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 dierlijke studies werden uitgevoerd in overeenstemming met de aanbevelingen van de Canadese Raad over Animal Care, in het kader van protocollen door de Western University Animal Gebruik Subcommissie goedgekeurd. 1. Organ Isolation (Lung, hersenen, botten, lymfkliertest) <ol><li> Neem vier steriele 50 ml conische buizen (een voor elk orgaan te isoleren) die ongeveer 30 ml steriel fosfaatgebufferde zoutoplossing (PBS). Pre-wegen elk buisje PBS met behulp van een elektronische weegschaal...

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Metastase is een complex proces waarbij een reeks cellulaire gebeurtenissen uiteindelijk veroorzaken weefselinvasie en verre tumor inrichting 4,30,31. De ex vivo modelsysteem gepresenteerde kunnen worden gebruikt om twee belangrijke aspecten van metastatische progressie studeren: kankercel homing of migratie naar een specifiek orgaan ( &quot;daarheen&quot;) en groei van dat orgaan ( &quot;daar groeien&quot;). Vele studies hebben eerder gericht op het identificeren van mol specifieke eigenschappen van...

Borstkanker is de meest gediagnosticeerde kanker bij vrouwen en de tweede belangrijke oorzaak van kanker-gerelateerde sterfgevallen 1. Borstkanker hoge sterftecijfer is vooral te wijten aan het falen van conventionele therapie te verminderen en te elimineren gemetastaseerde ziekte; ongeveer 90% van kanker-gerelateerde sterfgevallen te wijten metastase 2. Het begrijpen van de onderliggende moleculaire mechanismen van de metastatische cascade grootste belang voor de ontwikkeling van therapeutische effectief zowel vroege als late stadium van borstkanker. Verleden onderzoek heeft geholpen verhelderen de meerdere stappen aard van de uitzaaiing van borstkanker en het wordt verondersteld dat het resultaat van zowel de progressie van kanker en metastase is grotendeels afhankelijk van interacties tussen kankercellen en de nieuwe omgeving 3. Klinische observaties tonen aan dat veel kankers tonen orgaan tropisme, dwz., De neiging om bij voorkeur metastaseren specifieke organs.In case borstkanker, ziekte van een patiënt typisch verspreidt of uitzaaiing 5 belangrijkste plaatsen, zoals het been, longen, lymfeknopen, lever en hersenen 4-6. Veel theorieën zijn ontwikkeld om dit proces uit te leggen, maar slechts een paar hebben de tand des tijds doorstaan. Ewing&#39;s theorie van metastase, in de jaren 1920 voorgesteld, de hypothese datde verdeling van metastase was strikt door mechanische factoren; waarbij tumorcellen in het lichaam via de normale fysiologische gedefinieerd bloedstroom patronen worden uitgevoerd en eenvoudig te arresteren in het eerste capillaire bed die ze tegenkomen 7. Daarentegen Stephen Paget 1889 &quot;zaad en aarde&quot; hypothese geopperd dat bijkomende moleculaire interacties verantwoordelijk waren voor overleving en groei van metastasen, waarbij kankercellen ( &quot;seeds&quot;) zich alleen kunnen oprichten en proliferatein orgaan microenvironments passende moleculaire factoren produceren ( &quot;bodem &quot;) 8. Bijna een eeuw later, Leonard Weiss ondernam een ​​meta-analyse van eerder gepubliceerde data autopsie bevestigd Ewing voorspelling dat vele uitgezaaide tumoren waargenomen bij de autopsie werden gevonden in de verwachte verhoudingen die zou worden verwacht wanneer orgaan met metastasen tropisme alleen bepaald door de bloedstroom patronen. In manyinstances waren er minder of meer metastasen gevormd op bepaalde plaatsen dan zou Ewing voorgestelde mechanische factoren 9 verwachten. Deze rekeningen en theorieën suggereren dat specifiek orgaan microenvironments een cruciale rol in de verspreiding patronen en de daaropvolgende groei en overleving van vele vormen van kanker, zoals borstkanker spelen. Eerder onderzoek inspanningen zijn voornamelijk gericht op tumor- cellen afgeleide factoren en hun bijdrage aan het orgaan tropisme waargenomen bij borstkanker metastase 10-12, maar weinig onderzoek is onderzocht factoren afgeleid van het micro orgaan dat kan gunstig niche in de vaststellingborstkanker metastasen. Dit is grotendeels te danken aan de technische uitdagingen van het bestuderen componenten van het orgel in vitro micro-omgeving. De huidige artikel beschrijft een gedetailleerd ex vivo model voor het bestuderen van de invloed van de oplosbare bestanddelen van de lymfeknoop, bot, long en hersenen op het metastatisch gedrag van humane borstkankercellen. Eerdere studies hebben dit modelsysteem bevestigd door aan te tonen dat verschillende borstkankercellijnen tonen cellijn specifieke en orgaanspecifieke maligne gedrag in reactie op geconditioneerde media-orgaan dat overeenkomt met de in vivo metastatisch potentieel 13. Dit model kan worden gebruikt voor het identificeren en specifieke organen afgeleide oplosbare factoren die een rol spelen bij het metastatische gedrag van borstkanker en andere soorten kankercellen, met inbegrip invloeden op de groei, migratie evalueren, stam-achtig gedrag en genexpressie en de identificatie vanpotentiële nieuwe therapeutische doelwitten voor kanker. Dit is de eerste ex vivo model dat kan worden gebruikt om orgaanspecifieke metastatisch gedrag in detail te onderzoeken en de rol van organen afgeleide oplosbare factoren voor het proces van metastase te beoordelen.

<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.

Generatie van Organ geconditioneerde Media Overzichtsdiagrammen / schema van het proces van orgaan isolatie en productie van geconditioneerd medium wordt getoond in Figuur 1, met representatieve fotografische beelden van de in figuur 2 procedure. Opgemerkt wordt dat bij dit protocol werd voor het eerst in ontwikkeling is lever inbegrepen in onze analyse omdat het een gemeensch...

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.

Genereren van Organ-geconditioneerde media en toepassingen voor het bestuderen van Organ-specifieke invloeden op Borstkanker Gemetastaseerde Behavior

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

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Medicine

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