Name: generation of organ-conditioned media and applications for studying organ-specific influences on breast cancer metastatic behavior
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This work was supported by grants from the Canadian Breast Cancer Foundation-Ontario Region, the Canada Foundation for Innovation (No. 13199), and donor support from John and Donna Bristol through the London Health Sciences Foundation (to A.L.A.). Studentship and fellowship support were provided by the Ontario Graduate Scholarship program (Province of Ontario, to G.M.P. and J.E.C.), the Canada Graduate Scholarship-Master's program (to M.M.P), the Canadian Institutes of Health Research (CIHR)-Strategic Training Program (to M.M.P., G.M.P and J.E.C.) and the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit at the London Regional Cancer Program (to M.M.P., G.M.P., J.E.C. and Y.X.). A.L.A. is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.

Alle Tierversuche wurden in Übereinstimmung mit den Empfehlungen des Canadian Council on Animal Care durchgeführt, unter Protokolle, die von der Western University Tiergebrauch Subcommittee genehmigt. 1. Organ Isolation (Lunge, Gehirn, Knochen, Lymphknoten) <ol><li> Bereiten vier sterile 50 ml konische Röhrchen (eine für jedes Organ isoliert werden), die etwa 30 ml steriler phosphatgepufferter Kochsalzlösung (PBS). Pre-wiegen jedes Röhrchen PBS eine elektronische Waage. </li><li> Eut...

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Metastasierung ist ein komplexer Prozess , bei dem eine Reihe von zellulären Ereignissen letztlich für Gewebe verantwortlich sind , Invasion und entfernten Tumor Einrichtung 4,30,31. Die ex vivo Modellsystem hier präsentiert werden können genutzt zu studieren zwei wichtige Aspekte des metastasierten Progression: Krebszelle Homing oder Migration auf ein bestimmtes Organ ( &quot;getting there&quot;) und das Wachstum in diesem Organ ( &quot;dort wachsen&quot;). Viele Studien haben sich auf die Ermit...

Brustkrebs ist die am häufigsten diagnostizierte Krebs bei Frauen und die zweithäufigste Ursache der durch Krebs verursachten Todesfälle 1. Brustkrebs hohe Mortalitätsrate ist vor allem auf das Scheitern der konventionellen Therapie zu lindern und Metastasen zu beseitigen; etwa 90% der durch Krebs verursachten Todesfälle sind auf Metastasen 2. die zugrunde liegenden molekularen Mechanismen der metastatischen Kaskade zu verstehen, ist sowohl von größter Bedeutung für die Entwicklung von Therapeutika in wirksamen frühen und späten Stadium von Brustkrebs. Frühere Forschung hat die vielstufige Natur der Metastasierung von Brustkrebs geholfen aufzuklären und es wird vermutet , dass das Ergebnis sowohl der Tumorprogression und Metastasierung auf Wechselwirkungen zwischen Krebszellen weitgehend abhängig ist und der Host - Umgebung 3. Klinische Beobachtungen zeigen , dass viele Krebsarten Organtropismus anzuzeigen, dh., Die Neigung vorzugsweise an spezifischen metastasieren organs.In der case von Brustkrebs breitet sich ein Patient die Krankheit in der Regel oder metastasiert bis 5 wichtigsten Sehenswürdigkeiten, wie der Knochen, Lunge, Lymphknoten, Leber und Gehirn 4-6. Viele Theorien wurden entwickelt, um diesen Prozess zu erklären, aber nur wenige haben den Test der Zeit überstanden. Ewing Theorie der Metastasierung, in den 1920er Jahren vorgeschlagen, die Hypothese aufgestellt dassdie Verteilung der Metastasierung auf mechanische Faktoren streng zurückzuführen war; wodurch Tumorzellen im ganzen Körper durch normale definierten physiologischen Blutflussmuster und einfach verhaften in der ersten kapillaren Bett getragen werden , stoßen sie auf 7. Im Gegensatz dazu 1889 Stephen Paget &quot;Saatgut und Boden&quot;, schlug Hypothese, dass zusätzliche molekulare Wechselwirkungen für das Überleben und das Wachstum von Metastasen verantwortlich waren, wobei Krebszellen ( &quot;Samen&quot;) sich nur etablieren können und proliferatein Organ Mikroumgebungen, die entsprechenden molekularen Faktoren ( &quot;Boden erzeugen 8 &quot;). Fast ein Jahrhundert später, Leonard Weiss unternahm eine Meta-Analyse von zuvor Autopsie Daten veröffentlicht und bestätigt Ewing-Vorhersage, dass viele Metastasen zum Zeitpunkt der Autopsie festgestellt wurden in den zu erwartenden Verhältnissen gefunden, dass, wenn metastasierendem Organtropismus wurde durch Blutflussmuster allein bestimmt zu erwarten wäre. Doch in manyinstances gab es weniger oder mehr Metastasen an bestimmten Stellen gebildet , dann würde durch Ewing vorgeschlagene mechanische Faktoren 9 zu erwarten. Diese Konten und Theorien besagen, dass bestimmte Organ Mikroumgebungen spielen eine entscheidende Rolle bei der Verbreitung Muster und das anschließende Wachstum und das Überleben von vielen Krebsarten, einschließlich Brustkrebs. Frühere Forschungsanstrengungen haben sich hauptsächlich auf Tumorzellen abgeleitet Faktoren konzentriert und ihren Beitrag zur Organtropismus bei Brustkrebs Metastasen beobachtet 10-12, jedoch wenig Forschung erforscht Faktoren aus dem Organ Mikro abgeleitet , die eine günstige Nische für die Einrichtung zur Verfügung stellen kannvon Brustkrebs Metastasen. Dies ist im Wesentlichen auf die technischen Herausforderungen der Komponenten des Organs Mikroumgebung in vitro zu studieren. Der aktuelle Artikel beschreibt ein umfassendes ex vivo Modellsystem zur Untersuchung des Einflusses von löslichen Bestandteilen des Lymphknotens studieren, Knochen, Lunge und Gehirn auf dem metastatischen Verhalten von menschlichen Brustkrebszellen. Frühere Studien haben dieses Modellsystem validiert durch den Nachweis , dass verschiedene Brustkrebszelllinien Zelllinie spezifische und organspezifische bösartige Verhalten als Reaktion auf organ konditionierten Medien angezeigt werden , die auf ihre in vivo Metastasepotential 13 entspricht. Dieses Modellsystem kann verwendet werden, um lösliche Faktoren zu identifizieren und zu bewerten spezifischen organ abgeleitet, die eine Rolle bei der metastatischen Verhaltens von Brustkrebs und anderen Arten von Krebszellen, einschließlich Einflüsse auf das Wachstum, der Migration spielen kann, stielartigen Verhalten und Genexpression, sowie die Identifizierung vonmögliche neue therapeutische Ziele für Krebs. Dies ist das erste ex vivo Modellsystem , das verwendet werden kann , organspezifische metastatischen Verhalten im Detail zu untersuchen und die Rolle der organ abgeleiteten löslichen Faktoren in den Antrieb der Prozess der Krebsmetastasen zu bewerten.

<table border="0" cellpadding="0" cellspacing="0" width="1195">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">50 ml conical tubes</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td style="width:223px;">339652</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">1X Phosphate-buffered saline</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:223px;">10010-023</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Nude mice</td>
			<td style="width:159px;">Harlan Laboratories</td>
			<td style="width:223px;">Hsd:Athymic Nude-Foxn1nu</td>
			<td>Use at 6-12 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:339px;">Polystyrene foam pad</td>
			<td style="width:159px;">N/A</td>
			<td style="width:223px;">N/A</td>
			<td style="width:475px;">The discarded lid (~4 x 8 inches or larger) of a polystyrene foam shipping container can be used for this purpose. Sterilize by wiping with ethanol.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:339px;">Forceps</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:223px;">11050-10</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:339px;">Scissors</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:223px;">14058-11</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:339px;">Gauze pads</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:223px;">22-246069</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:339px;">60 mm2 glass petri dishes</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">CLS7016560</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:339px;">Scalpel blades</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:223px;">S95937A</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM:F12</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">21331-020</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">1 x Mito+ Serum Extender</td>
			<td style="width:159px;">BD Biosciences</td>
			<td style="width:223px;">355006</td>
			<td style="width:475px;">Referred to as &#34;concentrated mitogen supplement&#34; in the manuscript. Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">15140-122</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Rosewell Park Memorial Institute 1640 (RPMI 1640)</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">11875-093</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Fetal Bovine Serum</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">F1051-500ML</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Trypsin/EDTA solution</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:223px;">R-001-100</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">6-well tissue culture plates</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td>140675</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">0.22 &#956;m syringe filters</td>
			<td>Sigma-Aldrich</td>
			<td>Z359904</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">T75 tissue culture flasks</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td style="width:223px;">178905</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Transwells</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">CLS3464</td>
			<td>Keep sterile, use for migration assays</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse Sca-1</td>
			<td>R&#38;D Systems</td>
			<td>FAB1226P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD105</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB1320P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD29</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB2405P-025</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD73</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB4488P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD44</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">MAB6127-SP</td>
			<td>use at 0.25 &#181;g/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD45</td>
			<td>eBioscience</td>
			<td style="width:223px;">11-0451-81</td>
			<td>use at 5 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse gp38</td>
			<td>eBioscience</td>
			<td style="width:223px;">12-5381-80</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">M6250&#160;</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Protein arrays</td>
			<td style="width:159px;">RayBiotech Inc.</td>
			<td style="width:223px;">AAM-BLM-1-2</td>
			<td>Use 1 array per media condition (including negative control), in triplicate</td>
		</tr>
	</tbody>
</table>

generation of organ-conditioned media and applications for studying organ-specific influences on breast cancer metastatic behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

Die Erzeugung von Orgelanlage Medien Ein Übersichtsdiagramm / schematische Darstellung des Prozesses der organ Isolierung und Erzeugung von konditionierten Medien ist in Abbildung 1, mit repräsentativen photographischen Bildern des Verfahrens in Figur 2 gezeigt dargestellt. Es ist zu beachten , dass , wenn dieses Protokoll zunächst in der Entwicklung war, Leber aufgenommen ...

Watch this Scientific Journal Video about Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior at JoVE.com

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Die Erzeugung von Orgelanlage Medien und Anwendungen für das Studium Organspezifische Einflüsse auf Brustkrebs Metastasiertem Verhalten

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Research

JoVE Journal

Medicine

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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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