Name: orthotopic implantation and peripheral immune cell monitoring in the ii-45 syngeneic rat mesothelioma model
Uploaded: 2015-10-02T14:00:00
Description: Watch this Scientific Journal Video about Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model at JoVE.com

The syngeneic rat mesothelioma II-45 cell line was a kind gift from A/Prof. Emanuela Felley-Bosco, Zurich University.

Alle procedurer, der involverer dyr blev udført i overensstemmelse med anbefalingerne i den australske Code of Practice for pasning og anvendelse af dyr til videnskabelige formål. Protokollen for denne undersøgelse blev godkendt af Royal North Shore Hospital Animal Care og etiske komité. Kvindelige Fischer 344 rotter (F344, 150-200 g) blev fastholdt på Kearns Facility, Kolling Institute under standardbetingelser (12 t lys / mørke cykler og fri adgang til mad og vand). Bemærk: et ruted...

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Dette papir beskriver en fremgangsmåde til generering af en rotte syngen model af ortotopisk pleural mesotheliom og en simpel metode til overvågning af sygdomsprogression ved langsgående blodprøvetagning. Den II-45 modellen blev udviklet ved at udsætte Fischer 344 rotter for asbestfibre 13. Selv om denne eksponering repræsenterer den sande dynamik host-asbest-immunsystem interaktioner for lungehindekræft patogenese, det har en lang forsinkelse tid (under år at generere), o...

Malignt mesotheliom (MM) er en aggressiv malignitet, der opstår fra transformerede celler i membranen (mesothelium) at linjer lungerne og bughule, hjerte og indre kønsorganer, og er den mest almindelige primære tumor i lungerne hulrum eller lungehinden 1,2 . Udsættelse for asbestfibre udgør 80% af al MM, og mens forbud mod brug af asbest blev indført årtier siden i de fleste vestlige lande, har den udbredte anvendelse i samfundet efterladt en dødelig arv. WHO har anslået, at 107.000 mennesker verden over dør hvert år af asbestrelaterede sygdomme, med dødelighed fortsætter med at stige. En ny ikke-erhvervsmæssig forekomst bølge også at opstå, og der er lidt forståelse af, hvornår og på hvilket niveau det vil toppe 3. De fleste mennesker med MM er diagnosticeret sent, når systemisk kemoterapi udgør en af de eneste levedygtige muligheder 4. De fleste effective kemoterapi og nuværende &quot;standard for pleje« (pemetrexed sammen med cisplatin 5) blev identificeret over 10 år siden. Men svigt af denne behandling er uundgåelig, og der er ingen dokumenterede anden linje muligheder, der forlader patienter med en grum prognose og median overlevelse på kun 12 måneder 2. Derfor er der et presserende uopfyldt behov for mere effektive behandlinger. På trods af den undersøgelse af en række nye behandlinger i kliniske forsøg ingen har medført ændringer i praksis. Dette skyldes til dels lav (5%) overførsel af prækliniske resultater, udføres generelt i xenograft musemodeller, til den kliniske indstilling 6-8. Sådanne modeller ikke trofast rekapitulere de komplekse aspekter af tumormikromiljøet forekommer i ikke-fysiologiske steder, ofte i fravær af et fungerende immunsystem 9. Syngene ortotopisk modeller skaber et betydeligt mere realistisk tumor miljøet end commonly anvendes subkutane xenograftmodeller som forekommer tumorer i den korrekte fysiologiske placering med et intakt immunsystem 10,11. Den større størrelse af rotte forbedrer dets anvendelse som en gnaver sygdomsmodel, især i narkotika undersøgelser, hvor serielle blodprøver skal vurdere behandlingsrespons og toksicitet 12. Endvidere i modeller, hvor overvågning sygdomsprogression er vanskelig på grund af placeringen af ​​tumorer (såsom i pleurahulen), muligheden for at overvåge sygdomsprogression ved hjælp af faktorer findes i cirkulationen, er yderst attraktiv. Frembringelsen af ​​en syngen ortotopisk model af pleural mesotheliom hjælp immun-kompetente rotter er beskrevet. Desuden er en let og relativt ikke-invasiv metode til overvågning af pleural sygdomsprogression ved at måle cirkulerende immunceller også beskrevet.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EDTA Collection tube (0.5ml)</td>
			<td>Greiner Bio One GmbH</td>
			<td>450480</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat T/B/NK Cell cocktail</td>
			<td>BD Pharmingen</td>
			<td>558509</td>
			<td>anti-Rat CD3&#160; APC (IgM clone 1F4), anti-Rat CD45RA Fitc (IgG1 clone OX-33), anti-Rat CD161a PE (IgG1 Clone 10/78)</td>
		</tr>
		<tr>
			<td>anti-RAT CD8a PE&#160;</td>
			<td>Biolegend</td>
			<td>200608</td>
			<td>(IgG1vClone G28)</td>
		</tr>
		<tr>
			<td>anti-Rat CD4 FITC (Domain 1)&#160;</td>
			<td>Biolegend</td>
			<td>203406</td>
			<td>(IgG1 Clone OX-38)</td>
		</tr>
		<tr>
			<td>anti-Rat CD45 PE/Cy7&#160;</td>
			<td>Biolegend</td>
			<td>202214</td>
			<td>(IgG1 Clone OX-1)</td>
		</tr>
		<tr>
			<td>TruCount&#8482; Tubes</td>
			<td>Becton Dickinson</td>
			<td>340334</td>
			<td>Box of 50 absolute counting tubes</td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>foetal bovine serum (FBS)</td>
			<td>Scientifix</td>
			<td>FBS500-S (lot# 010101-1)</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Medicago AB</td>
			<td>09-9400-100</td>
			<td></td>
		</tr>
		<tr>
			<td>23Gx1&#188; Needle</td>
			<td>Becton Dickinson</td>
			<td>302008</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>302 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fischer 344 Rat</td>
			<td>Animal Resources Centre, Perth Australia</td>
			<td>F344</td>
			<td></td>
		</tr>
		<tr>
			<td>I.S.O (Isoflurane USP)</td>
			<td>Veterinary Companys Australia (VCA)&#160;</td>
			<td>B7058</td>
			<td></td>
		</tr>
		<tr>
			<td>II-45 Rat Mesothelioma line</td>
			<td>Zurich University</td>
			<td></td>
			<td>Note: The cell line was given as a gift and is not commercially available at the ATCC</td>
		</tr>
		<tr>
			<td>FACSCalibur&#8482; 4 colour</td>
			<td>Becton Dickinson</td>
			<td>342975</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS-HCL</td>
			<td>SIGMA</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>SIGMA</td>
			<td>9718</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaesthetic Machine (The stinger)</td>
			<td>Advanced Anaesthesia specialists</td>
			<td>#00449</td>
			<td></td>
		</tr>
	</tbody>
</table>

orthotopic implantation and peripheral immune cell monitoring in the ii-45 syngeneic rat mesothelioma model

Den enorme stigning i interesse i immun-baserede behandlinger for cancer, såsom vacciner og immune checkpoint inhibitorer og øget forståelse af den rolle, tumormikromiljøet i behandlingsrespons, tilsammen peger på behovet for immun-kompetente ortotopisk modeller for præklinisk afprøvning af disse nye behandlinger. Dette papir viser, hvordan at etablere en orthotopisk immun-kompetent rotte model af pleura malignt mesotheliom. Overvågning sygdomsprogression i ortotopisk modeller er forvirret af den interne placering af tumorer. Til længderetningen overvåge sygdommen og dens virkning på cirkulerende immunceller i denne og andre rottemodeller af cancer, et enkelt rør flowcytometriassayet kræver kun 25 pi helblod beskrives. Dette giver nøjagtig kvantificering af syv immunparametre: Samlet lymfocytter, monocytter og neutrofiler, samt T-celle-undergrupper CD4 og CD8, B-celler og naturlige dræberceller. Forskellige subsets af disse parametre er nyttige i forskellige forhold og modeller, med neutrofil til lymfocyt-forhold har den største anvendelighed til overvågning sygdomsprogression i mesotheliom model. Analyse cirkulerende immun celle niveauer ved hjælp af denne enkelt rør metode kan også hjælpe med at overvåge responsen på immun-baserede behandlinger og forstå de underliggende mekanismer, der fører til succes eller fiasko af behandlingen.

Den anvendte metode i dette dokument til frembringelse af en ortotopisk model af pleural mesotheliom hjælp II-45-celler resulterede i dyr bukke under for mesotheliom på en reproducerbar og hurtig tidsramme, med ingen rotter døde på grund af implantation metode. Titrering af antallet af celler implanteret fastslået, at 1x 10 3 celler var den krævede minimum for en fuldt penetrerende model (100% engraftment). De forskellige antal celler implanteret i rotterne ændret tidsforløbet af sygdommen uden at syn...

Watch this Scientific Journal Video about Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model at JoVE.com

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

Dannelsen af ​​en ortotopisk rottemodel for pleural malignt mesotheliom efter implantation af II-45 mesotheliom celler i pleurahulen af ​​immunkompetente rotter præsenteres. En flowcytometrisk metode til at analysere syv immuncelleundergrupper i disse dyr fra en 25 pi blodprøve er også beskrevet.

Orthotopisk Implantation og perifert immuncelle Overvågning i II-45 Syngene Rat mesotheliom Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

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