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

Dette arbejde støttes af NIH/NCI R01 CA164335-01A1 (K. F. Staveley-O'Carroll, PI) og NIH/NCI R01CA208396 (Mark Kester, Guangfu Li, Kevin F. Staveley-O'Carroll).

Bemærk: alle de procedurer, herunder dyr emner er blevet godkendt af IACUC på University of Missouri. Alle mus fik human behandling i henhold til de kriterier, som er skitseret i "vejledning i pleje og anvendelse af forsøgsdyr". Følgende procedure for celle isolering og inokulation bør udføres i en hætte. Alle udøvende kunstnere bør bære standard personlige værnemidler til håndtering af mus og væv.
1. induktion af lever fibrose og cirrhose med IP-injektion af Tetrachlorid (CCl4...

<ol>
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Med denne protokol har vi etableret en pålidelig og reproducerbar murine model af HCC, der efterligner Human HCC initiering og progression. Klinisk, mange risikofaktorer successivt inducere leverskade, lever fibrose, cirrhose og den sidste fase af HCC. I vores protokol, IP injektion af CCL4 bruges til først at producere lever fibrose i vildtype mus, som gør det muligt for de efterfølgende onkogene hepatocytter til dannelse af tumorer i fastsættelsen af lever fibrose. Vi fandt, at tumordannelse forekom mes...

Hepatocellulær cancer (HCC) er den mest hastigt voksende type kræft i USA (US)1,2,3. Hvert år diagnosticeres ca. 850.000 nye tilfælde4,5 og 700.000 patienter dør af denne dødeligesygdom 6,7,8,9,10 , hvilket gør den til den næsthøjeste årsag til kræft relateret død verden over. Administration af HCC omfatter kirurgisk resektion, transplantation, ablation, chemoembolization eller systemiske terapier, såsom sorafenib11. Tidlig diagnose og administration med kirurgisk resektion eller transplantation har den højeste samlede overlevelsesfordel4. Desværre, de fleste patienter til stede på et senere tidspunkt og kræver administration med ablation, chemoembolization eller sorafenib12. Sorafenib, en receptor tyrosinkinasehæmmer (RTKI), blev godkendt af Food and Drug Administration i 2008 som den eneste systemiske lægemiddelbehandling, der var tilgængelig til behandling af inoperabel HCC. Selv om stoffet kun giver en beskeden stigning i den samlede overlevelse, fra 7,9 til 10,7 måneder13, det gav en ny terapeutisk strategi, der kunne udnyttes til at forvalte HCC.
Manipulation af immunsystemet for at eliminere etablerede kræftformer er et hastigt voksende felt inden for kræftforskning14. Immun check point studier har betydeligt avanceret immunotherapeutisk lægemiddeludvikling i kræftbehandling15,16. FDA godkendte brugen af antistoffer (ABS) mod cytotoksisk T-lymfocyt antigen 4 (CTLA-4), programmeret celledød protein 1 (PD-1) og dets ligand PD-L1 til behandling af melanom, lungekræft, hoved-og nakke kræft og blærecancer17, 18 , 19 , 20. kliniske forsøg med monoterapi eller kombinationsbehandling med et eller flere antistoffer mod PD-1, PD-L1 eller ctla-4 til behandling af avanceret HCC er i gang21,22,23og nogle forsøg har vist gunstige resultater. I 2017, FDA givet fremskyndet godkendelse for anti-PD-1 antistof til behandling af HCC patienter, der er resistens over for sorafenib, men den samlede responsrate af denne behandling er kun 14,3%. Andre strategier er ikke blevet oversat til klinisk praksis på dette tidspunkt24,25. Overvinde tumor-induceret dyb immuntolerance for at forbedre immun check point terapi26; forudsigelse af effekten af immun check point terapi; forebyggelse af immunrelaterede uønskede hændelser optimering af administrationsvej, dosering og hyppighed; og finde effektive kombinationer af terapier27,28,29 alle forbliver yderst udfordrende opgaver.
Der er flere konventionelle tilgange, der anvendes til at inducere HCC i musemodeller i øjeblikket og udnyttes afhængigt af investigators særlige forskning spørgsmål30. Kemisk inducerede HCC-musemodeller med genotoksiske forbindelser efterligner skade forårsagede maligniteter. Xenograft-modeller gennem enten ektopisk eller ortotopisk implantation af HCC-cellelinjer er velegnede til lægemiddel screening. En række genetisk modificerede mus er blevet konstrueret til at undersøge Patofysiologi af HCC. Transgene mus, der udtrykker virale gener, onkogener og/eller vækstfaktorer gør det muligt at identificere veje involveret i hepatocarcinogenese. På grund af iboende begrænsninger, kan disse modeller ikke rekapitulere de typiske immun karakteristika, der ses i human HCC, som har betydeligt hæmmet belysning af de underliggende mekanismer og udvikling af innovative immunotherapeutiske strategier14 ,15. Vi har for nylig skabt en klinisk relevant murine model. Denne roman model ikke kun efterligner Human HCC initiering og progression, men også afspejler de fleste typiske træk ved sygdomme hos mennesker, herunder immun dysfunktion. Vi har karakteriseret dets biologiske og immunologiske egenskaber. Ved at udnytte denne nye model har vi undersøgt forskellige immun terapeutiske strategier til behandling af HCC31,32,33,34,35,36, 37. denne unikke platform giver os mulighed for at studere mekanismer af tumor-induceret immunotolerance og udvikle proof-of-koncept terapeutiske strategier for HCC mod eventuel klinisk oversættelse.

<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

Fraværet af en klinisk relevant dyremodel, der adresserer de typiske immun karakteristika ved hepatocellulær cancer (HCC), har i væsentlig grad forhindret elucidationen af de underliggende mekanismer og udviklingen af innovative immunterapeutiske strategier. For at udvikle en ideel dyremodel til at rekapitulere Human HCC, immunkompetente mandlige C57BL/6J mus først modtage en Carbon tetrachlorid (CCl4) injektion for at fremkalde lever fibrose, derefter modtage histologisk-normale onkogene hepatocytter fra unge mandlige SV40 T antigen (TAg)-Transgene mus (MTD2) ved intra-splenisk (ISPL) inokulation. Androgen genereret i recipient mandlige mus i puberteten indleder TAg udtryk under kontrol af en lever-specifik promotor. Som et resultat, de overførte hepatocytter bliver kræftceller og danne tumor masser i fastsættelsen af lever fibrose/cirrhose. Denne nye model efterligner Human HCC initiering og progression i forbindelse med lever fibrose/cirrhose og afspejler de mest typiske træk ved humant HCC herunder immun dysfunktion.

Onkogene hepatocytter isoleret fra TAg-Transgene mus (figur 2) blev seedet i leveren af vilde type mus ved intra-splenisk injektion (figur 3). Den transplanterede hepatocytter succesfuldt og pålideligt voksede ortotopisk HCC tumorer (figur 4) med tumor specifikke antigen SV40 TAg (figur 5) i indstillingen af hepatisk inflammation og fibrose (figur 1).
<img alt="Figure 1" class...

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An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

Her beskriver vi udviklingen af en klinisk relevant murine model af leverkræft rekapitulere de typiske immun funktioner af hepatocellulær cancer (HCC).

En onkogen Hepatocyte-induceret Ortotopisk muse model af Hepatocellulær cancer, der opstår i fastsættelsen af hepatisk inflammation og fibrose

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

Ved hjælp af blot en model til at udvikle en hepatocellulær kræft masse model kan hjælpe folk belyse deres tumor-induceret immuntolerance og specifik behandling-induceret, enhver tumor immunrespons. Den ortopiske tumor, der udvikler sig med leveren fibrose teknik efterligner de kliniske træk ved human leverkræft. Denne metode kan give et nyttigt værktøj i hepatocellulær kræft immunologisk undersøgelse.Visuel demonstration af denne metode kan illustrere de mere teknisk udfordrende trin, såsom intrasplenic injektion, der er afgørende for en vellykket udførelse af denne protokol. Dernæst manuelt begrænse en mandlig, seks til otte uger gamle C57 sort 6 mus dorsal-side op. Brug alternative 70% ethanol og Betadine scrubs til at rengøre injektionsstedet tre gange.Derefter leveres 160 mikroliter tetrachlormetr ded til musen ved intraperitoneal injektion og returnere dyret til sit hjem bur. Efter at have bekræftet en manglende reaktion på tå knivspids, i en fem måneder gammel simian virus 40 T antigen transgen mus, placere musen i liggende position og sikre lemmer med tape. Lav en midline laparotomi snit langs længden af linea alba, stor nok til at give en tilstrækkelig eksponering af leveren, og fortrænge tarmene til venstre.Dissekere over leveren for at få den ringere vena cava. Inducer en arterieklemme til at lasere beholderen. Brug en sommerfuglål til at få intravenøs adgang til portalvenen og fastgør kateteret manuelt.Forsigtigt skifte sprøjter til hver opløsning, med succes levere 15 milliliter opløsning en, 15 milliliter 0,75% kollagenopløsning to, og 15 milliliter opløsning to uden kollagenase via kateter, på en omtrentlig 8,9 milliliter opløsning per minut strømningshastighed. I slutningen af perfusionen høstes tumormassen i et 50-milliliter konisk rør indeholdende 10 til 15 milliliter PBS. Punktafgifter tumoren fra den næste MTD2 mus som netop demonstreret.Brug saks til at skære leveren i mindre stykker. Vævet overføres til en ny beholder med frisk PBS for at fjerne resten af blodet fra vævet, før vævsstykkerne overføres til et konisk rør med 50 milliliter, der indeholder fem milliliter komplet RPMI-medium. Hakke de vaskede leverprøver, indtil stykkerne kan passe let gennem spidsen af en fem-milliliter pipette.Tilsæt nok medium til røret til at nå et endeligt volumen på 30 milliliter. Brug en fem-milliliter pipette til at triturere vævsfragmenterne, før opløsningen filtreres gennem en 70-mikrometer si i et nyt 50-milliliterrør. Syrlakt flere gange med komplet medium.Den endelige lydstyrke justeres til 50 milliliter med yderligere komplet medium. Drej hurtigt suspensionen ved centrifugering til maksimalt 50 gange g, før centrifugen stoppes, og supernatanten dekanteres. Derefter resuspend pellet i 20 milliliter PBS til optælling.Til intrasplenisk injektion af MTD2 hepatocytterne skal en en millilitersprøjte være udstyret med en 27-gauge nål pr. hvert recipientdyr med 220 mikroliter hepatocytter pr. mus. Bekræft manglende reaktion på tå knivspids i en bedøvet, carbon tetrachlorid-behandlede dyr. Begynder lige under rygsøjlen muskel, næste, foretage en en-centimeter snit på venstre flanke parallelt med den trettende ribben fra rygsøjle ekstreme og bruge stump-pegede kraftbefæstet til at eksteriør milten.Klip milten med to mellemstore titanium clips mellem milt arterie og vene, og levere 200 mikroliter af celler i ringere pol af milten. Klip den ringere gren af pedicle med en mellemstor klip og skære milten mellem de to oprindeligt placerede klip. Fjern ringere pol af milten, der blev direkte injiceret med tumorceller og bruge 3-0 polyglactin 910 afbrudt suturing at lukke den indre muskel lag.Brug derefter steriliserede stålsårclips til at lukke det ydre hudlag og subkutanet administrere fem milligram pr. kg carprofen. Efter isolering fra T antigen transgene mus som påvist, både hepatocytter og tumor infiltrerende celler kan observeres. Cellerne viser typisk en næsten 100% levedygtighed efter behandling.Efter intrasplenic inokulering i vilde-type carbon tetrachlorid-behandlede dyr, de transplanterede hepatocytter med succes og pålideligt vokse ortopisk hepatocellulær karcinom tumorer, der udtrykker tumor-specifikke simian virus 40 T antigen. Forberedelsen af vores levedygtige og rene population af hepatocytter fra MTD2 masse og nøjagtig administration af intrasplenic injektion er vigtige for en vellykket procedure. Selv om vores team har testet hepatocyt inokulering i modtagermus via intravaskulær og intraperitoneal injektion, disse andre metoder var ikke i stand til at fremkalde ortopisk hepatocellulær karcinom.Denne model giver en unik platform gør det muligt for undersøgelsen for immunovervågning og tolerance i sin tidlige og de senere stadier af tumor progression. Vær forsigtig, når du bruger tetrachlormetorid, da det er giftigt.

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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.

Fluorescent ortotopisk Mouse Model af kræft i bugspytkirtlen

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.

Isolering af cirkulerende tumorceller i en ortotopisk musemodel af kolorektal cancer

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.

Immunofænotypning af Orthotopic Homograft (Syngeneic) af Murine primære KPC pancreas duktalt adenokarcinom ved flowcytometri

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.

Studere Triple Negative Breast Cancer Brug 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 ...

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.

Ultralyd-guidet Ortotopisk implantation af murin Pankreatisk 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 ...

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.

Generation af Ortotopiske tumorer i bugspytkirtlen og ex vivo karakterisering af tumor Infiltrerende T-celle cytotoksicitet

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 evaluering af onkogene transformation i humane mammary epitelceller

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.

En orthotopic Resectional Mouse Model af kræft i bugspytkirtlen

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.

En tredimensionel spheroid model til at undersøge tumor-stromal interaktion i hepatocellulær karcinom

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).

Ikke-invasiv PET/MR-billeddannelse i en ortopisk musemodel af hepatocellulært karcinom

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