות קרן המחקר הלאומי של קוריאה ממומנת על ידי משרד החינוך, המדע והטכנולוגיה (R31-10069) (PAF ), NIH IG20 RR025828-01 (ציוד מתקן גדר מכרסם), וNIH NCI 5P30CA051008 (מיקרוסקופי ומשאבי הדמיה ובעלי חיים משותפים).

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	<li>Jones, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+estrogen+signaling+pathways+collaborates+with+loss+of+Brca1+to+promote+development+of+ERalpha-negative+and+ERalpha-positive+mammary+preneoplasia+and+cancer.">Activation of estrogen signaling pathways collaborates with loss of Brca1 to promote development of ERalpha-negative and ERalpha-positive mammary preneoplasia and cancer.</a> Oncogene. 27, 794-802 (2008).</li><li>Burga, L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+proliferation+and+differentiation+properties+of+primary+mammary+epithelial+cells+from+BRCA1+mutation+carriers.">Altered proliferation and differentiation properties of primary mammary epithelial cells from BRCA1 mutation carriers.</a> Cancer Res. 69, 1273-1278 (2009).</li><li>Li, M., Wagner, K., Furth, P. A., Ip, M., Asch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+primary+mammary+epithelial+cells+and+transfection+by+viral+and+nonviral+methods.">Preparation of primary mammary epithelial cells and transfection by viral and nonviral methods.</a> Methods in Mammary Gland Biology and Breast Cancer. , (2000).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+stem-cell-driven+regeneration+in+mammals.">Imaging stem-cell-driven regeneration in mammals.</a> Nature. 453, 345-351 (2008).</li><li>Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+single-cell+imaging+of+mammalian+stem+cells.">Long-term single-cell imaging of mammalian stem cells.</a> Nature Methods. 8, S30-S35 (2011).</li><li>Eilken, H. M., Nishikawa, S. -. I., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+single-cell+imaging+of+blood+generation+from+haemogenic+endothelium.">Continuous single-cell imaging of blood generation from haemogenic endothelium.</a> Nature. 457, 896-900 (2009).</li><li>Kokkaliaris, K. D., Loeffler, D., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+tracking+hematopoiesis+at+the+single-cell+level.">Advances in tracking hematopoiesis at the single-cell level.</a> Current opinion in hematology. , (2012).</li><li>Rieger, M. A., Hoppe, P. S., Smejkal, B. M., Eitelhuber, A. C., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+cytokines+can+instruct+lineage+choice.">Hematopoietic cytokines can instruct lineage choice.</a> Science. 325, 217-218 (2009).</li><li>Rieger, M. A., Schroeder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instruction+of+lineage+choice+by+hematopoietic+cytokines.">Instruction of lineage choice by hematopoietic cytokines.</a> Cell Cycle. 8, 4019-4020 (2009).</li><li>Heinrich, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+subtype-specific+neurons+from+postnatal+astroglia+of+the+mouse+cerebral+cortex.">Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex.</a> Nat. Protoc. 6, 214-228 (2011).</li></ol>

החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

שלבים קריטיים חשוב לוודא שבלוטות חלב נקצרות מעכברי בנים אותו הגיל לשליטה לשונות הקשורים בגיל בהתנהגות תא אפיתל חלב. כאשר ציפוי התאים, אותו מספר התאים צריך להיות מצופה בכל אחד גם לכל ניסוי. תאים צריכים להיות דלילים יחסית כאשר ציפוי ...

מודלי עכבר מהונדס גנטי הם כלים כדי ללמוד ולהבין איך נגעים גנטיים שונים תורמים לסיכון להתפתחות סרטן השד. לדוגמה, עכברים מהונדסים גנטי הראו כי שילוב של שלושה גורמים: האובדן של השד באורך מלא 1 הסרטן, התחלה מוקדמת (BRCA1) גנים בתאי האפיתל חלב, haploinsufficiency ניבט קו החלבון p53 גידול (TP53), וחלב אפיתל תא ממוקד עד מוסדר אלפא קולטן אסטרוגן (עידן) תוצאות ביטוי בהתפתחות של סרטן השד ב100% של BRCA1 (ו) וירוס 11/f11/Mouse חלב גידול (MMTV) -Cre/p53 + / -/tetracycline-operator (floxed ט-op) -ER/MMTV-reverse טטרציקלין transactivator (rtTA עכברים על ידי גיל 12 חודשים בהשוואה לאחוזים הנמוכים יותר דיווחו בBRCA1 f11/f11/MMTV-Cre/p53 + / - עכברים ללא עידן על ביטוי (~ 50 - 60%) ועכברי BRCA1 f11/f11/MMTV-Cre ללא TP53 haploinsufficiency (&lt;5%). 1 הדמית זמן לשגות דינמית של ההתנהגות של תאי האפיתל חלב ראשוני preneoplastic מגלה הבדלים בהתנהגות תא המוערכים פחות בקלות בסעיפי רקמות סטטיות. שינויים בשגשוג והתמיינות תאים הם נצפו בחלב ראשוניים מנישאי מוטצית BRCA1 אדם. 2 יצירת השעיות תא הבודד של תאי האפיתל חלב ראשוניים מרגיל ועכברים מהונדסים גנטי נוצרות דרך התנערות האנזימטית של רקמת בלוטת חלב resected. 3 זמן לשגות תמונות נתפסות להעריך מנגנון ועיתוי של הופעה ושכיחות של שינויים מורפולוגיים בתאי אפיתל כולל מעבר-mesenchymal (EMT) ואפופטוזיס מושבת תא. דור של מפות גורל תא, כימות של משך הזמן בין חלוקות תא (גלגולים סלולריים), וקביעת הדפוסים של חלוקת תאים מתאפשרים על ידי שימוש במעקב מהתא בודד. טיםכלי מעקב של &#39;(TTT) הוא תוכנה זמינה בפומבי משמשת ליצירת מפות גורל תא בודד. השירות שלה בהבהרת מנגנונים של גורל תא נקבע 4,5 בחינת התפתחות תקינה תא גזע hematopoietic 6-9 והדור של נוירונים. 10

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> עכבר בינוני אסאל EpiCult-B </td><td> טכנולוגיות StemCell </td><td> 05610 </td><td></td></tr><tr><td> EpiCult-B מאוס תוספים למניעת הפצת נשק </td><td> טכנולוגיות StemCell </td><td> 05612 </td><td></td></tr><tr><td> רקומביננטי עוריות Growth Factor האנושי (rhEGF) </td><td> טכנולוגיות StemCell </td><td> 02633 </td><td></td></tr><tr><td> Collagenase / Hyaluronidase </td><td> טכנולוגיות StemCell </td><td> 07912 </td><td></td></tr><tr><td> Scapels חד פעמי </td><td> נוצה </td><td> 2975 # 10 </td><td></td></tr><tr><td> תמיסת המלח המאוזן &quot;הנקס </td><td> טכנולוגיות StemCell </td><td> 37150 </td><td></td></tr><tr><td>כלוריד mmonium </td><td> טכנולוגיות StemCell </td><td> 07800 </td><td></td></tr><tr><td> Tryspin-EDTA </td><td> טכנולוגיות StemCell </td><td> 07901 </td><td></td></tr><tr><td> Dispase </td><td> טכנולוגיות StemCell </td><td> 07913 </td><td></td></tr><tr><td> אני DNase </td><td> טכנולוגיות StemCell </td><td> 07900 </td><td></td></tr><tr><td> מסננת תא 40 מיקרומטר </td><td> טכנולוגיות StemCell </td><td> 27305 </td><td></td></tr><tr><td> FBS </td><td> טכנולוגיות StemCell </td><td> 06100 </td><td></td></tr><tr><td> PenStrep </td><td> Gibco </td><td> 15140 </td><td></td></tr></tbody></table>

time-lapse imaging of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer

הדמית זמן לשגות ניתן להשתמש כדי להשוות התנהגות של תאים בתרבית ראשונית preneoplastic חלב האפיתל הנגזרים ממודלי עכבר מהונדס גנטי שונים של סרטן השד. לדוגמה, זמן בין חלוקות התא (גלגולים סלולריים), מספרים סלולריים אפופטוטיים, אבולוציה של שינויים מורפולוגיים, ומנגנון של יצירת מושבות ניתן לכמת והשוואה בתאים שנשאו נגעים גנטיים ספציפיים. תרביות תאים ראשוניות אפיתל חלב נוצרות מבלוטות חלב ללא גידול מוחשי. בלוטות resected זהירות עם הפרדה ברורה משרירים סמוכים, בלוטות לימפה הוסרו, והשעיות תא בודד של תאי האפיתל חלב מועשר נוצרות על ידי רקמת שד ריסוק ואחרי ניתוק וסינון האנזימטית. השעיות תא בודד מצופות וממוקמות ישירות מתחת למיקרוסקופ בתוך תא חממה עבור הדמית חית תאים. שישה עשרה 650 x 700 מיקרומטר מיקרומטר שדות בתצורת 4x4 מכל well של צלחת 6-גם הם צלמו כל 15 דקות במשך 5 ימים. תמונות זמן לשגות נבדקות ישירות למדוד התנהגויות סלולריות שיכול לכלול מנגנון ותדירות של יצירת מושבות תאים בתוך 24 השעות הראשונות של ציפוי לתאים (לעומת צבירת התפשטות תאים), שכיחות של אפופטוזיס, וביטול הדרגתי של שינויים מורפולוגיים. מעקב תא בודד משמש ליצירת מפות גורל תא למדידה של חיי תא בודדים וחקירות של דפוסי חלוקת תא. נתונים כמותיים מנותחים סטטיסטי לאמוד את ההבדל משמעותי בהתנהגות מתואמת עם נגעים גנטיים ספציפיים.

תאי אפיתל ופיברובלסטים יכולים להיות מכובדים על ידי מורפולוגיה של תאים. תאי האפיתל יש צורת cuboidal (איור 1 א-B) ומושבות תאי טופס (איור 1 א). Fibroblasts, סוג של תא סטרומה, יש מורפולוגיה מוארכת (האיור 1C). תאים היו מעוגל...

Watch this Scientific Journal Video about Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer at JoVE.com

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

הדמית זמן לשגות משמשת כדי להעריך את ההתנהגות של תאים ראשוניים preneoplastic חלב האפיתל הנגזרים ממודלי עכבר מהונדס גנטי של סיכון לסרטן שד על מנת לקבוע אם יש קשר בין פרמטרים התנהגותיים ספציפיים ונגעים גנטיים מובחנים.

הדמית זמן לשגות של תאי אפיתל חלב Preneoplastic ראשוניים הנגזרת ממודלי עכבר מהונדס גנטי של סרטן השד

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

time-lapse-imaging-primary-preneoplastic-mammary-epithelial-cells

Research

JoVE Journal

Medicine

11.7K Views.  Georgetown University. הדמית זמן לשגות משמשת כדי להעריך את ההתנהגות של תאים ראשוניים preneoplastic חלב האפיתל הנגזרים ממודלי עכבר מהונדס גנטי של סיכון לסרטן שד על מנת לקבוע אם יש קשר בין פרמטרים התנהגותיים ספציפיים ונגעים גנטיים מובחנים.

Video: Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

בתחום ההדמיה vivo של פליטת אור Dynamics היפוקסיה גידול של גרורות סרטן השד המוח במודל של עכברים

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

הדור ניסויית של קרצינומה-Associated Fibroblasts (CAFs) מ Fibroblasts החלב אנוש

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

חי הדמיה של תגובות סמים בmicroenvironment הגידול במודלים עכבריים של סרטן השד

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

תרבות מטופל נגזר סלולרי ובידוד של CD133<sup&gt; +</sup&gt; תאי גזע סרטני משוערים מלנומה

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

חניכה של סרטן השד גרורתי על ידי מיקוד של האפיתל ductal עם Adenovirus-Cre: דגם רומן מהונדס עכבר של סרטן השד

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

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.

פרופיל של מיקרו RNA המוסדר אסטרוגן בתאי סרטן השד

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

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

שיטה לקבלת תאי סרטן השחלות ראשיים מתוך דגימות מוצקים

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

הזרקת orthotopic של תאי סרטן השד לחלב שומן Pad של עכברים לחקר התפתחות גידול.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

סרטן שד מעקב מאפיינים אדריכליים החלב וזיהוי עם דימות מותחת דיפוזי תהודה מגנטית

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.