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

Dit werk wordt ondersteund door NIH/NCI R01 CA164335-01A1 (K. F. Staveley-O'Carroll, PI) en NIH/NCI R01CA208396 (Mark Kester, Guangfu Li, Kevin F. Staveley-O'Carroll).

Opmerking: alle procedures inclusief dierproef personen zijn goedgekeurd door de IACUC aan de Universiteit van Missouri. Alle muizen kregen humane verzorging volgens de criteria die zijn uiteengezet in de "gids voor de verzorging en het gebruik van proefdieren". De volgende procedure voor celisolatie en inoculatie moet worden uitgevoerd in een afzuigkap. Alle performers moeten de standaard persoonlijke beschermingsmiddelen dragen voor het hanteren van de muizen en weefsels.
1. inductie van lever...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma+-+United+States.">Hepatocellular carcinoma - United States.</a> Morbidity and Mortality Weekly Report. 59, 517-520 (2010).</li><li>Petrick, J., Kelly, S., Altekruse, S., McGlynn, K., Rosenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+of+Hepatocellular+Carcinoma+Incidence+in+the+United+States+Forecast+Through+2030.">Future of Hepatocellular Carcinoma Incidence in the United States Forecast Through 2030.</a> Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 34, 1787-1794 (2016).</li><li>Greten, T., Lai, C., Li, G., Staveley-O'Carroll, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+and+Immune-based+Therapies+for+Hepatocellular+Carcinoma.">Targeted and Immune-based Therapies for Hepatocellular Carcinoma.</a> Gastroenterology. 156, 510-524 (2019).</li><li>Llovet, J., Zucman-Rossi, J., Pikarsky, E., Sangro, B., Schwartz, M., Sherman, M., Gores, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+Carcinoma.">Hepatocellular Carcinoma.</a> Nature reviews. Disease primers. 2, 16018 (2016).</li><li>Ding, X., 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=Precision+medicine+for+hepatocellular+carcinoma:+driver+mutations+and+targeted+therapy.">Precision medicine for hepatocellular carcinoma: driver mutations and targeted therapy.</a> Oncotarget. 8, 55715-55730 (2017).</li><li>Colombo, M., Maisonneuve, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+liver+cancer+mortality+on+a+global+scale:+still+a+long+way+to+go.">Controlling liver cancer mortality on a global scale: still a long way to go.</a> Journal of Hepatology. 67, 216-217 (2017).</li><li>Llovet, J., Burroughs, A., Bruix, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma.">Hepatocellular carcinoma.</a> Lancet. 362, 1907-1917 (2003).</li><li>Parkin, D., Bray, F., Ferlay, J., Pisani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+world+cancer+burden:+Globocan+2000.">Estimating the world cancer burden: Globocan 2000.</a> International Journal of Cancer. 94, 153-156 (2001).</li><li>Thomas, M., Zhu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepocellular+carcinoma:+the+need+for+progress.">Hepocellular carcinoma: the need for progress.</a> Journal of Clinical Oncology. 23, 2892-2899 (2005).</li><li>Llovet, J., Bruix, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+targeted+therapies+in+hepatocellular+carcinoma.">Molecular targeted therapies in hepatocellular carcinoma.</a> Hepatology. 48, 1312-1327 (2008).</li><li>Bruix, J., Sherman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+hepatocellular+carcinoma:+an+update.">Management of hepatocellular carcinoma: an update.</a> Hepatology. 53, 1020-1022 (2011).</li><li>Pang, T., Lam, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+management+of+hepatocellular+carcinoma.">Surgical management of hepatocellular carcinoma.</a> World Journal of Hepatology. 7, 245-252 (2015).</li><li>Llovet, J., 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=Design+and+endpoints+of+clinical+trials+in+hepatocellular+carcinoma.">Design and endpoints of clinical trials in hepatocellular carcinoma.</a> Journal of the National Cancer Institution. 100, 698-711 (2008).</li><li>Mueller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunology+and+immunotherapy.+Realizing+the+promise.+Introduction.">Cancer immunology and immunotherapy. 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Met dit protocol hebben we een betrouwbaar en reproduceerbaar muriene model van HCC opgezet dat menselijke HCC initiatie en progressie nabootst. Klinisch, veel risicofactoren achtereenvolgens induceren leverletsel, leverfibrose, cirrose en de laatste fase van HCC. In ons protocol wordt IP-injectie van CCl4 gebruikt om eerst leverfibrose te produceren in wild type muizen, waardoor de daaropvolgende oncogene hepatocyten de tumoren vormen in de setting van leverfibrose. We ontdekten dat tumorvorming het meest suc...

Hepatocellulaire kanker (HCC) is de snelst groeiende vorm van kanker in de Verenigde Staten (VS)1,2,3. Elk jaar worden ongeveer 850.000 nieuwe gevallen gediagnosticeerd4,5 en 700.000 patiënten sterven aan deze dodelijke ziekte6,7,8,9,10 , waardoor het de op één na hoogste oorzaak van kanker-gerelateerde dood wereldwijd. Het beheer van HCC omvat chirurgische resectie, transplantatie, ablatie, chemoembolisatie of systemische therapieën, zoals sorafenib11. Vroegtijdige diagnose en behandeling met chirurgische resectie of transplantatie hebben het hoogste totale overlevingsvoordeel4. Helaas, de meerderheid van de patiënten aanwezig in een later stadium en vereisen Management met ablatie, chemoembolisatie of sorafenib12. Sorafenib, een receptor tyrosine kinase remmer (rtki), werd goedgekeurd door de Food and Drug Administration in 2008 als de enige systemische medicamenteuze therapie beschikbaar voor de behandeling van inoperabel HCC. Hoewel het medicijn alleen een bescheiden toename van de totale overleving biedt, van 7,9 tot 10,7 maanden13, voorzag het in een nieuwe therapeutische strategie die kon worden gebruikt om HCC te beheren.
Het manipuleren van het immuunsysteem om gevestigde kankers te elimineren is een snel groeiend veld in kankeronderzoek14. Immuun Checkpoint studies hebben aanzienlijk geavanceerde immunotherapeutische drug ontwikkeling in de behandeling van kanker15,16. De FDA keurde het gebruik van antilichamen (ABS) goed tegen cytotoxisch T-lymfocyten antigeen 4 (CTLA-4), geprogrammeerde celdood proteïne 1 (PD-1), en zijn ligand PD-L1 voor de behandeling van melanoom, longkanker, hoofd-en nekkanker, en blaaskanker17, 18 , 19 , 20. klinische studies met monotherapie-of combinatietherapie waarbij gebruik wordt gemaakt van één of meerdere antilichamen tegen pd-1, PD-L1 of ctla-4 voor de behandeling van Advanced HCC zijn aan de gang21,22,23, en sommige proeven hebben gunstige resultaten opgeleverd. In 2017, de FDA verleende versnelde goedkeuring voor anti-PD-1 antilichaam voor de behandeling van HCC patiënten, die resistentie tegen sorafenib zijn, maar de totale responspercentage van deze therapie is slechts 14,3%. Andere strategieën zijn niet vertaald in de klinische praktijk op dit moment24,25. Overwinnen van tumor-geïnduceerde diepgaande immuun tolerantie ter verbetering van immune Checkpoint therapie26; voorspellen van de werkzaamheid van immuuncontrolepunt therapie; preventie van immuungerelateerde ongewenste voorvallen; het optimaliseren van de toedieningsroute, dosering en frequentie; en het vinden van effectieve combinaties van therapieën27,28,29 blijven allemaal uiterst uitdagende taken.
Er zijn verschillende conventionele benaderingen gebruikt voor het induceren van HCC in muismodellen momenteel en worden gebruikt, afhankelijk van de onderzoeker specifieke onderzoek vraag30. Chemisch geïnduceerde HCC-Muismodellen met genotoxische verbindingen bootsen letsel geïnduceerde maligniteit na. Xenograft-modellen via ectopische of orthotopic implantatie van HCC-cellijnen zijn geschikt voor geneesmiddelen screening. Een aantal genetisch gemodificeerde muizen is ontwikkeld om de pathofysiologie van HCC te onderzoeken. Transgene muizen die virale genen, oncogenen en/of groeifactoren uitdrukken, maken de identificatie mogelijk van trajecten die betrokken zijn bij hepatocarcinogenese. Vanwege inherente beperkingen, deze modellen niet recapituleren de typische immuunkenmerken gezien in humaan HCC, die aanzienlijk heeft belemmerd verheldering van de onderliggende mechanismen en ontwikkeling van innovatieve immunotherapeutische strategieën14 ,15. Onlangs hebben we een klinisch relevant Murine model gecreëerd. Dit nieuwe model bootst niet alleen menselijke HCC initiatie en progressie na, maar weerspiegelt ook de meest typische kenmerken van menselijke ziekte, waaronder immuun disfunctie. We hebben de biologische en immunologische kenmerken ervan gekenmerkt. Gebruikmakend van dit nieuwe model hebben we verschillende immunotherapeutische strategieën onderzocht voor de behandeling van HCC31,32,33,34,35,36, 37. dit unieke platform stelt ons in staat om mechanismen van tumor-geïnduceerde immunotolerance te bestuderen en om proof-of-concept therapeutische strategieën te ontwikkelen voor HCC naar uiteindelijke klinische vertaling.

<table border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="847">
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	</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

De afwezigheid van een klinisch relevant diermodel dat de typische immuunkenmerken van hepatocellulaire kanker (HCC) aanpakt, heeft de verheldering van de onderliggende mechanismen en de ontwikkeling van innovatieve immunotherapeutische strategieën aanzienlijk belemmerd. Om een ideaal diermodel te ontwikkelen dat het humaan HCC samenvoert, krijgen immunocompetente mannelijke C57BL/6J-muizen eerst een koolstoftetrachloride (CCl4)-injectie om leverfibrose te induceren, waarna ze histologisch-normale oncogene hepatocyten van jonge mannen krijgen SV40 T antigeen (TAg)-transgene muizen (MTD2) door intra-splenic (ISPL) inoculatie. Androgeen gegenereerd in de ontvangende mannelijke muizen in de puberteit initieert TAg expressie onder controle van een lever-specifieke promotor. Als gevolg daarvan, de overgedragen hepatocyten worden kankercellen en vorm tumor massa's in de setting van leverfibrose/cirrose. Dit nieuwe model bootst Human HCC initiatie en progressie na in de context van leverfibrose/cirrose en weerspiegelt de meest typische kenmerken van humaan HCC inclusief immuun disfunctie.

Oncogene hepatocyten die zijn geïsoleerd van TAg-transgene muizen (Figuur 2) werden in de lever van wild type muizen geënt door intra-milt injectie (Figuur 3). De getransplanteerde hepatocyten succesvol en betrouwbaar groeide orthotopic HCC tumoren (Figuur 4) met tumor specifieke antigeen SV40 TAg (Figuur 5) in de setting van hepatische ontsteking en fibrose (Figuur 1).
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An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

Hier beschrijven we de ontwikkeling van een klinisch relevant muriene model van leverkanker die de typische immuunkenmerken van hepatocellulaire kanker (HCC) recapituleren.

Een oncogene hepatocyte-geïnduceerde Orthotopic muis model van hepatocellulaire kanker ontstaan in de setting van leverontsteking en fibrose

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

Met behulp van slechts een model om een hepatocellulaire kanker massa model te ontwikkelen kan mensen helpen hun tumor-geïnduceerde immunotolerantie en specifieke behandeling-geïnduceerde, elke tumor immunoresponses. De orthotopische tumor die zich ontwikkelt met de leverfibrose techniek bootst de klinische kenmerken van menselijke leverkanker na. Deze methode kan een nuttig hulpmiddel bieden in hepatocellulaire kanker immunologische studie.Visuele demonstratie van deze methode kan illustreren de meer technisch uitdagende stappen, zoals de intrasplenic injectie, die essentieel zijn voor een succesvolle uitvoering van dit protocol. Vervolgens handmatig beperken een mannetje, zes tot acht weken oude C57 zwarte 6 muis dorsal-side omhoog. Gebruik alternatieve 70% ethanol en Betadine scrubs om de injectieplaats drie keer schoon te maken.Lever vervolgens 160 microliter koolstoftetrachloride aan de muis door intraperitoneale injectie en breng het dier terug naar zijn kooi thuis. Na bevestiging van een gebrek aan respons op teen knijpen, in een vijf maanden oude simian virus 40 T antigeen transgene muis, plaats de muis in de supine positie en zet de ledematen met tape. Maak een midline laparotomie incisie langs de lengte van de linea alba, groot genoeg om een adequate blootstelling van de lever te bieden, en verdringen de darmen naar links.Ontleden boven de lever aan blootstelling aan de inferieure vena cava. Induceer een slagaderklem om het vat te binden. Gebruik een vlindernaald om intraveneuze toegang tot de poortader te krijgen en de katheter handmatig vast te stellen.Zorgvuldig schakelen spuiten voor elke oplossing, met succes leveren 15 milliliter oplossing een, 15 milliliter van 0,75%collagenase oplossing twee, en 15 milliliter oplossing twee zonder collagenase via katheter, bij een geschatte 8,9 milliliter oplossing per minuut stroomsnelheid. Aan het einde van de perfusie, oogst de tumormassa in een 50-milliliter conische buis met 10 tot 15 milliliter PBS. Accijns de tumor van de volgende MTD2 muis zoals net aangetoond.Gebruik een schaar om de levers in kleinere stukken te snijden. Breng de weefsels in een nieuwe container van verse PBS om de rest van het bloed uit het weefsel te verwijderen voordat u de weefselstukken in een 50-milliliter conische buis met vijf milliliter van volledige RPMI medium. Hak de gewassen levermonsters tot de stukken gemakkelijk door de punt van een pijp van vijf milliliter kunnen passen.Voeg voldoende medium aan de buis om een uiteindelijk volume van 30 milliliter te bereiken. Gebruik een pipet van vijf milliliter om de weefselfragmenten te tritureren, voordat u de oplossing door een zeef van 70 micrometer in een nieuwe buis van 50 milliliter filtert. Was de zeef meerdere malen met compleet medium.Pas het uiteindelijke volume aan op 50 milliliter met extra compleet medium. Draai de vering snel door centrifugeren tot maximaal 50 keer g voordat u de centrifuge stopt en de supernatant decanteert. Dan resuspend de pellet in 20 milliliter pbs voor het tellen.Voor intrasplenic injectie van de MTD2 hepatocyten, laad een een-milliliter spuit uitgerust met een 27-gauge naald per elk ontvanger dier met 220 microliters van hepatocyten per muis. Bevestig een gebrek aan respons op teenknijpen in een verdoofd, met koolstoftetrachloride behandeld dier. Begin net onder de wervelkolom spier, vervolgens, maak een een-centimeter incisie op de linkerflank parallel aan de dertiende rib van de dorsale extreme en gebruik botte-puntige tangen om de milt exterioriseren.Knip de milt met twee middelgrote titaniumclips tussen de miltslagader en ader en lever 200 microliter cellen in de inferieure pool van de milt. Knip de inferieure tak van de pedicle met één middelgrote clip en snijd de milt tussen de twee in eerste instantie geplaatste clips. Verwijder de inferieure pool van de milt die direct werd geïnjecteerd met tumorcellen en gebruik 3-0 polyglactine 910 onderbroken hechten om de binnenste spierlaag te sluiten.Gebruik vervolgens gesteriliseerde stalen wondclips om de buitenste huidlaag te sluiten en onderhuids vijf milligram per kilogram carprofen toe te dienen. Na isolatie van T-antigeentransgene muizen zoals aangetoond, kunnen zowel hepatocyten als tumorinfiltrerende cellen worden waargenomen. De cellen tonen meestal een bijna 100% levensvatbaarheid na verwerking.Na intrasplenische inenting in in het wild-type koolstoftetrachloride-behandelde dieren, de getransplanteerde hepatocyten met succes en betrouwbaar groeien orthopedische hepatocellulaire carcinoom tumoren die de tumor-specifieke simian virus 40 T antigeen uitdrukken. De voorbereiding van onze levensvatbare en zuivere populatie hepatocyten uit MTD2 massa en nauwkeurige toediening van de intrasplenic injectie zijn belangrijk voor het succes van de procedure. Hoewel ons team hepatocyte-inenting in ontvangende muizen heeft getest via intravasculaire en intraperitoneale injectie, waren deze andere methoden niet in staat om orthopedisch hepatocellulair carcinoom te induceren.Dit model biedt een uniek platform dat het onderzoek mogelijk maakt voor immuunbewaking en de tolerantie in de vroege en latere stadia van tumorprogressie. Wees voorzichtig bij het gebruik van de koolstoftetrachloride, want het is giftig.

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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.

Fluorescerende orthotope Muis Model van pancreaskanker

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 ...

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.

Isolatie van circulerende tumorcellen in een orthotopische muismodel van colorectale kanker

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.

Immunophenotyping van Orthotopic Homograft (Syngeneic) van de alvleesklier ductaal adenocarcinoom lymfkliertest primaire KPC door Stroom Cytometry

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.

Studeren Triple Negatieve Borstkanker met behulp van Orthotopische Borstkanker Model

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.

Echografie-geleide Orthotopic implantatie van Murine pancreatic Ductal adenocarcinoom

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.

Generatie van Orthotopic alvleesklier tumoren en ex vivo karakterisering van tumor-Infiltrerende T cel cytotoxiciteit

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 Evaluatie van Oncogene Transformatie 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 ...

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.

Een orthotopisch resectiemuismodel van alvleesklierkanker

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.

Een driedimensionaal sferoïde model om de tumor-stromale interactie bij hepatocellulair carcinoom te onderzoeken

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).

Niet-invasieve PET/MR-beeldvorming in een orthotopisch muismodel van hepatocellulair carcinoom

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