Name: fluorescence molecular tomography for in vivo imaging of glioblastoma xenografts
Uploaded: 2018-04-26T14:00:00
Description: Watch this Scientific Journal Video about Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts at JoVE.com

Vi takker Dr. Frederick Lang, MD Anderson Cancer Center for GBM-PDX neurospheres. Dette arbejde blev støttet af nederlag GBM forskning Collaborative, et datterselskab af National hjerne Tumor Society (Frank Furnari), R01-NS080939 (Frank Furnari), James S. McDonnell Foundation (Frank Furnari); Jorge Benitez blev støttet af en award fra den amerikanske hjerne Tumor Association (ABTA); Ciro Zanca blev delvist støttet af en amerikansk-italienske Cancer Foundation postdoc research fellowship. Frank Furnari modtager løn og supplerende støtte fra Ludwig Institut for kræftforskning.

Anvendelsen af eksperimentel forskning dyr og smitsomme agenser, såsom lentivirus at transduce kræftceller, kræver forudgående godkendelse af programmet institutionelle dyrs pleje og institutionelle biosikkerhed udvalget. Denne protokol følger retningslinjerne dyrs pleje af University of California San Diego (UCSD).
1. mærkning af glioblastom celler med FP635 eller FP720 konstruktion
<ol><li>Producere og rense lentivirus i henhold til protokollen, beskrevet af Tiscornia et al....

<ol>
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Tumor xenografts har været flittigt brugt i kræftforskning og har udviklet en række veletablerede Billeddannende teknikker: BLI; magnetisk resonans imaging (MR); Positron emissions tomografi (PET), beregnet tomografi (CT); FMT. Hver af disse tilgange kommer med fordele og ulemper, men i sidste ende supplerer hinanden med typen af oplysningerne. En af de mest almindeligt anvendte i vivo imaging-teknologi er BLI5,6,7<s...

Kræft er en af de førende årsager til sygdom-relaterede dødsfald blandt mennesker i den industrialiserede verden. Med en ekstremt høje dødstal er nye behandlinger tvingende nødvendigt. Glioblastom multiforme (GBM) er en ekstremt dødelige form for kræft i hjernen, sammensat af heterogene befolkninger af hjernen tumor, stromale og immun celler. Ifølge Central hjerne tumor registreringsdatabasen i USA er forekomsten af primære maligne og ikke-maligne hjernetumorer ca 22 tilfælde pr. 100.000. Ca 11.000 nye sager forventes at blive diagnosticeret i USA i 20171.
Prækliniske undersøgelser undersøge sandsynligheden for et stof, procedure eller behandling for at være effektiv inden prøvningen i mennesker. Et af de tidligste laboratorium skridt i prækliniske undersøgelser er at identificere potentielle molekylære mål for narkotikabehandling ved hjælp af kræftceller implanteret i en værtsorganisme, defineret som menneskelige xenograft modeller. I denne kontekst, har intrakraniel hjerne tumor xenograft modeller ved hjælp af patient-afledte xenografts (PDXs) været meget anvendt til at studere hjernen tumor biology, progression, evolution og terapeutiske reaktion, og for nylig for biomarkører udvikling, narkotika screening, og personlig medicin2,3,4.
En af de mest overkommelige og ikke-invasiv i vivo imaging metoder til at overvåge intrakranielle xenografts er bioluminescens imaging (BLI)5,6,7,8. Men nogle BLI begrænsninger omfatter substrat administration og tilgængelighed, enzym stabilitet, og lys dæmper og spredning under imaging erhvervelse9. Her rapporterer vi den infrarøde FMT som alternativ tænkelig metode til at overvåge prækliniske glioblastom modeller. I denne metode, signal erhvervelse og kvantificering af intracranially implanterede PDXs, udtrykker en nær-infrarødt fluorescerende protein iRFP72010,11 (herefter betegnet som FP720) eller turboFP635 (herefter benævnt som FP635), udføres med en FMT imaging system. Ved hjælp af FMT teknologi, overvåget orthotopic tumorer kan være i vivo før, under eller efter behandling, i en ikke-invasiv, substrat-fri og kvantitative måde for prækliniske observationer.

<table border="0" cellpadding="0" cellspacing="0" width="1489">
	<tbody>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">DMEM/High Glucose&#160;</td>
			<td style="width:191px;">HyClone/GE</td>
			<td style="width:213px;">SH30022.1</td>
			<td style="width:744px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">DMEM/F12 1:1&#160;</td>
			<td style="width:191px;">Gibco</td>
			<td>11320-082</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">FBS</td>
			<td style="width:191px;">HyClone/GE</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Accutase</td>
			<td style="width:191px;">Innovative cell technologies</td>
			<td style="width:213px;">AT-104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Trypsin</td>
			<td style="width:191px;">HyClone/GE</td>
			<td style="width:213px;">SH30236.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">B27 supplement</td>
			<td style="width:191px;">Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">human recombinant EGF&#160;</td>
			<td>Stemcell Technologies</td>
			<td style="width:213px;">2633</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">human recombinant FGF</td>
			<td>Stemcell Technologies</td>
			<td style="width:213px;">2634</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">DPBS</td>
			<td style="width:191px;">Corning</td>
			<td style="width:213px;">21-031-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">FACS tubes</td>
			<td style="width:191px;">Falcon</td>
			<td style="width:213px;">352235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">DAPI</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:213px;">62248</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Blasticidin</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:213px;">A1113903</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">p24 ELISA&#160;</td>
			<td style="width:191px;">Clontech</td>
			<td style="width:213px;">632200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Xylazine</td>
			<td style="width:191px;">Akorn</td>
			<td style="width:213px;">NDC 59399-110-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Ketamine</td>
			<td style="width:191px;">Zoetis</td>
			<td style="width:213px;">NADA 043-403</td>
			<td>Controlled substance</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Ointment</td>
			<td style="width:191px;">Dechron</td>
			<td style="width:213px;">NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Absorbable suture</td>
			<td style="width:191px;">CpMedical</td>
			<td style="width:213px;">VQ392</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">5 ul syringe</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:213px;">26200-U</td>
			<td>Catalog number as sold by Sigma-Aldrich</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Cell Sorter</td>
			<td style="width:191px;">Sony</td>
			<td style="width:213px;">SH8007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Mouse stereotaxic frame&#160;</td>
			<td style="width:191px;">Stoelting</td>
			<td style="width:213px;">51730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Motorized stereotaxic injector</td>
			<td style="width:191px;">Stoelting</td>
			<td style="width:213px;">53311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Micromotor hand-held drill</td>
			<td style="width:191px;">Foredom</td>
			<td style="width:213px;">K1070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Mouse warming pad&#160;</td>
			<td style="width:191px;">Ken Scientific Corporation</td>
			<td style="width:213px;">TP-22G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Fluorescence Tomography System&#160;</td>
			<td>PerkinElmer</td>
			<td style="width:213px;">FMT 2500 XL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">TrueQuant Imaging Software&#160;</td>
			<td>Perkin Elmer&#160;</td>
			<td style="width:213px;">7005319</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Ultra-centrifuge Optima L-80 XP</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:213px;">392049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Tissue Culture 100mm Dishes</td>
			<td style="width:191px;">Olympus Plastics</td>
			<td style="width:213px;">25-202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Tissue Culture 150mm Dishes</td>
			<td style="width:191px;">Olympus Plastics</td>
			<td style="width:213px;">25-203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Tissue Culture Flasks T75</td>
			<td style="width:191px;">Corning</td>
			<td style="width:213px;">430720U</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">50 mL conical tubes</td>
			<td style="width:191px;">Corning</td>
			<td style="width:213px;">430290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">15 mL conical tubes</td>
			<td style="width:191px;">Olympus Plastics</td>
			<td style="width:213px;">28-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Centrifuge Avanti J-20</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:213px;">J320XP-IM-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Tube, Polypropylene, Thinwall, 5.0 mL</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:213px;">326819</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Tube, Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:213px;">326823</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:343px;">Athymic nude mice</td>
			<td style="width:191px;">Charles River Laboratories</td>
			<td style="width:213px;">Strain Code&#160; 490 (Homozygous)</td>
			<td>Prior approval by the Institutional Animal Care Program and by the Institutional Biosafety Committee required.&#160;&#160;&#160;</td>
		</tr>
	</tbody>
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fluorescence molecular tomography for in vivo imaging of glioblastoma xenografts

Tumorigenisitetsforsøg er kræftceller evne til at danne en tumor masse. En udbredt tilgang til at afgøre, om cellerne er tumorfremkaldende er ved at indsprøjte immundefekte mus subkutant med kræftceller og måle tumor massen, når det bliver synlig og håndgribelig. Orthotopic injektioner af kræftceller har til formål at indføre xenograft i det mikromiljø, der mest ligner væv af oprindelsen af tumor undersøges. Hjernen kræftforskning kræver intrakraniel indsprøjtning af kræftceller til at tillade tumordannelse og analyse i den unikke mikromiljø i hjernen. I vivo billeddannelse af intrakranielle xenografts overvåger øjeblikkeligt tumor massen af orthotopically aflægger mus. Her rapporterer vi brugen af fluorescens molekylære tomografi (FMT) af hjernen tumor xenografts. Kræftcellerne er først transduced med nær infrarød fluorescerende proteiner og derefter injiceres i hjernen hos immunkompromitterede mus. Dyrene er derefter scannet for at få kvantitative oplysninger om tumor massen over en længere periode. Celle pre mærkning giver mulighed for omkostningseffektiv, reproducerbare og pålidelig kvantificering af tumor byrde inden for hver mus. Vi fjernede behovet for intravenøs imaging substrater, og således reduceret stress på dyrene. En begrænsning af denne tilgang er repræsenteret ved manglende evne til at detektere meget små masserne; Det har imidlertid bedre opløsning for større masse end andre teknikker. Det kan anvendes til at vurdere effekten af en behandling eller genetiske ændringer af gliom cellelinjer og patient-afledte prøver.

Glioblastom celler U87EGFRvIII (U87 celler over udtrykker EGF receptoren variant III) blev kulturperler ifølge trin 1.2. Lentivirus blev produceret og renset ifølge trin 1.1. Viral koncentrationen var bestemt af p24 ELISA analyse. Celler var transduced med lentivirus transporterer infrarød fluorescerende proteiner ifølge trin 1.8. Plasmid kodning FP72010,11 blev venligst leveret af Dr. V.V. Verkhusha og FP635 vektor blev købt...

Watch this Scientific Journal Video about Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts at JoVE.com

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intrakraniel injektion af tumorceller har været anvendt i kræftforskning for at studere hjernen tumor biology, progression, evolution og terapeutisk respons. Her præsenterer vi fluorescens molekylære tomografi af tumor xenografts, der giver real-time intravital imaging og kvantificering af en tumor masse i prækliniske glioblastom modeller.

Fluorescens molekylære tomografi for In Vivo billeddannelse af glioblastom Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

The overall goal of this experimental procedure is to establish a preclinical brain tumor imaging model using fluorescence molecular tomography or FMT. This method can help answer key questions in the fields of brain tumor biology, cancer research, and drug discovery about tumor aggressiveness, heterogeneity, and treatment responsiveness. The main advantage of this technique is that the tumors can be monitored in vivo before, during, or after treatment in a non-invasive, suture free, and quantitative manner.Another advantage of this technique is the possibility to apply to other types of xenograft models implanted in different organs of the animal. Begin by seeding one times 10 to the sixth glioblastoma cells in five milliliters of medium into a 10 centimeter dish for 24 hour incubation in a cell culture incubator. The next morning, transduce the cells with lentivirus, expressing an appropriate fluorescent protein of interest at a multiplicity of infection of five and return the cells to the incubator.After 24 hours, replace the supernatant with five milliliters of fresh medium and incubate the culture for another 48 hours. At the end of the incubation, replace the medium with three to five milliliters of tripson for 10 to 15 minutes at 37 degrees Celsius followed by careful pipetting to fully dissociate the detached cells. Collect the cells by centrifugation and re-suspend the pellet in 500 microliters of sorting solution.Split the cells into the appropriate number of fax tubes and co-stain the cells with DAPI for dead cell exclusion. Then load the tubes onto the flow cytometer and sort the live cells according to their fluorescent construct expression into a 15 milliliter conical tube containing fresh sorting solution. After centrifugation, re-suspend the construct positive, DAPI negative cells in five milliliters of culture medium and seed them onto a new 10 centimeter dish for their culture at 37 degrees Celsius for 48 to 72 hours.At the end of the incubation, split the cells for seeding into multiple dishes for subsequent in vivo experiments. On the day of the injection, dissociate the fluorescent positive gliomacells into a single cell suspension and re-suspend them at a 0.5 or one times 10 to the sixth cells per two to five microliters of PBS per injection per animal concentration in a 1.5 milliliter microcentrifuge tube on ice. After confirming a lack of response to toe pinch, apply ointment to the eyes of the immuno-deficient Athymic Nude recipient mouse and load a five microliter Hamilton syringe equipped with a blunt tipped needle with the cells for the injection.Mount the syringe into a probe holder and place the mouse in a small, animal sterotactic frame. Fix the head using the ear bars and the incisor adaptor and disinfect the head with 70%ethanol and batadine solution. Using a small scalpel, make a middle incision and separate the skin and connective tissues.Using the micromanipulator, place the Hamilton syringe on the bregma point and move the probe holder one millimeter anteroposterior and two millimeters lateral from the bregma point. After marking the position with a pencil, use a micromotor handheld drill to carefully make a hole in the skull, applying a slight downward pressure until the blood vessels become visible. Introduce the needle into the burr hole three millimeters below the pial surface and inject the cells at a one microliter per minute flow right.When the full experimental cell volume has been injected, gradually remove the needle at a one millimeter per minute ascension rate and clean the injection site with 70%ethanol. Then close the skin wound according to standard protocols and monitor the animal on a warming pad until it has fully recovered. To image the injected cells, place the anesthetized recipient animal in the imaging cassette of a fluorescence molecular tomography imager, head adapter first in the prone position, with the head in the center of the cassette.With the cassette closed, tighten the adjustment knobs to 17 millimeters. When the animal is secure, insert the cassette into the internal docking station and open the imager and analyzer software. In the experimental tab window, select the appropriate database and study group and open the scan tab window.Click select subject to select the subject to image and select the laser channel in the laser channel panel. Click capture to acquire an image and click and drag that scan field in the captured image to identify the source locations. Check the add to reconstruction que option and click scan in the scan tab window.When the scanning is complete, remove the imaging cassette from the docking station and return the animal to its cage with monitoring until full recovery. To analyze the images, in the imager and analyzer software open the analysis tab window and click the plus sign button in the data set selection panel to load the data set and subject for analysis. Then use the ellipsoid icon to select the region of interest and right click the threshold column to adjust the threshold to zero in the statistic data panel.Glioblastoma cells labeled with near-infrared fluorescent proteins, as just demonstrated, exhibit distinct fluorescent profiles that can be distinguished by fluorescence molecular tomography, even up to five days after injection. Further, the lack of background signal between the constructs facilitates dual in vivo imaging of cancer cell populations of interest. In this experiment, fluorescent construct labeled glioblastoma spheres co-transduced with lentivirus encoding SHRNA targeting death-domain associated protein were injected into recipient immunodeficient Athymic Nude mice, as just demonstrated.Reverse transcription quantitative preliminaries chain reaction confirmed the efficacy of the SHRNA targeting in both cancer cell lines and fluorescence molecular tomography revealed a decrease in fluorescence signal in the mice engrafted with SHRNA glioblastoma spheres compared to animals implanted with SH control transduced cells. After watching this video, you should have a good understanding of how to generate preclinical data for brain cancer research using a fluorescence molecular tomography system.

Research

JoVE Journal

Cancer Research

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

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Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...