Supported by grants from the Department of Defense (W81XWH-09-1-0127), the NIH (2-R01-CA089569), the Elsa U. Pardee Foundation, a Marsha Rivkin Center for Ovarian Cancer Research Pilot Study Award, and generous philanthropic support from Section of Urology and Section of Research in the Department of Surgery, University of Chicago.

<ol>
	<li>Bast, R. C., Hennessy, B., Mills, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+ovarian+cancer:+new+opportunities+for+translation.">The biology of ovarian cancer: new opportunities for translation.</a> Nature Reviews Cancer. 9 (6), 415-428 (2009).</li><li>Bowtell, D. D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genesis+and+evolution+of+high-grade+serous+ovarian+cancer.">The genesis and evolution of high-grade serous ovarian cancer.</a> Nature Reviews Cancer. 10 (11), 803-808 (2010).</li><li>Colgan, T. J., Chang, M. 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A., 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=Preferential+Attachment+of+Peritoneal+Tumor+Metastases+to+Omental+Immune+Aggregates+and+Possible+Role+of+a+Unique+Vascular+Microenvironment+in+Metastatic+Survival+and+Growth.">Preferential Attachment of Peritoneal Tumor Metastases to Omental Immune Aggregates and Possible Role of a Unique Vascular Microenvironment in Metastatic Survival and Growth.</a> The American Journal of Pathology. 169 (5), 1739-1752 (2010).</li><li>Liebermann-Meffert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+greater+omentum.+Anatomy,+embryology,+and+surgical+applications.">The greater omentum. Anatomy, embryology, and surgical applications.</a> The Surgical Clinics of North America. 80 (1), 275-293 (2000).</li><li>Collins, D., Hogan, A. M., O&#8217;Shea, D., Winter, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Omentum:+Anatomical,+Metabolic,+and+Surgical+Aspects.">The Omentum: Anatomical, Metabolic, and Surgical Aspects.</a> Journal of Gastrointestinal Surgery. 13 (6), 1138-1146 (2009).</li><li>Khan, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fallopian+tube+precursors+of+ovarian+low-+and+high-grade+serous+neoplasms.">Fallopian tube precursors of ovarian low- and high-grade serous neoplasms.</a> Histopathology. 62 (1), 44-58 (2012).</li><li>Folkins, A. K., Jarboe, E. A., Roh, M. H., Crum, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precursors+to+pelvic+serous+carcinoma+and+their+clinical+implications.">Precursors to pelvic serous carcinoma and their clinical implications.</a> Gynecologic Oncology. 113 (3), 391-396 (2009).</li><li>Li, J., Fadare, O., Xiang, L., Kong, B., Zheng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+serous+carcinoma:+recent+concepts+on+its+origin+and+carcinogenesis.">Ovarian serous carcinoma: recent concepts on its origin and carcinogenesis.</a> Journal of Hematology, &amp; Oncology. 5 (1), 8 (2012).</li><li>Kurman, R. J., Shih, I. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+pathogenesis+of+epithelial+ovarian+cancer:+a+proposed+unifying+theory.">The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory.</a> The American Journal Of Surgical Pathology. 34 (3), 433-443 (2010).</li><li>Crum, C. 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=The+distal+fallopian+tube:+a+new+model+for+pelvic+serous+carcinogenesis.">The distal fallopian tube: a new model for pelvic serous carcinogenesis.</a> Current Opinion In Obstetrics, &amp; Gynecology. 19 (1), 3-9 (2007).</li><li>Crum, C. 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=Lessons+from+BRCA:+The+Tubal+Fimbria+Emerges+as+an+Origin+for+Pelvic+Serous+Cancer.">Lessons from BRCA: The Tubal Fimbria Emerges as an Origin for Pelvic Serous Cancer.</a> Clinical Medicine, &amp; Research. 5 (1), 35-44 (2007).</li><li>Foulkes, W. D., Narod, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+risk+and+family+history.">Ovarian cancer risk and family history.</a> The Lancet. 349, 878 (1997).</li><li>Antoniou, A., 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=Average+Risks+of+Breast+and+Ovarian+Cancer+Associated+with+BRCA1+or+BRCA2+Mutations+Detected+in+Case+Series+Unselected+for+Family+History:+A+Combined+Analysis+of+22+Studies.">Average Risks of Breast and Ovarian Cancer Associated with BRCA1 or BRCA2 Mutations Detected in Case Series Unselected for Family History: A Combined Analysis of 22 Studies.</a> The American Journal of Human Genetics. 72 (5), 1117-1130 (2003).</li><li>King, M. -. C., Marks, J. H., Mandell, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+York+Breast+Cancer+Study+Group+Breast+and+ovarian+cancer+risks+due+to+inherited+mutations+in+BRCA1+and+BRCA2.">New York Breast Cancer Study Group Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2.</a> Science. 302 (5645), 643-646 (2003).</li><li>Kj&#230;r Kr&#252;ger, S., Gayther, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+and+genetic+susceptibility+in+relation+to+the+BRCA1+and+BRCA2+genes.+Occurrence,+clinical+importance+and+intervention.">Ovarian cancer and genetic susceptibility in relation to the BRCA1 and BRCA2 genes. Occurrence, clinical importance and intervention.</a> Acta Obstetricia et Gynecologica Scandinavica. 85 (1), 93-105 (2006).</li><li>Easton, D. F., Ford, D., Bishop, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+and+ovarian+cancer+incidence+in+BRCA1-mutation+carriers.+Breast+Cancer+Linkage+Consortium.">Breast and ovarian cancer incidence in BRCA1-mutation carriers. Breast Cancer Linkage Consortium.</a> American Journal of Human Genetics. 56 (1), 265-271 (1995).</li><li>Rebbeck, T. R., 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=Prophylactic+Oophorectomy+in+Carriers+of+BRCA1or+BRCA2Mutations.">Prophylactic Oophorectomy in Carriers of BRCA1or BRCA2Mutations.</a> New England Journal of Medicine. 346 (21), 1616-1622 (2002).</li><li>Olivier, R. I., 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=Clinical+outcome+of+prophylactic+oophorectomy+in+BRCA1/BRCA2+mutation+carriers+and+events+during+follow-up.">Clinical outcome of prophylactic oophorectomy in BRCA1/BRCA2 mutation carriers and events during follow-up.</a> British Journal Of Cancer. 90 (8), 1492-1497 (2004).</li></ol>

The authors have nothing to disclose.

פיתוח של טיפולים למקד תאים הפיצו דורש הבנה מכניסטית של קולוניזציה גרורתית, הצעד הראשון הקריטי בהתפתחות של מחלה הצפק. כדי לטפל בבעיות אלה אנו מדווחים גישות שניתן להשתמש כדי להבחין איך הרכב של omentum ייחודי רקמה והאדריכלות לקדם קולוניזציה גרורתית סרטן השחלות. מאפיי...

שלא כמו רוב הגידולים מוצקים, גרורות מסרטן בדרגה גבוהה השחלות הצפק (HGSC) מוגבלות לחלל הצפק 1. לפיכך, טיפולי הצפק יעילים עלולים לשלוט או לחסל HGSC. נכון לעכשיו, גישה טיפולית סטנדרטית היא cytoreduction כירורגית בשילוב עם כימותרפיה 1-3. למרבה הצער, הרוב המכריע של מטופלים חווים ולהיכנע לסיבוכים של הישנות מחלה. סטטיסטיקה העגומה אלה מראה את הצורך בהבנה משופרת של קולוניזציה גרורתית, התהליך שבו תאי הסרטן בתרגום ל, לנצל, ולהתרבות בתוך רקמות מארח כדי ליצור גרורות 2. Omentum הוא אתר מוקדם המועדף HGSC גרורות 4-7. שלא כמו שומן הצפק אחר, רקמת שומן omental מכילה מבנים חיסוניים חריגים ידועים ככתמים חלביים, המכילים B, T, ותאי NK ומקרופאגים, אשר ממלאים תפקידי מפתח בhomeos הצפקtasis 8,9. בנוסף לפונקציות הפיזיולוגיות שלהם, מצאנו כי כתמים חלביים למלא תפקיד פעיל בקולוניזציה גרורתית סרטן השחלות 4. במבחני גרורות ניסיוניים, SKOV3ip.1, CaOV3, וHeyA8 (אדם) וID8 (עכברי; C57BL / 6) תאי סרטן השחלות במהירות הביתה לכתמים חלביים, המצביע על כך שהתאים לנוע לעבר גורם chemotactic מופרש. מעניין לציין, תאי סרטן לא ליישב שומן הצפק חסר כתמים חלביים (כלומר, האשכים ושומן רחם) 4. על מנת לזהות מנגנוני ויסות קולוניזציה מקום חלבי, יש לנו מודלים מותאמים xenograft המאפשרים החקירה של אירועים תאיים ומולקולריים במהלך קולוניזציה גרורתית 4 הזמן. יתרון ספציפי של הגישות המתוארות במסמך זה דגש על ארכיטקטורה ותפקוד רקמות, המאפשר למשתמשים לבחון השערות במשולבות במלואו ובמודלי vivo לשעבר vivo של מטר4,10 קולוניזציה etastatic. על ידי השוואת לוקליזציה של תאי הסרטן וגידול במחסנים שומן הצפק אשר גם להכיל או חסרי כתמים חלביים, חוקרים יכולים לבדוק את תרומתו היחסית (ים) של תאי שומן ותאים בתוך כתמים חלביים ללינה והצמיחה ההדרגתית של תאי סרטן השחלה ברקמות רלוונטיות מבחינה פיזיולוגית.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dissection Tools</td>
			<td>Fine Science Tools</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Geimsa</td>
			<td>Fluka/Sigma Aldrich</td>
			<td>48900</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Formalin</td>
			<td>Sigma</td>
			<td>HT501320</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>Without calcium and Magnesium</td>
		</tr>
		<tr>
			<td>26 gauge needle</td>
			<td>BD</td>
			<td>329652</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Stomacher</td>
			<td>Seward Labsystems</td>
			<td>Stomacher 80 Biomaster</td>
			<td></td>
		</tr>
		<tr>
			<td>Microstomacher bag</td>
			<td>Stomacher Lab Systems</td>
			<td>BA6040/Micro</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysis Buffer</td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell culture plate insert</td>
			<td>Millipore</td>
			<td>PICM01250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Tak</td>
			<td>BD</td>
			<td>354240</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo and ex vivo approaches to study ovarian cancer metastatic colonization of milky spot structures in peritoneal adipose

בדרגה גבוהה לסרטן השחלות הצפק (HGSC), הגורם לגרורות הצפק נרחבות, ממשיכה להיות פרוגנוזה גרועה מאוד; פחות מ -30% מהנשים בחיים 5 שנים לאחר האבחון. Omentum הוא אתר מועדף של היווצרות גרורה HGSC. למרות החשיבות הקלינית של מייקרו-סביבה זו, התרומה של רקמת שומן omental להתקדמות סרטן השחלות נותרת understudied. שומן Omental יוצא דופן בכך שהוא מכיל מבנים הידועים ככתמים חלביים, המורכבים מB, T, ותאי NK, מקרופאגים, ותאים המקיפים את הקנים צפופים של כלי דם. כתמי החלב לשחק תפקיד מפתח בפונקציות הפיזיולוגיות של omentum, אשר נדרשות להומאוסטזיס הצפק. הראינו כי כתמים חלביים גם לקדם התישבות השחלות סרטן גרורתי של שומן הצפק, צעד מפתח בפיתוח של גרורות הצפק. כאן אנו מתארים את הגישות שפיתחנו כדי להעריך ולכמת כתמים חלביים לitoneal שומן וללמוד התרומה הפונקציונלית שלהם לקולוניזציה גרורתית תאים סרטניים בשחלות של רקמות omental שני in vivo לשעבר vivo. גישות אלה הן להכליל לדגמים נוספים עכבר ושורות תאים, ובכך לאפשר את חקר היווצרות גרורות סרטן השחלות מלוקליזציה ראשונית של תאים למבני מקום חלביים להתפתחות של גרורות הצפק נרחבות.

זיהוי ובדיקה היסטולוגית מאגרי שומן הצפק נתיחה אנטומיים גולמית מאפשרת זיהוי של ארבעה מחמשת המקורות העיקריים של שומן הצפק (איור 1 א). נע בכיוון השעון מהמרכז העליון הם: omentum (OM; התווה) ממוקם על הבטן והטחול, שומן האשכי...

Watch this Scientific Journal Video about In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose at JoVE.com

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.

In vivo לשעבר vivo גישות לחקר סרטן השחלות גרורתי התיישבות של מבני ספוט החלב בהצפק שומני

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

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

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12.9K Views.  The University of Chicago. 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.

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

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex Vivo זיהום של רקמה חיה עם וירוסים oncolytic

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

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.

במבחנה מערכת לחקר תרדמת גידול ואת החלף ל גידול גרורתי

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 ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

רומן ברזולוציה גבוהה<em&gt; בvivo</em&gt; טכניקת הדמיה כדי לחקור את התגובה דינמית של מבנים תוך גולגולתי לצמיחה גידול וTherapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

אדם<em&gt; Ex Vivo</em&gt; טרשת רובד מודל ללמוד ביולוגיה נגע

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

בF-MRI 19 vivo למעקב סלולרי

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

מולקולרי פרופיל של microenvironment הגידול פולש במודל 3 ממדים של תאי סרטן המעי הגס ו<em&gt; Ex vivo</em&gt; פיברובלסטים

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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.

<em&gt; Ex vivo</em&gt; דגם לחקר הורמוני פעולה בשד האדם

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 ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

אימות של Nanobody ונוגדנים מבוסס<em&gt; In vivo</em&gt; ניסויי גידול xenograft NIRF הדמיה בעכברים באמצעות<em&gt; Ex Vivo</em&gt; Cytometry הזרימה ומיקרוסקופי

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

אדם Dupuytren של<em&gt; Ex Vivo</em&gt; תרבות לחקר myofibroblasts והתאי מטריקס האינטראקציות

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

טלמטריה תוך רחמי למדידת לחץ עכבר התכווצות<em&gt; In vivo</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...