This work was supported by NCI Grants RO1CA157664 and RO1CA124515, and a Breast Cancer Alliance award. We would like to thank Jeffrey Faust, David Ambrose, and Scott Weiss from the Wistar Flow Cytometry core facility, James Hayden from the Wistar&#160;Imaging facility, and the entire staff of the Wistar Institute Animal Facility for their invaluable technical support.

Alle Tierversuche wurden von der Wistar-Institut Animal Care und Verwenden Committee genehmigt. 1. Erstellung und Wartung von transgenen Mäusen <ol><li> Rasse LSL-K-ras tm4Tyj 26 und Trp53 tm1Brn 27 (von NGI-Maus-Modellen der menschlichen Krebs Konsortium auf eine gemischte Hintergrund erhalten wird) zu einem vollen C57BL / 6 Hintergrund 28 durch Rückkreuzung mindestens 10 Generationen mit C57BL / 6 Mäusen. So verfolgen Tumormetastasen, Rasse B6.129X1-Gt (ROSA) 26Sor tm1 (EYFP) Cos / J (LSL-EYFP, von The Jackson Laboratory auf Voll C57BL / 6 Hintergrund erhalten) mit Doppel transgenen LSL-K-ras G12D / + p53 loxP / loxP Mäusen. Hinweis: Transgene LSL-K-ras G12D / + p53 loxP / loxP Mäuse haben loxP-Stellen flankieren eine transkriptionell Schweigen Allel des onkogenen K-ras und der endogenen p53-Locus, so dass bei Cre-vermittelte Exzision, die Überexpression eines onkogenen K-ras Mutante und Ablation von p53 ist achiEVED. Hinweis: Die LSL-EYFP Maus enthält ein Stopcodon flankieren, ein Gen für eine verstärkte gelb fluoreszierendes Protein (YFP), das nach Cre-vermittelter Exzision Ergebnisse der Expression von YFP in den Geweben, in denen die YFP Stop-Kassette wird herausgeschnitten. <ol><li> Rasse transgenen Mäusen zu LSL-K-ras G12D / + p53 loxP / loxP Mäusen oder LSL-K-ras G12D / + p53 loxP / loxP LSL-EYFP-Mäuse für intraduktale Injektionen erhalten. Hinweis: Die Mäuse werden als homozygot für p53 loxP / loxP und heterozygot für rassigen LSL-K-ras G12D / +, da Mäuse mit einer homozygote Deletion des K-ras sterben in utero. Verwenden naiv jungfräulichen Weibchen mindestens sechs Wochen alt für intraduktale Injektionen. Hinweis: Die Primer für die Genotypisierung floxed p53 Allel homozygot sind p53 - T010-fwd (5&#39;-AAGGGGTATGAGGGACAAGG-3 &#39;) und p53-T011-rev (5&#39;-GAAGACAGAAAAGGGGAGGG-3&#39;). Sie produzieren eine Wildtyp-Allel bei 391 bp und das p53-Allel floxed bei 461 bp 29,30. Hinweis: Die Primer, die mutierte Form des K-ras detektieren oIMR8273 (5&#39;-CGCAGACTGTAGAGCAGCG-3 &#39;) und oIMR8274 (5&#39;-CCATGGCTTGAGTAAGTCTGC-3&#39;). Sie produzieren das mutierte Bande bei 600 bp detektiert. Hinweis: Für die Dreifach-Reporter YFP transgenen Mäusen, um die Primer die ROSA Kassette erkennen (5&#39;-AAGACCGCGAAGAGTTTGTC-3 &#39;), die das Wildtyp-Allel (5&#39;-GGAGCGGGAGAAATGGATATG-3&#39;) und eine gemeinsame Allel (5&#39;-AAAGTCGCTCTGAGTTGTTAT -3 &#39;) führen zur Verstärkung der Banden bei 320 bp für floxed Allel und 600 bp für das Wildtyp-Allel. </li></ol></li></ol> 2. OP-Vorbereitung <ol><li> Saubere chirurgischen Materialien mit 75% EtOH und Autoklaven sie vor und nach den Injektionen. </li><li> Führen Operation auf einem Labortisch ordentlich sauber desinfiziert in einem Zimmer in einer Tieranlage. Wischen Sie alle Oberflächen, einschließlich der Bühne und Zifferblätter des Operationsmikroskops mit einem Breitspektrum-Desinfektionslösung, gefolgt von 75% EtOH. </li><li> Wiegen und Mäuse anästhesiert durch intraperitoneale Injektion einer Mischung aus Ketamin (80-100 mg / kg) und Xylazin (8-10 mg / kg) in steriler Kochsalzlösung. </li><li> Sanft legen Mäuse wieder in ihre Käfige für 5 Minuten ungestört, während sie unter Narkose zu gehen. Während dieser Zeit erzeugen Virus Niederschläge (siehe Protokoll Nr. 3). </li><li> Überprüfen Sie, ob Mangel an Reaktion auf Schmerzen, die durch Quetschung Zehe. Die Augen der narkotisierten Mäusen vorsichtig Abdeckung mit Tier Salbe, um übermäßige Hornhaut Austrocknen zu verhindern. </li><li> Um Unterkühlung zu vermeiden, legen narkotisierten Mäusen auf einem Heizkissen auf kleiner Flamme während des chirurgischen Eingriffs eingestellt und bis sie sich zu erholen beginnen. </li><li> Für die Behandlung von Schmerzen, verwalten Mäusen subkutan Meloxicam bei 1 mg / kg vor der Operation und 24 Stunden nach. </li></ol> 3. Erzeugung von Virus Niederschläge VORSICHT: Adenovirus-Vektoren, wenn sie modifiziert worden sind und sich nicht replizieren, besteht das Risiko,Infektion. Griff Adenovirus mit Vorsicht. Alle Mitarbeiter sollten entsprechend den Richtlinien des Instituts für den Umgang mit BSL2 Agenten Nach intraduktale Injektion, entsorgen Adenovirus nach BSL2 Richtlinien geschult werden. <ol><li> Speicher Adenovirus konzentrierte Virusbestände bei -80 ° C in Aliquots von 4 x 10 8 pfu jedes ausreichend gefroren zum Einspritzen 16 Tiere mit 3 ml 2,5 x 10 7 pfu des Adenovirus-Partikel. </li><li> Bewahren Adenovirus aliquoten auf Trockeneis, bis ca. 15-20 min vor Beginn der Injektionen. Hinweis: Vermeiden Sie wiederholtes Einfrieren und Auftauen als Virus-Titer sinkt deutlich zwischen jedem Zyklus. Hinweis: Adenovirus Niederschläge werden durch Modifizieren eines zuvor beschriebenen Protokolls 31 gebildet. </li><li> Rekonstituieren 504 mg Pulver MEM mit 50 ml steriler Molekular Grad Wasser, Nahrungsergänzungsmittel mit 244 mg Natriumbicarbonat und Filter in sterilen Bedingungen und bei 4 ° C <ol> &lt;li&gt; Bereiten Sie die Calciumchlorid-Lösung durch Zugabe von 1,5 g Calciumchlorid in 50 ml molekular reines Wasser und Filter unter sterilen Bedingungen und bei 4 ° C </li><li> Mischen Aliquots mit 4 x 10 8 pfu Adenovirus-Cre mit ausreichender 3% Saccharose in sterilem Wasser auf ein Endvolumen von 10 ml gelöst. In 34 ml MEM, um das Virus und vorsichtig mischen. Dann fügen Sie 4 ml der CaCl 2-Lösung, vorsichtig mischen, und bei Raumtemperatur für 15-20 min. </li><li> Shop Adenovirus auf Trockeneis bis bereit, um Niederschläge zu bilden. Vermeiden Auftauen des Adenovirus und Speichern auf Eis oder bei Raumtemperatur für längere Zeiträume, sofern Niederschläge gebildet werden. Anmerkung: Es ist auch möglich, die Saccharose, MEM und CaCl 2 vor den Operationen mischen, wenn es nicht möglich ist, den Adenovirus-Aliquot aufgetaut und beginnen, fällt unmittelbar nach dem Entfernen von -80 ° C. Dieses Aliquot von Saccharose, MEM, und Kalzium kann auf Trockeneis, bis bereit, den Adenovirus hinzufügen gespeichert werden. Hinweis: Viruspartikel werden etwa 1 h stabil. </li><li> Vor jeder Injektion, sanft Flick das Rohr, um sicherzustellen, Viruspartikel werden gemischt. Zeichnen bis 3 ml (2,5 x 10 7 pfu) von Viruspartikeln in den 10-ml-Spritze und bereiten Sie die Maus für die intraduktale Injektion. </li></ol></li></ol> 4. Intraduktale Injektion von Viruspartikeln <ol><li> Sanft statt mit der Maus auf dem Rücken auf den beleuchteten Bühne von einem sauberen Dissektionsmikroskop. Beleuchten Sie die Bauchseite mit einer zusätzlichen Lichtquelle und suchen Sie die links oder rechts, 4. 9. Leistenbrustdrüse durch die kleinen weißen Flecken von Pelz (sichtbar auf C57BL / 6 Frauen) jeden Nippel umgibt. </li><li> Reiben Sie die Brustwarze vorsichtig mit einer sterilen Ethanol getränkten Wattestäbchen, um Haare aus der Brustwarze zu löschen und die Injektionsstelle zu sterilisieren. Wenn sie schwer zu finden sind, vorsichtig eine dicke Schicht einer Enthaarungscreme oder Verwendung Schere, um die Brustwarzen freizulegen. </li&gt; <li> Entfernen Sie das Keratin Stecker, eine Schicht aus dichtem abgestorbene Hautzellen, die Abdeckung wird die Brustwarze. Sobald die Nippel ausgesetzt ist, sollte das Keratin Stecker gut sichtbar unter der Dissektionsmikroskop sein. </li><li> Sichern Sie die Brustwarze mit feinen chirurgischen Pinzette und ziehen Sie mit wenig Kraft um das Keratin Stecker entfernen. </li><li> Stabilisieren Sie die Brustwarze zwischen der Zange. </li><li> Setzen Sie vorsichtig die Nadel zwischen den Zangen, Kanülieren den Kanal Kanal bei 90 °. Geben Sie die Sauger etwas hinter der Abschrägung der Nadel (nicht mehr als 2 mm), um das Eindringen durch das Brustgewebe und in die serösen Membranen der ventralen Körperhöhle zu verhindern. <ol><li> Sie die Nadel zu tief einführen. Um die richtige Tiefe der Injektion zu gewährleisten, ziehen Sie die Nadel bis nach dem Einlegen in das Lumen des Kanals, zeichnen die Brustwarze bis an den Rändern der Nadel, wie es oben gezogen. Hinweis: Visualisierung der Injektion schwierig, deshalb practicE für diesen Schritt wird unter Verwendung von Trypanblau empfohlen. </li></ol></li><li> Wenn die Nadel in geeigneter Weise in der Brustkanal platziert, lassen Sie die 3 ml Virus Niederschläge (2,5 x 10 7 pfu des Adenovirus-Cre), indem Sie vorsichtig stürzen die Spritze mit dem Daumen der Hand hält die Spritze. Die Brustwarze sollte etwas aufblasen, wenn die Flüssigkeit aufgenommen ist. </li></ol> 5. Wiederherstellung von Mäusen <ol><li> Platzieren Sie die Maus wieder auf das Heizkissen nach der Injektion, bis sie beginnt, sich von der Narkose zu erholen. </li><li> Sobald die Maus gewonnen, setzen Sie ihn wieder in einen sauberen Käfig und Monitor für vollständige Erholung und Bewegung. </li><li> 24 Stunden nach der Injektion intraductal, subkutan verabreichen Meloxicam bei 1 mg / kg. </li></ol> 6. Überwachung Tumorprogression <ol><li> Tasten Sie die Brustdrüse injiziert am Tag 30 für die Erweiterung und Schwellungen. <ol><li> Überwachung der Tumorprogression alle 5-7 Tage einmal geschwollen und vergrößerte Brust Gland beobachtet. </li></ol></li><li> Messen Tumorvolumina alle 3-4 Tage für das Tumorwachstum Kinetik einmal tastbaren Tumoren erscheinen (ca. 50-60 Tage nach der Adenovirus-Injektion). </li><li> Euthanize Mäusen als Tumorvolumen 10% des Körpergewichts der Mäuse überschreiten. </li></ol>

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Exp. , e4030 (2012).</li><li>Murata, S., 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=Ductal+access+for+prevention+and+therapy+of+mammary+tumors.">Ductal access for prevention and therapy of mammary tumors.</a> Cancer Res. 66, 638-645 (2006).</li></ol>

The authors declare no competing financial interests.

The success of this procedure hinges on proper technique during intraductal injections, which will be difficult for untrained experimenters. Experienced researchers in our laboratory typically obtain breast tumor masses 79% of the times they administer adenoviral injections. Problems with the injection can result in significantly delayed, variable, or absent tumor development. If the needle is inserted too deep or at an inappropriate angle, the ductal canal may be missed. It is important to enter the nipple slightly past the bevel of the needle (not more than 2 mm), to prevent penetration through the mammary tissue and into the serous membranes of the ventral body cavity. Also, too shallow placement of the needle or the injection of greater than 3 ml of virus precipitates can result in spillage of viral prep outside the mammary gland and the induction of unintentional tumors. One way to overcome these issues is to insert the needle into the nipple slightly deeper than 3 mm, and slowly draw the syringe back up out of the duct until 2 mm from the tip of the bevel. This will ensure that mammary tissue is targeted instead of the muscle surrounding the peritoneal cavity of mice (Figure 1A). This will also stretch the nipple along the edges of the syringe so that when the virus is expelled into the duct, there is no spillage and loss of viral precipitates.
Visualization of the injection is difficult and practice for this step is recommended. We have observed an increase in successful injections following practice, resulting in a higher penetrance of tumor development. Because this technique uses nonlactating virgin females, it is critical to remove the keratin plug covering the nipple to reveal the underlying duct canal. We recommend practicing this step by injecting trypan blue or some other sterile traceable dye inside of the duct and preparing whole mammary mounts to confirm targeting of the ductal tree. Additionally, other protocols describing intraductal injection of reagents have been published33,34, which may be useful for developing proper technique. Issues with viral preparation or infection of the ductal lumen can also be investigated by using a mCherry expressing adenovirus. Nontransgenic mice can be used for each of these purposes until the injection technique is optimized.
Although any mammary gland can be used to initiate tumors, we have achieved the most consistent growth rates by targeting mammary gland 4 or 9, which we believe is because it is easier to perform proper injections on these glands, resulting in more efficient targeting of the duct. The proximity of the left 4th or right 9th inguinal mammary glands to the draining inguinal lymph node is also useful to examine anti-tumor immune responses during different temporal points of tumor progression. To model and track latent metastasis to distal sites, transgenic mice were crossed with LSL-EYFP mice. As the tumor progresses, vasculature connecting the tumor to the axillary lymph node will become slightly engorged at approximately 5 weeks, before the tumor begins to grow exponentially (Figure 3A). Eventually, after 7-8 weeks, lymphovascular invasion will result in tumor growth within the axillary lymph node (Figures 3A and 3B). Using reporter mice and Cre-loxP technology, incorporation of YFP creates a platform to track tumor cells metastasizing to distal sites throughout tumor progression. This can facilitate studies aimed at elucidating the cellular and epigenetic mechanisms that promote latent metastasis. In our hands, breast tumor cell lines expressed cytokeratin-8, mesothelin, estrogen receptor-&#945;, and Her2/neu, confirming targeting of the ductal epithelium. However, depending upon the mutations induced and due to the difficulty and the variability of injections, we recommend histological characterization of tumors once the model is well established in the laboratory.
Because breast cancer is such a deadly and pervasive disease1,2, it is important to use animal models that accurately recapitulate the complex interplay between tumor and host. Here we describe a fully backcrossed C57BL/6 murine model of breast tumor. First, by inducing tumors from native cells, we allow the tumor to evolve naturally in a full immune microenvironment. The immune microenvironment in the advanced mouse breast tumors recapitulates the populations of &#945;&#946; and &#947;&#948; T cells, myeloid derived suppressor cells, and macrophages commonly observed in human breast cancer (Figure 4). As we have published previously using adenovirus-Cre to induce ovarian tumors, we found that the viral injection had negligible effect on the corresponding tumor infiltrates and tumor progression28. Second, endocrine independent expression of oncogenes ensures that tumor cells have persistently high levels of target gene expression. Third, by taking advantage of latent mutations, we can control the timing of tumorigenesis to facilitate precise temporal tracking of tumor evolution. Applications of this model include research on tumor cell biology, studies on factors in the tumor microenvironment, anti-tumor immune responses, and even efficacy evaluation of new therapeutics. Through the availability of the Cre-loxP system, this technique can be used as a platform for investigating a diverse array of additional mutations in the initiation and progression of breast tumors. We hope that the use of this model will enhance the understanding of breast cancer biology and eventually lead to new therapeutics aimed at treating metastatic breast cancer.

A correction was made to <a href="/video/51171">Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer</a>. Protocol sections 3.1, 3.3.2, 3.4, and 4.7 were updated. The materials table was also updated.
Protocol section 3.1 was updated from:
3.1) Store adenovirus concentrated virus stocks at -80&#176;C frozen in aliquots of 4 x 108 pfu each, sufficient for injecting 16 animals with 3 ml of 2.5 x 107 pfu of adenovirus particles.
to:
3.1) Store adenovirus concentrated virus stocks at -80&#176;C frozen in aliquots of 4 x 108 pfu each, sufficient for injecting 16 animals with 3 &#181;l of 2.5 x 107 pfu of adenovirus particles.
Protocol section 3.3.2 was updated from:
Mix aliquots containing 4 x 108 pfu adenovirus-Cre with sufficient 3% sucrose in sterile water for a final volume of 10 ml. Add 34 ml of MEM to the virus and gently mix. Then add 4 ml of the CaCl2 solution, gently mix, and incubate at room temperature for 15-20 min.
to:
Mix aliquots containing 4 x 108 pfu adenovirus-Cre with sufficient 3% sucrose in sterile water for a final volume of 10 &#181;l. Add 34 &#181;l of MEM to the virus and gently mix. Then add 4 &#181;l of the CaCl2 solution, gently mix, and incubate at room temperature for 15-20 min.
Protocol section 3.4 was updated from:
Prior to each injection, gently flick the tube to make sure virus particles are mixed. Draw up 3 ml (2.5 x 107 pfu) of virus particles into the 10 ml syringe and prepare the mouse for the intraductal injection.
to:
Prior to each injection, gently flick the tube to make sure virus particles are mixed. Draw up 3 &#181;l (2.5 x 107 pfu) of virus particles into the 10 &#181;l syringe and prepare the mouse for the intraductal injection.
Protocol section 4.7 was updated from:
When the needle is appropriately placed into the mammary duct, release the 3 ml of virus precipitates (2.5 x 107 pfu of adenovirus-Cre) by gently plunging the syringe with the thumb of the hand holding the syringe. The nipple should slightly inflate as the liquid is added.
to:
When the needle is appropriately placed into the mammary duct, release the 3 &#181;l of virus precipitates (2.5 x 107 pfu of adenovirus-Cre) by gently plunging the syringe with the thumb of the hand holding the syringe. The nipple should slightly inflate as the liquid is added.
Item 15 in the Materials table under Comments was updated from:
10ml syringe, RN series Autoclave before and after each use. Clean with PBS and 75% ethanol.
to:
10&#181;l syringe, RN series Autoclave before and after each use. Clean with PBS and 75% ethanol.

A correction was made to <a href="/video/51171">Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer</a>. Protocol sections 3.1, 3.3.2, 3.4, and 4.7 were updated. The materials table was also updated.

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

Brustkrebs ist die am häufigsten auftretende Krebserkrankung bei Frauen in der ganzen Welt 1,2 und die zweithäufigste Ursache der durch Krebs verursachten Todesfälle 2. Komplexe genetische 3,4, 5 histologische und klinische Phänotypen 6 werden verwendet, um die verschiedenen Subtypen von Brustkrebs zu charakterisieren und werden oft als ein Mittel, um das Überleben vorherzusagen. Die Analyse einer großen Kohorte von Frauen mit Brustkrebs zeigte, dass die meisten (ca. 80%) der Patienten, die starben innerhalb von 10 yr Beitrag Entfernung des Primärtumors 7 wiedergekehrt war. Für die Mehrheit der invasiven Mammakarzinome hat lymphovascular Invasion gezeigt worden, um stark zu einem schlechten Ergebnis und aggressiveren klinischen Verlauf der Erkrankung korreliert werden 8. Aufgrund der genetischen und phänotypischen Komplexität von Brustkrebs, gibt es keine Tiermodell, das den gesamten Verlauf der Krankheit rekapituliert. Menschliche Muttertumorzelllinien wurden mehrals Xenotransplantat oder orthotopen 9 Modelle von invasiven und metastatischen Brustkrebs bei immungeschwächten Mäusen verwendet. Obwohl informative treten diese Modelle in Abwesenheit von Immun Druck und weil es eine Quer Arten Transplantat, verzerren die Wirkung des gesamten Tumor-Mikroumgebung. Induzierbare genetische Mutationen durch Brust spezifische Promotoren, wie Maus-Mammatumorvirus (MMTV) und Molke saures Protein (WAP) angetrieben haben eine enorme Menge an Wissen über die genetische Beschaffenheit von Brustkrebs beigetragen. Jedoch ist die gewebespezifische Expression dieser Promotoren durch ihre Reaktionsfähigkeit mit dem endokrinen System 10-16 beeinträchtigt, was zu einer variablen Expression der induzierten genetischen Mutationen, spiegeln nicht die Expression von Onkogenen in der Regel in menschlichem Brustkrebs überexprimiert. Um endokrine Kontrolle des MMTV gesteuerte Expression von Onkogenen zu überwinden, Moody et al. Erzeugt eine bedingte, Doxycyclin induzierbaren Überexpression Modell Neu in der BrustEpithel 17. Dieses Modell ist nützlich für deinducing Neu nach Tumorbildung zu Regression und Wiederholung zu studieren, aber erfordert ständige Doxycyclin Verwaltung für konsistente, langfristige Onkogenexpression. Eine umfassende Diskussion der vielen relevanten Brusttumor-Modelle in der Übersicht von Vargo-Gogola et al. 10 gefunden werden Unser Ziel war es, ein Mausmodell für Brustkrebs rückführbar auf Voll C57BL / 6 Hintergrund zu entwickeln, dass nach der ständigen Induktion der Mutationsereignisse, Modelle die Bildung einer entstehenden Tumor in der Anwesenheit von Immundruck. Wir führten ein Adenovirus Cre-Rekombinase in die Milchgänge von transgenen Mäusen enthält floxed Allele Tp53 und eine onkogene Form von K-ras und YFP. Cre-Expression Tp53 abträgt, eine häufig mutierte Gen in vielen Brustkrebs 18 und induziert eine onkogene Allel des K-ras neben YFP Ausdruck spezifischin der Brust duktalen Epithel. Obwohl Mutationen in K-ras sind selten bei Brustkrebs, in nur 6,5% der Brustkrebspatientinnen 19,20 auftritt, die Überexpression von stromaufwärtigen Kinasen wie Her2/neu und EGFR bewirken die konstitutive Aktivierung des Ras-Signalwegs in menschlichen Brusttumoren 21-23. Aktivierung des Ras-Signalwegs in vielen Brusttumorzelllinien wurde auch berichtet, 24,25. Wir werden den Beginn der Tumorbildung und die Technik der intraduktale Injektion eines Adenovirus Cre-Rekombinase in sexuell reifen, Jungfrau weiblichen Mäusen zu beschreiben. Dieses Modell von Brustkrebs entwickelt offene Läsionen, die exponentiell nach etwa 8 Wochen der langsamen Tumorprogression zu wachsen, mit lymphovascular Invasion und Metastasierung in die axillären Lymphknoten von 7-8 Wochen. Da diese Mäuse sind auf Voll C57BL / 6 Hintergrund und YFP-exprimierenden Tumorzellen sind rückführbar in distalen Lymphknoten, bietet dieses Modell eine entsprechende Werkzeug, um die ce studierenllular und immunologischen Mechanismen der Metastasierung latent und wird dazu beitragen, neue therapeutische Ansätze für die Behandlung von metastasierendem duktalen Brustkrebs zu entwickeln.

<table border="0" cellpadding="0" cellspacing="0" width="825">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:251px;">Trp53tm1Brn transgenic mice 
			Krastm4Tyj transgenic mice</td>
			<td style="width:145px;">Obtained from NCI mouse models of human cancer consortium</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">Mice were backcrossed ten times to a full C57BL/6 background</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:251px;">B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J Transgenic mice</td>
			<td style="width:145px;">Jackson labs</td>
			<td style="width:141px;">006148</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers p53loxp/loxp</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-AAGGGGTATGAGGGACAAGG-3&#39; 
			5&#39;-GAAGACAGAAAAGGGGAGGG-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers LSL-K-ras G12D/+</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-CGCAGACTGTAGAGCAGCG-3&#39; 
			5&#39;-CCATGGCTTGAGTAAGTCTGC-3&#39;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Primers for LSL-EYFP to detect Rosa promoter</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-AAGACCGCGAAGAGTTTGTC-3&#39; 
			5&#39;-GGAGCGGGAGAAATGGATATG-3&#39; 
			5&#39;-AAAGTCGCTCTGAGTTGTTAT-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Mesothelin expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-TTGGGTGGATACCACGTCTG-3&#39; 
			5&#39;-CGGAGTGTAATGTTCTTCTGTC-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Progesterone Receptor expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-GCAATGGAAGGGCAGCATAA-3&#39; 
			5&#39;-TGGCGGGACCAGTTGAATTT-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Cytokeratin 8 expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-ATCAGCTCTTCCAGCTTTTCCC-3&#39; 
			5&#39;-GAAGCGCACCTTGTCAATGAAGG-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Erbb2 expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-ACCTGCCCCTACAACTACCT-3&#39; 
			5&#39;-AAATGCCAGGCTCCCAAAGA-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Estrogen Receptor A expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-ATGAAAGGCGGCATACGGAA-3&#39; 
			5&#39;-GCGGTTCAGCATCCAACAAG-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Estrogen Receptor B expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-ACCCAATGTGCTAGTGAGCC-3&#39; 
			5&#39;-TGAGGACCTGTCCAGAACGA-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			B-Actin expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-GCCTTCCTTCTTGGGTATGG-3&#39; 
			5&#39;-CAGCTCAGTAACAGTCCGCC-3&#39;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Adenovirus-CRE</td>
			<td style="width:145px;">Gene Transfer Vector Core from the University of Iowa</td>
			<td style="width:141px;">Ad5CMVCre</td>
			<td style="width:289px;">Store aliquots of virus (4 x 108 pfu/aliquot) at -80C to avoid repeated freeze thaw cycles</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Adenovirus-mCherry</td>
			<td style="width:145px;">Gene Transfer Vector Core from the University of Iowa</td>
			<td style="width:141px;">Ad5CMVmCherry</td>
			<td style="width:289px;">Store aliquots of virus (4 x 108 pfu/aliquot) at -80C to avoid repeated freeze thaw cycles</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Hamilton syringe</td>
			<td style="width:145px;">Hamilton company</td>
			<td style="width:141px;">701RN</td>
			<td style="width:289px;">10ml syringe, RN series 
			Autoclave before and after each use. Clean with PBS and 75% ethanol.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Custom needle</td>
			<td style="width:145px;">Hamilton company</td>
			<td style="width:141px;">7803-05</td>
			<td style="width:289px;">33 gauge 0.5 inch long RN needle, with a 12 degree bevel. Autoclave before and after each use. Clean with PBS and 75% ethanol.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Surgical forceps</td>
			<td style="width:145px;">Dumont</td>
			<td style="width:141px;">52100-58</td>
			<td style="width:289px;">Dumostar No5 forceps. Clean with 75% ethanol after each use, followed by autoclaving</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">MEM powder</td>
			<td style="width:145px;">Cellgro</td>
			<td style="width:141px;">50 012 PB</td>
			<td style="width:289px;">Store at 4C in powder and reconstituted form</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">Sodium Bicarbonate</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:141px;">S233</td>
			<td style="width:289px;">Add to MEM and filter stearilize</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">Calcium Chloride</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:141px;">C4901</td>
			<td style="width:289px;">Minimum 96%, anhydrous</td>
		</tr>
	</tbody>
</table>

initiation of metastatic breast carcinoma by targeting of the ductal epithelium with adenovirus-cre: a novel transgenic mouse model of breast cancer

Brustkrebs ist eine heterogene Erkrankung, die komplexen zellulären Interaktionen zwischen der Entwicklungs Tumor und Immunsystems, was schließlich in der exponentiellen Tumorwachstum und Metastasen in Geweben distalen und dem Zusammenbruch der Anti-Tumor-Immunität. Viele nützliche Tiermodelle existieren, um Brustkrebs zu studieren, aber keine, die das Fortschreiten der Krankheit beim Menschen auftritt vollständig rekapitulieren. Um ein besseres Verständnis der zellulären Interaktionen, die in der Bildung von latenten Metastasierung und verminderter Überleben zu gewinnen, haben wir einen induzierbaren transgenen Mausmodell von YFP-exprimierenden duktales Karzinom, die nach der Geschlechtsreife entwickelt in immunkompetenten Mäusen und wird angetrieben wird durch konsequente, endokrine unabhängige Onkogenexpression. Die Aktivierung von YFP, Ablation von p53, und Ausdruck einer onkogenen Form von K-ras wurde von der Lieferung eines Adenovirus Cre-Rekombinase in der Milchkanal von sexuell reifen, rein weiblichen Mäusen erreicht. Tumorenbeginnen zu 6 Wochen nach der Initiierung der onkogenen Ereignisse angezeigt. Nach Tumoren deutlich wird, Fortschritte sie sich langsam für etwa zwei Wochen, bevor sie, exponentiell zu wachsen beginnen. Nach 7-8 Wochen nach der Adenovirus-Injektion wird Gefäßsystem beobachtet Anschluss der Tumormasse zu distalen Lymphknoten, mit eventuellen lymphovascular Invasion von YFP + Tumorzellen mit den distalen axillären Lymphknoten. Infiltrierenden Leukozyten-Populationen sind ähnlich zu denjenigen in der menschlichen Brustkarzinome, einschließlich der Anwesenheit von αβ und γδ T-Zellen, Makrophagen und MDSCs gefunden. Dieses einzigartige Modell wird die Untersuchung der zellulären und immunologischen Mechanismen in latenter Metastasen und Ruhe zusätzlich zu nützlich für die Entwicklung neuer immun invasive Interventionen zur Behandlung von Brustkrebs beteiligt sind, erleichtern.

Successful targeting of the mammary ductal tree can be visualized by preparing whole mounts of the mammary gland as previously described32 after injection of trypan blue (to verify proper injection technique (Figure 1A) or an adenovirus expressing mCherry (to verify proper viral preparation and infection of ductal epithelial cells, Figure 1B).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig1highres.jpg" src="/files/ftp_upload/51171/51171fig1.jpg" /> 
Figure 1. Intraductal targeting of mammary glands by injection with trypan blue or adenovirus-mCherry. A) A whole mount, as previously reported32, of the mammary gland after injection of mammary gland #4 with trypan blue was prepared 3 hr post injection to visualize/confirm targeting of the entire ductal tree. Images are 4X magnification. B) Infection of the ductal epithelium with adenovirus by injecting 2.5 x 107 pfu of adenovirus expressing mCherry. Mice were injected intraductally, and 4 days post-injection, a whole mount of the mammary gland was prepared to confirm viral infection of the ductal tree. Image is 4X&#160;magnification. MG is mammary gland, LD is the lactiferous, or main duct, and TD is the terminal duct. <a href="https://www.jove.com/files/ftp_upload/51171/51171fig1highres.jpg" target="_blank">Click here to view larger image.</a>
When tumors are induced in p53loxP/loxP LSL-K-rasG12D/+ transgenic mice, tumors will not be apparent until around day 40 when the mammary glands will become enlarged and swollen. It is necessary to begin palpations when this is observed, monitoring tumor growth every 5-7 days. In our hands, hardening of the mammary gland always precedes the onset of tumor development. Tumors will progress slowly for an additional 2 weeks. Beginning around day 56, tumors will begin to grow exponentially (Figure 2B). At this point, it is critical to measure tumor volumes every 3 days if kinetic studies are desired because there will be slight mouse to mouse variability in tumor progression, which is normal (Figures 2A&#160;and&#160;3A). Large abdominal masses will be apparent by day 80 (Figure 2A, 2B, and 3A), after which mice should be euthanized if tumors exceed more than 10% of their body weight. cDNA analysis of three clones from a tumor-bearing mouse revealed expression of mesothelin, cytokeratin-8, Her2/neu, and estrogen receptor-&#945; (Figure 2C).
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig2highres.jpg" src="/files/ftp_upload/51171/51171fig2.jpg" /> 
Figure 2. Tumor development in p53loxP/loxP LSL-K-rasG12D/+ mice injected intraductally with adenovirus-Cre. A) Two examples of tumors 80 days after injection with adenovirus expressing Cre. Mice were given 2.5 x 107 PFU of adenovirus expressing Cre and 80 days post tumor-initiation, large palpable masses can be visualized protruding from the abdominal side of the animal. B) Typical tumor kinetics and palpation schedule for tumors induced in p53loxP/loxP LSL-K-rasG12D/+ mice. C) Characterization of three tumor cell clones derived from the same homogenized tumor of a p53loxP/loxP LSL-K-rasG12D/+ mouse. RNA was extracted and cDNA synthesized for RT-PCR analysis using primers specific to &#946;-actin, mesothelin, cytokeratin-8, Her2/neu, progesterone receptor, estrogen receptor-&#945;, and estrogen receptor-&#946;. <a href="https://www.jove.com/files/ftp_upload/51171/51171fig2highres.jpg" target="_blank">Click here to view larger image.</a>
Similar to the cellular microenvironment in human breast cancer, we have observed infiltration of &#945;&#946; and &#947;&#948; T cells as well as myeloid derived suppressor cells and macrophages into the tumors (Figure 4). Vasculature draining to the axillary lymph node will begin to engorge before tumors have grown to encompass the entire mammary tissue where the injection was performed (Figure 3A). Lymphovascular invasion and metastasis of tumor cells can be tracked by crossing LSL-K-rasG12D/+ p53loxP/loxP mice with LSL-EYFP mice. After Cre-mediated excision, tumor cells expressing YFP (both high and low) are detected in the tumor and can be traced metastasizing to the draining axillary lymph node (Figure 3B). Metastasis to the distal axillary lymph node was confirmed by successfully culturing a tumor cell line from this site in a tumor-bearing LSL-K-rasG12D/+ p53loxP/loxP mouse (data not shown).
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig3highres.jpg" src="/files/ftp_upload/51171/51171fig3.jpg" /> 
Figure 3. Formation of tumors and latent metastasis to the axillary lymph node. A) Example of three advanced breast tumors with different rates of tumor progression. A solid mass, indicated by the arrowhead, forms and eventually grows to the size of the entire abdominal mammary tissue. The tumor stays confined to the mammary tissue and is not observed to invade or attach to the muscle covering the peritoneal cavity. There is evident engorgement of the superficial epigastric vein between the inguinal and axillary lymph nodes, denoted by white arrowheads. After 7-8 weeks, the axillary lymph node begins to become enlarged due to lymphovascular invasion of the tumor cells, indicated by arrow. B) Metastasis of YFP positive tumor cells can be visualized in the axillary lymph node by flow cytometry. LSL-K-rasG12D/+ p53loxP/loxP LSL-EYFP mice were used to induce tumors and activation of YFP by intraductal delivery of adenovirus-Cre. To verify lymphovascular invasion of tumor cells into the axillary lymph node, 80 days post adenoviral injection, the indicated lymph nodes and organs were harvested and stained for lymphocyte markers and examined for YFP expression. CL represents contralateral nontumor draining lymph node. Results represent gating on CD45 negative tumor cells, indicating the tumor cells are invading the distal axillary lymph node. Numbers represent percent YFP positive cells from total population. <a href="https://www.jove.com/files/ftp_upload/51171/51171fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig4highres.jpg" src="/files/ftp_upload/51171/51171fig4.jpg" /> 
Figure 4. Immune infiltrates in mouse transgenic breast tumors. Mouse breast tumors were homogenized and stained for CD45, CD3, &#947;&#948;TCR, CD11b and GR1. Numbers represent percent of positive leukocytes in entire tumor (63.5), total CD3+ (46.7), total CD3 negative (40.1), total CD3+ &#947;&#948;+ (&#947;&#948; T cells, 13), CD3+ &#947;&#948;negative (24), total GR1 high CD11b (MDSC, 28.6) and total CD11b GR1 low (macrophages, 18.5). <a href="https://www.jove.com/files/ftp_upload/51171/51171fig4highres.jpg" target="_blank">Click here to view larger image.</a>
Due to the anatomy of the mouse and technique of intraductal injections, we find targeting of mammary glands 4 and 9 (Figure 5) yields the most consistent results and reliable injections. However, any gland can be targeted depending upon the preference of the technician performing the surgery.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig5highres.jpg" src="/files/ftp_upload/51171/51171fig5.jpg" /> 
Figure 5. Numbering of mammary ducts. Mammary ducts 4 and 9 are highlighted in red on the diagram and indicated with arrows on the mouse. In our hands, we found that injections were easiest to perform on these mammary ducts, however all other mammary tissues that we targeted developed tumors with similar kinetics. <a href="https://www.jove.com/files/ftp_upload/51171/51171fig5highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer at JoVE.c

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

Initiation Metastasierende Mammakarzinom durch Targeting des duktalen Epithel mit Adenovirus-Cre: Eine neue transgene Maus-Modell der Brustkrebs

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

initiation-metastatic-breast-carcinoma-targeting-ductal-epithelium

Research

JoVE Journal

Medicine

23.1K Aufrufe.  Wistar Institute. 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.

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

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.

Ein In Vitro System zur Tumor Vegetationsruhe und die Umstellung auf Metastasen Study

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 ...

Forschung

Medizin

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.

In-vivo-Biolumineszenz-Bildgebung von Tumorhypoxie Dynamics of Breast Cancer Hirnmetastasen in einem Mausmodell

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

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.

Live-Imaging of Drug Responses in der Tumor-Mikroumgebung in Mausmodellen von Brustkrebs

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

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Ultraschall-Imaging-guided Intra Injection zur Entwicklung eines Mausmodells von Brustkrebs Hirnmetastasen durch Längs MRI Gefolgt

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantifizierung der Brustkrebs-Zelle Invasivität Mit einem Drei-dimensionale (3D) Modell

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, 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.

Ein<em&gt; Ex-vivo-</em&gt; Modell um die Hormonwirkung in der menschlichen Brust Studieren

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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

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

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

Verfahren zur Kultivierung humaner Femur Gewebeexplantaten an Brustkrebs Zellbesiedlung des metastasierten Niche Studieren

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

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

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

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 und ex vivo Ansätze für Eierstockkrebs Metastatische Colonization von Milky Punktstrukturen in Peritoneal Fettgewebe Studieren

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

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Nutzung der Koloniebildung in Weichagar-Assay auf Inhibitoren der Tumorigenität in Brustkrebs-Zellen zu identifizieren

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

Systembiologie der Stoffwechselregulation von Estrogen Receptor Signaling bei Brustkrebs

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...