Name: an oncogenic hepatocyte-induced orthotopic mouse model of hepatocellular cancer arising in the setting of hepatic inflammation and fibrosis
Uploaded: 2019-09-12T13:00:00
Description: Watch this Scientific Journal Video about An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis at JoVE.com

Diese Arbeit wird unterstützt von NIH/NCI R01 CA164335-01A1 (K. F. Staveley-O'Carroll, PI) und NIH/NCI R01CA208396 (Mark Kester, Guangfu Li, Kevin F. Staveley-O'Carroll).

HINWEIS: Das gesamte Verfahren einschließlich tierischer Probanden wurde vom IACUC an der University of Missouri genehmigt. Alle Mäuse wurden nach den Kriterien des "Leitfadens für die Pflege und Verwendung von Labortieren" human gepflegt. Das folgende Verfahren zur Zellisolierung und -impfung sollte in einer Kapuze durchgeführt werden. Alle Darsteller sollten die standardmäßige persönliche Schutzausrüstung für den Umgang mit Mäusen und Gewebe tragen.
1. Induktion von Leberfibrose und ...

<ol>
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Mit diesem Protokoll haben wir ein zuverlässiges und reproduzierbares murines Modell von HCC etabliert, das die menschliche HCC-Initiierung und Progression imitiert. Klinisch, viele Risikofaktoren sukzessive induzieren Leberschäden, Leberfibrose, Zirrhose und die endk.-Phase von HCC. In unserem Protokoll wird die IP-Injektion von CCl4 verwendet, um zuerst Leberfibrose bei Wildtypmäusen zu produzieren, was es den nachfolgenden onkogenen Hepatozyten ermöglicht, die Tumoren bei der Einstellung von Leberfibros...

Hepatozellulärer Krebs (HCC) ist die am schnellsten zunehmende Krebsart in den Vereinigten Staaten (US)1,2,3. Jedes Jahr werden ca. 850.000 neue Fälle diagnostiziert4,5 und 700.000 Patienten sterben an dieser tödlichen Krankheit6,7,8,9,10 und ist damit weltweit die zweithäufigste Todesursache im Zusammenhang mit Krebs. Die Verwaltung von HCC umfasst chirurgische Resektion, Transplantation, Ablation, Chemoembolisierung oder systemische Therapien, wie Sorafenib11. Früherkennung und Management mit chirurgischer Resektion oder Transplantation haben den höchsten Gesamtüberlebensvorteil4. Leider, die Mehrheit der Patienten in einem späteren Stadium anwesend und erfordern Management mit Ablation, Chemoembolisierung oder Sorafenib12. Sorafenib, ein Rezeptor-Tyrosinkinase-Inhibitor (RTKI), wurde von der Food and Drug Administration im Jahr 2008 als einzige systemische medikamentöse Therapie zur Behandlung von nicht resezierbaren HCC zugelassen. Obwohl das Medikament nur eine bescheidene Erhöhung des Gesamtüberlebens bietet, von 7,9 auf 10,7 Monate13, es bot eine neue therapeutische Strategie, die verwendet werden könnte, um HCC zu verwalten.
Die Manipulation des Immunsystems zur Beseitigung etablierter Krebsarten ist ein schnell wachsendes Feld in der Krebsforschung14. Immun-Checkpoint-Studien haben erheblich fortgeschrittene immuntherapeutische Arzneimittelentwicklung in der Krebsbehandlung15,16. Die FDA genehmigte die Verwendung von Antikörpern (Abs) gegen zytotoxisches T-Lymphozyten-Antigen 4 (CTLA-4), programmiertes Zelltodprotein 1 (PD-1) und seinen Liganden PD-L1 zur Behandlung von Melanom, Lungenkrebs, Kopf- und Nackenkrebs und Blasenkrebs17, 18 , 19 , 20. Klinische Studien zur Monotherapie oder Kombinationstherapie mit einem oder mehreren Antikörpern gegen PD-1, PD-L1 oder CTLA-4 zur Behandlung fortgeschrittener HCC sind im Gange21,22,23und einige Studien haben positive Ergebnisse gezeigt. Im Jahr 2017 erteilte die FDA eine beschleunigte Zulassung für Anti-PD-1-Antikörper zur Behandlung von HCC-Patienten, die gegen Sorafenib resistenzen, aber die Gesamtansprechrate dieser Therapie beträgt nur 14,3%. Andere Strategien wurden zu diesem Zeitpunkt nicht in die klinische Praxis übersetzt24,25. Überwindung der tumorinduzierten tiefen Immuntoleranz zur Verbesserung der Immun-Checkpoint-Therapie26; Vorhersage der Wirksamkeit der Immun-Checkpoint-Therapie; Immunbedingte Nebenwirkungen zu verhindern; Optimierung der Verabreichung S-Route, Dosierung, und Häufigkeit; und die Suche nach effektiven Kombinationen der Therapien27,28,29 bleiben alle extrem herausfordernde Aufgaben.
Es gibt mehrere konventionelle Ansätze, die verwendet werden, um HCC in Mausmodellen zu induzieren und werden in Abhängigkeit von der speziellen Forschungsfrage des Forschersverwendet 30. Chemisch induzierte HCC-Mausmodelle mit genotoxischen Verbindungen imitieren verletzungsinduzierte Malignität. Xenograft-Modelle durch ektopische oder orthotopische Implantation von HCC-Zelllinien eignen sich für das Arzneimittelscreening. Eine Reihe von genetisch veränderten Mäusen wurden entwickelt, um die Pathophysiologie von HCC zu untersuchen. Transgene Mäuse, die virale Gene, Onkogene und/oder Wachstumsfaktoren exdrücken, ermöglichen die Identifizierung von Wegen, die an der Hepatocarcinogenese beteiligt sind. Aufgrund inhärenter Einschränkungen fassen diese Modelle nicht die typischen Immuneigenschaften des menschlichen HCC zusammen, was die Aufklärung der zugrunde liegenden Mechanismen und die Entwicklung innovativer immuntherapeutischer Strategien erheblich behindert hat14 ,15. Kürzlich haben wir ein klinisch relevantes murines Modell entwickelt. Dieses neuartige Modell imitiert nicht nur die Menschliche HCC-Initiierung und Progression, sondern spiegelt auch die meisten typischen Merkmale menschlicher Krankheiten wider, einschließlich Immunfunktionsstörungen. Wir haben seine biologischen und immunologischen Eigenschaften charakterisiert. Unter Der Nutzung dieses neuartigen Modells haben wir verschiedene immuntherapeutische Strategien zur Behandlung von HCC31,32,33,34,35,36, 37. Diese einzigartige Plattform ermöglicht es uns, Mechanismen der tumorinduzierten Immunverträglichkeit zu untersuchen und proof-of-Concept-Therapiestrategien für HCC für eine eventuelle klinische Translation zu entwickeln.

<table border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="847">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Anesthesia machine</td>
			<td style="width:123px;">VETEQUIP</td>
			<td style="width:344px;">IMPAC6</td>
			<td style="width:167px;">anesthesia machine for surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butterfly needle</td>
			<td>BD</td>
			<td>8122963</td>
			<td>Needle used for liver perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mice</td>
			<td>Jackson Lab</td>
			<td>000664&#160;</td>
			<td>mice used in prototol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carprofen</td>
			<td>CRESCENT CHEMICAL</td>
			<td>20402</td>
			<td>carprofen for pain release</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer&#160;</td>
			<td>CORNING</td>
			<td>REF 431751</td>
			<td>Cell strainer, 70&#181;m, for hepatocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-30R</td>
			<td>centrifuge for cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clips&#160;</td>
			<td>Teleflex Medical</td>
			<td>REF 523700</td>
			<td>Titanium Clips for spleen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Zeiss</td>
			<td>Primovert&#160;</td>
			<td>microscope for cell observation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Mtd2 mice</td>
			<td>N/A</td>
			<td></td>
			<td>Gift from Dr. William A Held at roswell Park Cancer Institute in 2002, maintained in our lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle</td>
			<td>BD</td>
			<td>REF 305109</td>
			<td>BD precisionglide needle, 27G x 1/2 (0.4mm x 13mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture</td>
			<td>ETHICON</td>
			<td>J303H</td>
			<td>coated VICRYL suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SV40 T Ag antibody</td>
			<td>Abcam</td>
			<td>ab16879</td>
			<td>anti-SV40 T-antigen antibody for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe</td>
			<td>BD</td>
			<td>REF 309626</td>
			<td>1 mL TB syringe for cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td>SIGMA</td>
			<td>T 8154</td>
			<td>Trypan blue solution for cell viability test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wound clips</td>
			<td>Reflex</td>
			<td>reflex9, Part. No. 201-1000</td>
			<td>stainless steel wound clips for wound close</td>
		</tr>
	</tbody>
</table>

an oncogenic hepatocyte-induced orthotopic mouse model of hepatocellular cancer arising in the setting of hepatic inflammation and fibrosis

Das Fehlen eines klinisch relevanten Tiermodells, das sich mit den typischen Immunmerkmalen von hepatozellulärem Krebs (HCC) befasst, hat die Aufklärung der zugrunde liegenden Mechanismen und die Entwicklung innovativer immuntherapeutischer Strategien erheblich behindert. Um ein ideales Tiermodell zu entwickeln, das menschliche HCC rekapituliert, erhalten immunkompetente männliche C57BL/6J-Mäuse zunächst eine Tetrachlorsäure-Injektion (CCl4), um Leberfibrose zu induzieren, und erhalten dann histologisch-normale onkogene Hepatozyten von jungen SV40 T Antigen (TAg)-transgene Mäuse (MTD2) durch intrasplenische (ISPL) Inokulation. Androgen erzeugt in Empfänger männlichen Mäusen in der Pubertät initiiert TAg-Expression unter Kontrolle eines Leber-spezifischen Promotor. Dadurch werden die übertragenen Hepatozyten zu Krebszellen und bilden Tumormassen bei der Einstellung von Leberfibrose/Zirrhose. Dieses neuartige Modell imitiert die Initiierung und Progression des menschlichen HCC im Kontext von Leberfibrose/Zirrhose und spiegelt die typischsten Merkmale des menschlichen HCC einschließlich Immunfunktionsstörungen wider.

Onkogene Hepatozyten, die von TAg-transgenen Mäusen isoliert wurden (Abbildung 2), wurden in der Leber von Wildtypmäusen durch intra-splenische Injektion ausgesät (Abbildung 3). Die transplantierten Hepatozyten wuchsen erfolgreich und zuverlässig orthotopische HCC-Tumoren (Abbildung 4) mit tumorspezifischem Antigen SV40 TAg (Abbildung 5) bei der Einstellung von Leberentzündungen und Fibrose ( Abbildung1).
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An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

Hier beschreiben wir die Entwicklung eines klinisch relevanten murinen Modells von Leberkrebs, das die typischen Immunmerkmale von hepatozellulärem Krebs (HCC) rekapituliert.

Ein onkogenes Hepatozyten-induziertes orthotopisches Mausmodell von hepatozellulärem Krebs, der bei der Einstellung von Leberentzündungen und Fibrose entsteht

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Mit nur einem Modell, um ein hepatozelluläres Krebs-Massenmodell zu entwickeln könnte Menschen helfen, ihre tumor-induzierte Immunverträglichkeit und spezifische Behandlung-induzierte, alle Tumor-Immunreaktionen zu klären. Der orthotopische Tumor, der sich mit der Leberfibrose-Technik entwickelt, imitiert die klinischen Merkmale von menschlichem Leberkrebs. Diese Methode könnte ein nützliches Werkzeug in hepatozellululären Krebs immunologischen Studie.Die visuelle Demonstration dieser Methode kann die technisch anspruchsvolleren Schritte, wie z. B. die intrasplenische Injektion, veranschaulichen, die für eine erfolgreiche Ausführung dieses Protokolls unerlässlich sind. Als nächstes, manuell zurückhalten ein Männchen, sechs bis acht Wochen alt C57 schwarz 6 Maus dorsal-Seite nach oben. Verwenden Sie abwechselnd 70% Ethanol und Betadine Peelings, um die Injektionsstelle dreimal zu reinigen.Dann 160 Mikroliter Tetrachlorkohlenstoff per intraperitonealer Injektion an die Maus liefern und das Tier in seinen Heimischenkäfig zurückbringen. Nach der Bestätigung eines Mangels an Reaktion auf Zehenkneifung, in einem fünf Monate alten Simian-Virus 40 T Antigen transgene Maus, legen Sie die Maus in die Supine-Position und sichern Sie die Gliedmaßen mit Klebeband. Machen Sie einen Mittellinien-Laparotomie-Einschnitt entlang der Länge der Linea alba, groß genug, um eine ausreichende Exposition der Leber zu bieten, und verdrängen Sie den Darm nach links.Sezieren Sie oberhalb der Leber die Exposition der unteren Vena cava. Induzieren Sie eine Arterienklemme, um das Gefäß zu ligieren. Verwenden Sie eine Schmetterlingsnadel, um intravenösen Zugriff auf die Portalvene zu erhalten und den Katheter manuell zu sichern.Sorgfältige Spritzen für jede Lösung zu schalten, liefern erfolgreich 15 Milliliter Lösung eins, 15 Milliliter 0,75%Kollagennase Lösung zwei und 15 Milliliter Lösung zwei ohne Kollagenase über Katheter, bei einer Lösung von ca. 8,9 Millilitern pro Minute Durchflussrate. Am Ende der Perfusion ernten Sie die Tumormasse in ein 50-Milliliter-Konustube mit 10 bis 15 Milliliter PBS. Verbrauchen Sie den Tumor von der nächsten MTD2-Maus, wie gerade gezeigt.Verwenden Sie eine Schere, um die Leber in kleinere Stücke zu schneiden. Übertragen Sie das Gewebe in einen neuen Behälter mit frischem PBS, um das Restdes des Blutes aus dem Gewebe zu entfernen, bevor Sie die Gewebeteile in eine 50-Milliliter-Konustube mit fünf Millilitern komplettem RPMI-Medium übertragen. Die gewaschenen Leberproben zerkleinern, bis die Stücke leicht durch die Spitze einer Fünf-Milliliter-Pipette passen können.Fügen Sie genügend Medium in die Röhre, um ein Endvolumen von 30 Millilitern zu erreichen. Verwenden Sie eine Fünf-Milliliter-Pipette, um die Gewebefragmente zu tritutrieren, bevor Sie die Lösung durch ein 70-Mikrometer-Sieb in ein neues 50-Milliliter-Rohr filtern. Waschen Sie das Sieb mehrmals mit komplettem Medium.Stellen Sie das Endvolumen auf 50 Milliliter mit zusätzlichem kompletten Medium ein. Drehen Sie die Suspension durch Zentrifugierung schnell auf maximal 50 mal g, bevor Sie die Zentrifuge stoppen und den Überstand dekantieren. Dann setzen Sie das Pellet in 20 Milliliter PBS zum Zählen wieder aus.Für die intrasplenische Injektion der MTD2-Hepatozyten eine Ein-Milliliter-Spritze mit einer 27-Spur-Nadel pro Empfängertier mit 220 Mikroliter Hepatozyten pro Maus beladen. Bestätigen Sie einen Mangel an Reaktion auf Zehenstiche bei einem anästhesierten, mit Tetrachlorkohlenstoff behandelten Tier. Beginnend direkt unter dem Wirbelsäulenmuskel, als nächstes, machen Sie einen Ein-Zentimeter-Einschnitt auf der linken Flanke parallel zur dreizehnten Rippe aus dem dorsalen Extrem und verwenden Stumpf-Spitzenzange, um die Milz zu exezieren.Schneiden Sie die Milz mit zwei mittelgroßen Titanclips zwischen Derstaubader und Vene ab und liefern Sie 200 Mikroliter Zellen in den unteren Pol der Milz. Schneiden Sie den unteren Zweig des Pedikles mit einem mittelgroßen Clip ab und schneiden Sie die Milz zwischen den beiden zunächst platzierten Clips. Entfernen Sie den unteren Pol der Milz, die direkt mit Tumorzellen injiziert wurde und verwenden Sie 3-0 Polyglactin 910 unterbrochenes Nähen, um die innere Muskelschicht zu schließen.Verwenden Sie dann sterilisierte Stahl-Wundclips, um die äußere Hautschicht zu schließen und subkutan fünf Milligramm pro Kilogramm Carprofen zu verabreichen. Nach der Isolierung von T-Antigen transgenen Mäusen, wie gezeigt, können sowohl Hepatozyten als auch Tumor infiltrierende Zellen beobachtet werden. Die Zellen weisen in der Regel eine fast 100%ige Lebensfähigkeit nach der Verarbeitung auf.Nach intrasplenischer Impfung in wildtype Tetrachlort-behandelte Tiere wachsen die transplantierten Hepatozyten erfolgreich und zuverlässig orthotopische hepatozelluläre Karzinomtumoren, die das tumorspezifische Simianvirus 40 T Antigen exprimieren. Die Vorbereitung unserer lebensfähigen und reinen Population von Hepatozyten aus MTD2 Masse und genaue Verabreichung der intrasplenischen Injektion sind wichtig für den Erfolg des Verfahrens. Obwohl unser Team die Hepatozytenimpfung bei Empfängermäusen mittels intravaskulärer und intraperitonealer Injektion getestet hat, waren diese anderen Methoden nicht in der Lage, orthotopisches hepatozelluläres Karzinom zu induzieren.Dieses Modell bietet eine einzigartige Plattform, die die Untersuchung der Immunüberwachung und der Toleranz in ihren frühen und späteren Stadien der Tumorprogression ermöglicht. Bitte seien Sie vorsichtig bei der Verwendung des Tetrachlorkohlenstoffs, da es giftig ist.

an-oncogenic-hepatocyte-induced-orthotopic-mouse-model-hepatocellular

Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Fluoreszierende orthotopen Mausmodell des Bauchspeicheldrüsenkrebs

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Forschung

Krebsforschung

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Isolierung von zirkulierenden Tumorzellen in einem orthotopischen Mausmodell des Darmkrebses

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Die Immunphänotypisierung der Orthotopen Homograft (Phänomen) der murinen primäre KPC duktales Pankreaskarzinom durch Durchflusszytometrie

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Studieren von dreifach negativem Brustkrebs mit orthotopischem Brustkrebsmodell

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Ultraschallgeführte orthotopische Implantation von murine Pankreas-Ductalsocarcinom

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Generierung von orthotopischen Pankreastumoren und Ex-vivo-Charakterisierung der tumorinfiltrierenden T-Zell-Zytotoxizität

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

In Vitro-Bewertung der onkogenen Transformation in menschlichen Mammaepithelzellen

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

Ein orthotopisches Resektions-Mausmodell für Bauchspeicheldrüsenkrebs

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

Ein dreidimensionales Sphäroidmodell zur Untersuchung der Tumor-Stroma-Wechselwirkung bei hepatozellulären Karzinomen

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Nicht-invasive PET/MR-Bildgebung in einem orthotopen Mausmodell des hepatozellulären Karzinoms

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...