Name: primary orthotopic glioma xenografts recapitulate infiltrative growth and isocitrate dehydrogenase i mutation
Uploaded: 2014-01-14T13:45:00
Description: Watch this Scientific Journal Video about Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation at JoVE.com

Vi står spesielt i gjeld til pasienter ved Vanderbilt University Medical Center som ga uvurderlig forskningsmateriale til Molecular Neurosurgical Tissue Bank. Vi takker de som etablerte og vedlikeholder Tissue Bank, Reid C. Thompson MD (prosjektleder), Cherryl Kinnard RN (forskningssykepleier) og Larry A. Pierce MS (leder). Histologiske tjenester ble delvis utført av Vanderbilt University Medical Center (VUMC) Translational Pathology Shared Resource (støttet av pris 5P30 CA068485 til Vanderbilt-Ingram Cancer Center). Dette arbeidet ble støttet av tilskudd til MKC fra NINDS (1R21NS070139), Burroughs Wellcome Fund og VMC utviklingsfond. MKC støttes av Institutt for veteransaker, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development gjennom tilskudd 1 I01 BX000744-01. Innholdet representerer ikke synspunktene til departementet for veteransaker eller USAs regjering.

1. Forberedelse av tumorcellefjæring
Merk: Hensiktsmessige institusjonelle godkjenninger for bruk av pasientmateriale og dyr er nødvendig for å etablere og vedlikeholde primære ortotopiske glioma xenografter. Ved Vanderbilt University Medical Center samles resected tumormateriale som er i overkant av det som kreves for diagnostiske formål, med pasientsamtykke for et forskningsvevslager. Prøver er merket med et randomisert 5-sifret REDcap-databasenummer og alle pasientspes...

<ol>
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Kultiverte cellelinjer, xenografter og genmodifiserte mus er de vanligste metodene for modellering av gliomer, og det er klare fordeler og begrensninger for hvert modellsystem3,13,14. Relevante fordeler med primære ortotopiske glioma xenografts inkluderer infiltrativ vekst som typifies diffuse gliomer og oppbevaring av genetiske endringer og viktige signalmekanismer som kan være svært vanskelig å opprettholde i dyrkede gliomaceller. For eksempel kan jegsocitrate dehydrogenase mutasjoner og Sonic p...

Ondartede gliomer er primære glialtumorer i sentralnervesystemet som forekommer i hjernen og noen ganger ryggmargen. Gliomas er klassifisert av Verdens helseorganisasjon (WHO) i henhold til histologiske likheter med astrocytter, oligodendrocytter eller ependymale celler og deretter numerisk gradert (I til IV) for patologiske egenskaper av malignitet. De vanligste histologiske undertypene er astrocytomer, oligodendrogliomer og blandede oligoastrocytomer. Ondartede gliomer som omfatter WHO-karakterer II til IV er preget av invasiv vekst og tilbakevendende til dagens terapier. Hvert år i USA diagnostiseres ca. 15 750 personer med ondartet gliom og anslagsvis 12 740 pasienter bukker under for denne sykdommen. Denne statistikken fremhever den spesielt dødelige karakteren av ondartede gliomer og viktig behov for forbedret terapeutisk effekt.
Kreftmodeller er avgjørende for å undersøke tumorbiologi og terapier. Cellelinjer for human kreft representerer et viktig første skritt for in vitro manipulasjoner og in vivo xenografting studier (sekundære xenografts)1. Imidlertid gjennomgår standard kreftcellekulturer fenotypisk og genotypisk transformasjon2-4 som kanskje ikke gjenopprettes i sekundære xenografts5. Videre kan genetiske endringer som isocitrate dehydrogenase (IDH) mutasjoner6, distinkte stamcellepopulasjoner7 og avhengighet av viktige signalveier8 gå tapt i kreftcellekulturer. Genomiske profiler kan opprettholdes i bedre grad i kreftsfærekultur, men klarer fortsatt ikke å speile genotypen av primærsvulster2,3. Direkte ortotopisk transplantasjon negerer påvirkningen av in vitrokultur, gir et riktig mikromiljø og bevarer integriteten til tumorinitierende celler9,10. Derfor representerer primære xenografts en kraftig og relevant preklinisk modell for grundig testing av målrettede agenter for å hjelpe til med rasjonell utforming av fremtidige kliniske studier5,11,12.

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			Phosphate buffered saline
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			<td style="width:142px;">
			Life Technologies
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			<td style="width:140px;">
			14040-133
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			<td style="width:211px;">
			Papain dissociation system
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			<td style="width:142px;">
			Worthington Biochemical Corp.
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			LK003150
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			<td style="width:97px;">
			
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			Trypan blue solution 0.4%
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			Life Technologies
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			15250061
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			Ketamine HCl
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			Obtained from institutional pharmacy or local veterinary supply company
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			Xylazine HCl
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			Ketoprofen
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			Ophthalmic ointment
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			Povidone-iodine
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			<td style="width:142px;">
			Fisher Scientific
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			<td style="width:140px;">
			190061617
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			Cryopreservation medium and proliferation supplement
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			StemCell Technologies
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			05751
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			0.2% Heparin sodium salt in PBS
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			<td style="width:142px;">
			StemCell Technologies
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			<td style="width:140px;">
			07980
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			Penicillin-streptomycin
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			<td style="width:142px;">
			Life Technologies
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			15140-122
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			Dimethyl sulfoxide
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			Sigma-Aldrich
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			D6250-5X10ML
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			NOD.Cg-Prkdcscid I/2rgtm1Wjl/SzJ mice
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			The Jackson Laboratory
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			005557
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			NSG mice
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			Anti-human vimentin antibody
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			<td style="width:142px;">
			Dako
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			M7020
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			Use 1:200 to 1:800
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			Anti-human IDH1 R132H antibody
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			<td style="width:142px;">
			Dianova
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			<td style="width:140px;">
			DIA-H09
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			<td style="width:97px;">
			Use 1:100 to 1:400
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			Comments
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			Centrifuge with swinging bucket rotor
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			Pipetter with dispensing speed control
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			Disposable hemocytometer
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			<td style="width:140px;">
			Fisher Scientific
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			<td style="width:143px;">
			22-600-100
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			<td style="width:210px;">
			Sterile surgical gloves
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			<td style="width:140px;">
			Fisher Scientific
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			<td style="width:143px;">
			11-388128
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			<td style="width:97px;">
			
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			Disposable gown
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			<td style="width:140px;">
			Fisher Scientific
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			18-567
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			Surgical mask
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			<td style="width:140px;">
			Fisher Scientific
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			<td style="width:143px;">
			19-120-1256
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			Tuberculin syringe
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			<td style="width:140px;">
			BD
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			305620
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			Alcohol pads
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			<td style="width:140px;">
			Fisher Scientific
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			<td style="width:143px;">
			22-246-073
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			Portable electronic scale
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			<td style="width:140px;">
			Fisher Scientific
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			<td style="width:143px;">
			01-919-33
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			Zoom stereomicroscope
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			Surgical clipper
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			<td style="width:140px;">
			Stoelting
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			<td style="width:143px;">
			51465
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			<td style="width:97px;">
			
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			Scalpel handle
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			<td style="width:140px;">
			Fine Science Tools
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			<td style="width:143px;">
			10003-12
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			Scalpel blades, #10
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			<td style="width:210px;">
			Stereotaxic instrument
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			<td style="width:140px;">
			Stoelting
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			<td style="width:143px;">
			51730
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			<td style="width:97px;">
			
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			<td style="width:210px;">
			High-speed drill
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			<td style="width:140px;">
			Stoelting
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			<td style="width:143px;">
			51449
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			<td style="width:97px;">
			
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		</tr>
		<tr>
			<td style="width:210px;">Drill bit, 0.6 mm</td>
			<td style="width:140px;">Stoelting</td>
			<td style="width:143px;">514552</td>
			<td style="width:97px;"></td>
		</tr>
		<tr>
			<td style="width:210px;">
			Hamilton syringe
			</td>
			<td style="width:140px;">
			Hamilton
			</td>
			<td style="width:143px;">
			80336
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Autoclip, 9 mm
			</td>
			<td style="width:140px;">
			BD
			</td>
			<td style="width:143px;">
			427630
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Circulating water warming pad
			</td>
			<td style="width:140px;">
			Kent Scientific
			</td>
			<td style="width:143px;">
			TP-700
			TP-1215EA
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Hot bead dry sterilizer
			</td>
			<td style="width:140px;">
			Kent Scientific
			</td>
			<td style="width:143px;">
			INS300850
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Surgical scissors
			</td>
			<td style="width:140px;">
			Fine Science Tools
			</td>
			<td style="width:143px;">
			14101-14
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Fine scissors
			</td>
			<td style="width:140px;">
			Fine Science Tools
			</td>
			<td style="width:143px;">
			14094-11
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Spring scissors
			</td>
			<td style="width:140px;">
			Fine Science Tools
			</td>
			<td style="width:143px;">
			15018-10
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Dumont forceps
			</td>
			<td style="width:140px;">
			Fine Science Tools
			</td>
			<td style="width:143px;">
			11251-30
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Semimicro spatulas
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			14374
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Mouse brain slicer matrix
			</td>
			<td style="width:140px;">
			Zivic Instruments
			</td>
			<td style="width:143px;">
			BSMAS002-1
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
			<td style="width:210px;">
			Cryogenic storage vials
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			12-567-501
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
	</tbody>
</table>

primary orthotopic glioma xenografts recapitulate infiltrative growth and isocitrate dehydrogenase i mutation

Ondartede gliomer utgjør en heterogen gruppe svært infiltrative glial neoplasmer med distinkte kliniske og molekylære egenskaper. Primære ortotopiske fremmedegrafter rekapitulerer histopatologiske og molekylære egenskaper av ondartede gliomaundertyper i prekliniske dyremodeller. For å modellere WHO grader III og IV ondartede gliomer i transplantasjonsanalyser, blir humane tumorceller xenograftert til et ortotopisk sted, hjernen, av immunkompromitterte mus. I motsetning til sekundære xenografter som bruker dyrkede tumorceller, blir menneskelige gliomaceller dissosiert fra resekterte prøver og transplantert uten forutgående passasje i vevskultur for å generere primære xenografter. Prosedyren i denne rapporten beskriver tumorprøvepreparering, intrakraniell transplantasjon til immunkompromitterte mus, overvåking for tumor engraftment og tumorhøsting for etterfølgende passasje til mottakerdyr eller analyse. Tumorcellepreparat krever 2 timer og kirurgisk prosedyre krever 20 min / dyr.

Dissosierte gliomaceller transplanteres direkte inn i hjernen til immunkompromitterte mus for å oppnå primære ortotopiske xenograftlinjer. Hver tumorprøve tildeles et randomisert tall før transplantasjon, som en del av deidentifiseringsprosessen for å fjerne beskyttet helseinformasjon. Vi bruker et 5-sifret REDcap databasenummer til dette formålet. Figur 1 illustrerer prosessen og nomenklaturen for å etablere en xenograftlinje fra et glioblastom (GBM 17182) med isocitrate dehydrogenase 1 (IDH1) m...

Watch this Scientific Journal Video about Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation at JoVE.com

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Ondartede gliomer utgjør en heterogen gruppe svært infiltrative glial neoplasmer med distinkte kliniske og molekylære egenskaper. Primære ortotopiske fremmedegrafter rekapitulerer histopatologiske og molekylære egenskaper av ondartede gliomaundertyper i prekliniske dyremodeller.

Primære ortotopiske glioma xenografter rekapitulere infiltrerende vekst og isokitrate dehydrogenase I mutasjon

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

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Medicine

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

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Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

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Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Imaging Glioma Initiation<em&gt; In Vivo</em&gt; Gjennom en polert og Forsterket Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal Ekkokardiografi og Pulsed-bølge Doppler ultralyd i et Rabbit Modell av intrauterine vekst begrensning

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

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Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

Generering av Subkutan og Intrahepatisk menneskeleverkreft xenografts i immunodeficient Mus

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Minimal Invasive Etablering av Murint orthotopic Blære xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Orthotopic Injeksjon av brystkreft celler inn i Mammary Fat Pad av mus å studere tumorvekst.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Opprette Anatomisk nøyaktige og reproduserbare Intrakraniell xenografts of Human Brain tumorer

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...