Diese Arbeit wurde durch die NIH P01 AI 068730 (SNC, NS), der NIH CA016520 / TAPITMAT (NS), die private Finanzierung durch die Claneil Foundation (NS), und die Eierstöcke SPORE zu gewähren FCCC und der University of Pennsylvania unterstützt ( P50 CA83638) und der Fox Chase Cancer Center-Core Grant (P30 CA06927) (DCC). Die Autoren bedanken sich die hervorragende technische Unterstützung der Optical / Biolumineszenz Core Facility von Dr. EJ Delikatny an der University of Pennsylvania, Anthony Secreto vom Stem Cell und Xenograft-Core von Dr. G. Danet-Desnoyers an der University of Pennsylvania Cancer gerichteten Center for Training SNC orthotopen Injektionstechnik und Denada Dangaj an der University of Pennsylvania / OCRC für die Unterstützung der Operation.

1. Cell Culture <ol><li> Vor orthotoper Injektion, Kultur MOV1 Kat-Zellen, aus DR26 Tumoren stammen, in einem T175 Flasche, bis sie 90% konfluent. Planen Sie 1 bis 5.000.000 Zellen pro Injektion, die 1 oder 2 T175 Flaschen erfordern verwenden. </li><li> Am Tag der Injektion Ernte der Zellen und Bestimmung der Zellzahl mit einer Zählkammer. </li><li> Sobald die Zellkonzentration bestimmt worden ist, Pellet die Zellen durch Zentrifugation für 5 Minuten bei 300 X g bei Raumtemperatur. </li><li> Nach dem Spin, resuspendieren zu 1 Million in 10 Mikroliter steriles PBS wurden mit 0,002 M EDTA </li></ol> 2. Pre-Chirurgie <ol><li> Vor der Operation, füllen Sie ein 3/10cc Insulinspritze mit 1 Million MOV1-Kat-Zellen in 10 Mikroliter PBS-EDTA. </li><li> Übertragen eines Isofluoran narkotisierten Maus ein Heizkissen. Add Augensalbe in Auge Austrocknung zu verhindern. Dann fügt unmittelbar den Kopf des Tieres in einem Nasenkonus System verbunden mit einem Isofluran Verdampfer auf die Anästhesie während der Operation zu liefern. </li><li> Nach Desinfektion der Einstichstelle mit Alkohol-Tupfer, subkutan injizieren 5 mg / kg Ketoprofen, eine präoperative Analgetikum mit einem 3/10cc Insulin Spritze. </li><li> Mit Klipper, rasieren die linke kaudale Teil des Rückens aus dem thorakolumbalen Übergang auf der Basis des Tieres Schwanz. Bewerben Enthaarungscreme vollständig zu entfernen die Haare. Dann entfernen Sie den Überschuss mit einem feuchten Papiertuch. </li><li> Sobald das Haar entfernt worden ist, sterilisieren die rasierte Fläche mit PVP-Jod und Alkoholtupfer. Legen Sie dann ein chirurgisches Abdecktuch rund um den Bereich des Einschnitts. </li></ol> 3. Chirurgie <ol><li> Kurz vor der Operation, passen Sie die Isofluran-Verdampfer Ebenen auf 1,5%. </li><li> Stellen Sie sicher, dass das Tier völlig betäubt durch Einklemmen des Fußes Pad ist. </li><li> Als nächstes suchen Sie die Milz unter die Haut. Dann mit chirurgischer Schere, einen dorsolateralen Schnitt 1-2 cm lange auf der oberen rechten Ecke der Milz. </li><li> Präparieren Sie die Retroperitoneum. Das Pad rund um die Maus Ovar beobachtet wird. Verwenden einer gebogenen Pinzette zu fassen und setzen die Fettpolster rund um die Maus Eierstock. </li><li> Hydrate der Orgel mit einigen Tropfen sterile PBS. </li><li> Verwenden einer gebogenen Pinzette zu fassen, zurückzunehmen ... Position ... und dann sichern den Eierstöcken zur Injektion </li><li> Während fest Greifen des Eierstocks mit der Zange, injizieren 10 Mikroliter MOV1 Kat Tumorzellen in den Eierstöcken. Ein fester Griff verhindert Flüssigkeit Aufstoßen oder Leckage. </li><li> Unmittelbar nach der Injektion, lassen Sie die Spannung, die durch die Zange ausgeübt wird. Die Perforation in den Eierstöcken sollte spontan zurückzuziehen und zu schließen. </li><li> Mit einem resorbierbaren Polyglykolsäure Naht an einer Nadel, in der Nähe der Retro-Peritoneum Wunde. </li><li> Lassen Sie die Tiere aus der Nase Kegel. </li><li> Ziehen Sie die Haut und dichten die dorsolateralen Wundränder mit einigen Tropfen Gewebekleber. </li><li> Schließlich oral zu verabreichen 100 Mikroliter von Antibiotika, um das Tier. Dann setzen Sie ihn wieder in seinem Käfig und Monitor für Erholung. Halten Sie das Tier auf Antibiotika im Trinkwasser für eine Woche. </li></ol> 4. In-vivo-Imaging <ol><li> Eine Woche nach der orthotopen Injektion von MOV1 Kat Tumorzellen, in-vivo-Bildgebung durchzuführen. Beginnen Sie mit der Übertragung eines Isofluoran narkotisierten Maus, um die Imaging-Kammer. </li><li> Schalten Sie die Isofluran Verdampfer Ebene bis hinunter zu 2%. </li><li> Führen Sie in-vivo-Bildgebung nach dem Imaging-System des Herstellers. In diesem Video werden die Lumina System verwendet werden. </li><li> Um das Bild, klicken Sie auf das Living Image Software Desktop-Symbol. Dann, am IVIS Acquisition Control Panel, wählen Sie &quot;Initialize&quot;. Die Geräteeinstellungen sind analog zu einem Kamera-Einstellungen </li><li> Auf den Erwerb Control Panel, das Instrument des Erwerbs Parameter. Für Fluoreszenz-Check &quot;Fluorescent&quot;. Klicken Sie auf das Foto-Box, um ein Foto mit jedem Bild zu erwerben. </li><li> Als nächstes legen Sie die Belichtungszeit Auto. Unter &quot;Pixel Binning-oder CCD-Auflösung&quot; aktivieren &quot;Medium&quot;. Dann unter F / Stop oder Blende, prüfen Sie den Wert von 2. Als nächstes wählen Sie den 535 Anregungsfilter und DsRed Emissionsfilter. </li><li> Unter Sehfeld auf B klicken, um Bild einem einzigen Mausklick. </li><li> Dann, am erwerben, um die Bildaufnahme zu beginnen. </li><li> Sobald das Bild Akquisition abgeschlossen ist, verwenden Sie die Region of Interest, oder ROI, Werkzeug des Signals messen. Klicken Sie auf die Messung Symbol, um das Signal-Werte und Umgebung veröffentlichen. </li><li> Schließlich auf &quot;Speichern&quot; klicken, um das Bild in den Benutzer-Ordner speichern </li><li> Nachdem die Bilder gespeichert wurden, stoppen Sie die Gabe von Isofluran und kehren Sie die Maus auf seinem Käfig. Die Maus sollte sofort aufwachen. </li></ol> 5. Repräsentative Ergebnisse -Mit diesem ProtokollKann das in vivo Wachstum von einem orthotopen Ovarialkarzinom für mindestens 3 Wochen mit einem nicht-invasive Verfahren überwacht werden. <img src="/files/ftp_upload/2146/2146fig1.jpg" alt="Abbildung 1" /> Abbildung 1. MOV1 Kat Zellen oder PBS als negative Kontrolle wurden orthotop in den Eierstock von Nicht-Tumor anfällig Mäusen (rechts und links Tier Tier-und bzw.) injiziert. In-vivo-Bildgebung wurde 2 Wochen später durchgeführt. Die Fluoreszenzemission von MOV1 Kat Zellen im Eierstock eingepflanzt generiert wurde gemessen und verglichen mit der von der negativen Kontroll-Maus. <img src="/files/ftp_upload/2146/2146fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Gefrierschnitte von MOV1 Kat Ovarialtumoren wurden mit biotinylierten anti-CD4 mAb von DAB-Substrat (dunkelbraun) für Tumor-infiltrierenden Lymphozyten (schwarzer Pfeil) zu erkennen gefolgt gefärbt. Die Zellen wurden mit Methyl-grün gegengefärbt, um Zellkerne (blau) zu visualisieren. Tumor-Zellen (rote Pfeile) wurden morphologisch unterscheidet sich von T-Zellen. Die Folie wird vergrößert 40x.

<ol>
	<li>Etzioni, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+case+for+early+detection.">The case for early detection.</a> Nat Rev Cancer. 3, 243-252 (2003).</li><li>Sasaroli, D., Coukos, G., Scholler, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+CA125:+the+coming+of+age+of+ovarian+cancer+biomarkers.+Are+we+there+yet.">Beyond CA125: the coming of age of ovarian cancer biomarkers. Are we there yet.</a> Future Medicine. 3, 275-288 (2009).</li><li>Anderson, G. L., 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=Assessing+Lead+Time+of+Selected+Ovarian+Cancer+Biomarkers:+A+Nested+Case+Control+Study.">Assessing Lead Time of Selected Ovarian Cancer Biomarkers: A Nested Case Control Study.</a> J Natl Cancer Inst. 102, 26-38 (2010).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocr Rev 22. , 255-288 (2001).</li><li>Dubeau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+of+origin+of+ovarian+epithelial+tumours.">The cell of origin of ovarian epithelial tumours.</a> Lancet Oncol. 9, 1191-1197 (2008).</li><li>Connolly, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Female+mice+chimeric+for+expression+of+the+simian+virus+40+TAg+under+control+of+the+MISIIR+promoter+develop+epithelial+ovarian+cancer.">Female mice chimeric for expression of the simian virus 40 TAg under control of the MISIIR promoter develop epithelial ovarian cancer.</a> Cancer Res. 63, 1389-1397 (2003).</li><li>Hensley, H., 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=Magnetic+resonance+imaging+for+detection+and+determination+of+tumor+volume+in+a+genetically+engineered+mouse+model+of+ovarian+cancer.">Magnetic resonance imaging for detection and determination of tumor volume in a genetically engineered mouse model of ovarian cancer.</a> Cancer Biol Ther. 6, (2007).</li><li>Deliolanis, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+the+red-shifted+fluorescent+proteins+in+deep-tissue+molecular+imaging+applications.">Performance of the red-shifted fluorescent proteins in deep-tissue molecular imaging applications.</a> J Biomed Opt. 13, (2008).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+better+fluorescent+protein+for+whole-body+imaging.">A better fluorescent protein for whole-body imaging.</a> Trends Biotechnol. 26, (2008).</li><li>Shcherbo, D., 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=far-red+fluorescent+protein+for+whole-body+imaging.">far-red fluorescent protein for whole-body imaging.</a> Nat Methods. 4, 741-746 (2007).</li><li>Fu, X., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ovarian+carcinoma+metastatic+models+constructed+in+nude+mice+by+orthotopic+transplantation+of+histologically-intact+patient+specimens.">Human ovarian carcinoma metastatic models constructed in nude mice by orthotopic transplantation of histologically-intact patient specimens.</a> Anticancer Res. 13, 283-286 (1993).</li><li>Kiguchi, K., 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=A+patient-like+orthotopic+implantation+nude+mouse+model+of+highly+metastatic+human+ovarian+cancer.">A patient-like orthotopic implantation nude mouse model of highly metastatic human ovarian cancer.</a> Clinical &amp; experimental metastasis 16. , 751-756 (1998).</li><li>Bao, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+cancer-specific+gene+expression+by+the+survivin+promoter.">Activation of cancer-specific gene expression by the survivin promoter.</a> J Natl Cancer Inst. 94, 522-528 (2002).</li><li>Connolly, D. 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Current Protocols in Pharmacology 45.</a> , 1-36 .</li><li>Milne, K., 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=Systematic+analysis+of+immune+infiltrates+in+high-grade+serous+ovarian+cancer+reveals+CD20,+FoxP3+and+TIA-1+as+positive+prognostic+factors.">Systematic analysis of immune infiltrates in high-grade serous ovarian cancer reveals CD20, FoxP3 and TIA-1 as positive prognostic factors.</a> , (2009).</li></ol>

Chirurgie und orthotopen Injektionen Orthotoper Injektion in Eierstock Bursa Anforderungen Ausbildung und Präzision. So <ol><li> Im Falle einer schlechten chirurgischen Erfahrung, Praxis mit Leichen zuerst. </li><li> Verwenden Sie bevorzugt multipare Frauen (ein oder zwei Würfe), da sie größere Eierstöcke im Laufe der Zeit die Injektion und die Überlebenszeit ermöglicht mit nulliparen Frauen vergleichen zu entwickeln. </li><li> Aufgrund der geringen Größe der Maus Eierstock Bursa,...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM-GLUTAMAX</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10564-011</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14040</td>
<td>&nbsp;</td>
<tr>
<td>Versene</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-711E</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Deltaphase</td>
<td>39 DP</td>
<td>&nbsp;</td>
<tr>
<td>Povidone pads</td>
<td>&nbsp;</td>
<td>Dynarex</td>
<td>1108</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol pads</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>06-669-62</td>
<td>&nbsp;</td>
<tr>
<td>Artificial tears ointment </td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>17845-153</td>
<td>&nbsp;</td>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Fort Dodge laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3cc/insulin syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309301</td>
<td>&nbsp;</td>
<tr>
<td>Polyg Polyglycolic Acid suture/needle (3/8 19mm)</td>
<td>&nbsp;</td>
<td>Syneture</td>
<td>9612-31</td>
<td>&nbsp;</td>
<tr>
<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>Vetbond</td>
<td>3M</td>
<td>&nbsp;</td>
<tr>
<td>Vet Bactrim/ oral suspension</td>
<td>&nbsp;</td>
<td>Hi-tech Pharmacal</td>
<td>840823</td>
<td>&nbsp;</td>
<tr>
<td>IVIS-Lumina</td>
<td>&nbsp;</td>
<td>Caliper lifesciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isofluorane</td>
<td>&nbsp;</td>
<td>Phoenix Pharma., Inc.</td>
<td>J108013</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum, Qualified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10437036</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>TurboFP635 mammalian vector</td>
<td>&nbsp;</td>
<td>Evrogen</td>
<td>FP721</td>
<td>&nbsp;</td>
<tr>
<td>T175 flasks</td>
<td>&nbsp;</td>
<td>cellstar</td>
<td>660-190</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an orthotopic model of serous ovarian cancer in immunocompetent mice for in vivo tumor imaging and monitoring of tumor immune responses

Hintergrund: Das Ovarialkarzinom ist in der Regel in einem fortgeschrittenen Stadium, wo der Fall / Todesfall Verhältnis hoch diagnostiziert und bleibt somit die tödlichste aller gynäkologischen Malignomen unter US-Frauen 1,2,3. Serösen Tumoren sind die häufigsten Formen von Eierstockkrebs und 4,5 der Tg-MISIIR-Tag transgenen stellt die einzige Maus-Modell, das sich spontan entwickelt diese Art von Tumoren. Tg-MISIIR-Tag Mäuse exprimieren SV40 transformiert Region unter die Kontrolle des Müllerschen Hemmstoff Typ-II-Rezeptor (MISIIR)-Gen-Promotor 6. Zusätzliche transgenen Linien wurden die Ausdruck der SV40 TAg Transgen identifiziert, aber nicht entwickeln Ovarialtumoren. Non-Tumor anfällig Mäuse zeigen typische Lebensdauer für C57Bl / 6 Mäusen und sind fruchtbar. Diese Mäuse können als syngenen Transplantat-Empfänger für Tumorzellen von Tg-MISIIR-Tag-DR26-Mäusen isoliert eingesetzt werden. Ziel: Obwohl Tumorimaging ist möglich, 7, Früherkennung von Tumoren tief in kleinen lebenden Tieren schwierig. Um präklinischen Studien in einer immunologisch intakten Tiermodell für seröse Ovarialkarzinom beschreiben wir eine syngenen Mausmodell für diese Art von Eierstock-Krebs, der in-vivo-Bildgebung, Studien über die Tumor-Mikroumgebung des Tumors Immunantwort ermöglicht. Methoden: Wir erste abgeleitete einem TAG + Maus-Zelllinie (MOV1) aus einem spontanen Ovarialtumor in einem 26 Wochen alten DR26 Tg-MISIIR-Tag weiblichen geerntet. Dann haben wir stabil transduzierten MOV1 Zellen mit TurboFP635 Lentivirus Säugetieren Vektor, Katuschka kodiert, eine weit-rot-Mutante der rot fluoreszierendes Protein aus Seeanemone Entacmaea quadricolor mit Anregung / Emissionsmaxima bei 588/635 nm 8,9,10. Wir orthotop implantiert MOV1 Kat in den Eierstöcken 11,12,13,14 von Nicht-Tumor anfällig Tg-MISIIR-Tag weiblichen Mäusen. Tumorprogression wurde in vivo optische Bildgebung und Tumor-Mikroumgebung wurde mittels Immunhistochemie analysiert gefolgt. Ergebnisse: orthotop implantiert MOV1 Kat-Zellen entwickelt serösen Ovarialtumoren. MOV1 Kat Tumoren konnten visualisiert werden in-vivo-Bildgebung bis zu drei Wochen nach der Implantation (Abb. 1) und wurden mit Leukozyten infiltriert, wie in menschlichen Eierstockkrebs 15 (Abb. 2) beobachtet. Schlussfolgerungen: Wir beschreiben einen orthotopen Modell des Ovarialkarzinoms geeignet für in-vivo-Bildgebung des frühen Tumoren aufgrund des hohen pH-Stabilität und Photostabilität Katuschka in tiefen Gewebeschichten. Wir schlagen vor, den Einsatz dieser neuartigen syngenen Modell seröser Eierstockkrebs für die in vivo Bildgebung und Überwachung der Tumor Immunantwort und Immuntherapien.

Watch this Scientific Journal Video about An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses at JoVE.com

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Zur Untersuchung<em&gt; In vivo</em&gt; Tumorwachstum und Tumor-Mikroumgebung, verwendeten wir eine syngenen und orthotopen Mausmodell des Ovarialkarzinoms in immunkompetenten Tieren. Wir transduzierten Maus Tumor-Zelllinie (MOV1) mit Katuschka fluoreszierende Protein (MOV1<sup&gt; KAT</sup&gt;) Und hier zeigen wir seine orthotopen Implantation in Ovar und<em&gt; In vivo</em&gt; Bildgebung.

Einem orthotopen Modell des seröse Ovarialkarzinom in immunkompetenten Mäusen für In vivo Tumor Imaging und Überwachung der Tumor Immunantworten

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

an-orthotopic-model-serous-ovarian-cancer-immunocompetent-mice-for

Research

JoVE Journal

Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

18.1K Aufrufe.  University of Pennsylvania-School of Medicine. Zur Untersuchung In vivo Tumorwachstum und Tumor-Mikroumgebung, verwendeten wir eine syngenen und orthotopen Mausmodell des Ovarialkarzinoms in immunkompetenten Tieren. Wir transduzierten Maus Tumor-Zelllinie (MOV1) mit Katuschka fluoreszierende Protein (MOV1 KAT) Und hier zeigen wir seine orthotopen Implantation in Ovar und In vivo Bildgebung.

Serie: An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Ein<em&gt; In-vitro-</em&gt; Modell zur Messung der Immunantwort auf Malaria im Kontext der HIV-Koinfektion

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Forschung

Immunologie und Infektion

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Ein IL-8 transientes transgenisiertes Mausmodell für die<em&gt; In vivo</em&gt; Langzeitüberwachung von entzündlichen Reaktionen

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolierung von Salmonella Typhimurium-mit Phagosom von Makrophagen

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Schneider In Vivo Cytotoxicity Assays zur Untersuchung von Immunodominanz in modularspezifischen CD8+ T Cell-Reaktionen

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Implantation und Überwachung eines orthotopischen Modells des menschlichen Pleuralmesothelioms bei athymischen Mäusen durch PET/CT

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

Visualisierung, Quantifizierung und Kartierung von Immunzellpopulationen in der Tumormikroumgebung

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

Präzise Gehirnkartierung zur Durchführung von Repetitiven In-Vivo-Bildgebungen der Neuro-Immun-Dynamik bei Mäusen

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Mikrofluidische Co-Kulturmodelle zur Analyse der Immunantwort in in vitro Tumormikroumgebungen

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Rezidivierende Escherichia coli Harnwegsinfektion, ausgelöst durch Gardnerella vaginalis Blasenexposition bei Mäusen

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.