In vivo billeddannelse og Kvantitation af værten Angiogen respons i Zebrafish tumor Xenografts
Summary
Formålet med denne metode er at generere en in vivo model af tumor angiogenese ved xenografting pattedyrs tumorceller i en zebra embryo, der har fluorescently-mærkede blodkar. Ved billeddannelse af xenograft og tilhørende beholdere kan der opnås en kvantitativ måling af angiogen respons.
Abstract
Tumor angiogenese er et centralt mål for anti-cancer terapi og denne metode er blevet udviklet til at give en ny model til at studere denne proces in vivo. En zebra xenograft er skabt ved at implantering pattedyr tumorceller i perivitelline rum af to dage-post-befrugtning Zebra embryoner, efterfulgt af måling af omfanget af den angiogene respons observeret ved et eksperimentelt endepunkt op til to dage efter implantation. Den vigtigste fordel ved denne metode er evnen til præcist at kvantificere Zebra Host angiogenetisk respons på transplantatet. Dette muliggør detaljeret undersøgelse af de molekylære mekanismer samt værten vs tumor bidrag til angiogen respons. De xenografede embryoner kan underkastes en bred vifte af behandlinger, såsom inkubation med potentielle anti-angiogenesis narkotika, for at undersøge strategier til at hæmme tumor angiogenese. Den angiogene respons kan også være levende-imaged for at undersøge mere dynamiske cellulære processer. Den relativt krævende eksperimentelle teknik, billige vedligeholdelsesomkostninger for Zebra og kort eksperimentel tidslinje gør denne model specielt nyttig til udvikling af strategier til at manipulere tumor angiogenese.
Introduction
Angiogenesis er en af de klassiske kendetegn for kræft og repræsenterer et mål for anti-cancer terapi1,2. For at studere denne proces, xenograft modeller af kræft er blevet skabt ved at implantering pattedyr tumorceller i dyr som mus3. En zebra xenograft model er også blevet udviklet, som involverer implantation af tumorceller i 2 dage efter befrugtning (dpi) Zebra som resulterer i hurtig vækst af Zebra blodkarrene ind i xenograft4.
Denne protokol beskriver en in vivo Zebra embryo tumor xenograft-model, hvor den angiogene respons kan nøjagtigt kvantiteres på tværs af hele xenograft. Denne metode gør det muligt for investigator at undersøge, in vivo, de molekylære mekanismer, der understøtter tumor angiogen respons. Den genetiske tracerbarhed af Zebra tillader værten bidrag, der skal afhøres, mens udvælgelsen af forskellige tumorcellelinjer tillader tumor bidrag til angiogenese også undersøges5,6,7. Da Zebra larver er gennemtrængelige for små molekyler, kan der desuden anvendes specifikke pathway-hæmmere, eller lægemiddelbiblioteker kan screenes for at identificere nye hæmmere af tumor angiogenese8,9,10, 11.
Zebra embryo xenograft-modellen præsenterer unikke fordele sammenlignet med andre pattedyr-xenograft-modeller. Zebrafish xenografter er billigere og lettere at udføre, et stort antal dyr kan undersøges og levende celle billeddannelse tillader detaljeret undersøgelse af celle adfærd4. I modsætning til andre in vivo-modeller, som kræver op til flere uger for at observere signifikant fartøjs vækst, kan angiogenese i Zebra xenografter observeres inden for 24 timer efter implantation3,4. Men manglen på et adaptivt immunsystem i embryonale zebrafish, mens det er gavnligt at vedligeholde xenograft, betyder, at det adaptive immunrespons og dets bidrag til tumor angiogenese ikke kan undersøges. Desuden manglen på tumor stromale celler, den manglende evne til at ortotopisk implantat tumoren og forskellen i vedligeholdelses temperatur mellem Zebra og pattedyrceller er potentielle svagheder ved denne metode. Ikke desto mindre, den modtagelighed af denne model for levende billeddannelse og evnen til præcist at kvantificere den angiogene respons gør det enestående gavnligt for at studere de cellulære processer, der regulerer tumor angiogenese in vivo.
Protocol
Representative Results
Discussion
Det første kritiske trin i protokollen er implantation af tumorceller. Det er vigtigt, at cellerne injiceres på et sted, der gør det muligt for xenograft at implantatet med succes i embryonet uden at gøre embryonet edematous. En injektion, der er for forreste kan tillade cellerne at bevæge sig mod hjertet, blokerer blodbanen og fører til ødem, mens en injektion, der er for posterior vil resultere i en dårligt implanteret xenograft. En forreste injektion er bedst undgås ved at indsætte nålen gennem blommesække…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker hr. alhad mahagaonkar for forvaltningen af University of Auckland Zebra facilitet og biomedicinsk billed forskning enhed, School of Medical Sciences, University of Auckland, for assistance i time-lapse confokal mikroskopi. Dette arbejde blev støttet af et sundheds Forskningsråd i New Zealands projekt bevilling (14/105), et kongeligt selskab i New Zealands Marsden fund-projekt bevilling (UOA1602) og et projekttilskud fra Auckland Medical Research Foundation (1116012) tildelt J.W.A.
Materials
| Air cylinder | BOC | 011G | Xenotransplantation |
| B16-F1 cells | ATCC | Cell culture | |
| BD Matrigel LDEV-free (extracellular matrix mixture) | Corning | 356235 | Xenotransplantation |
| Borosillicate glass capillaries | Warner Instruments | G100T-4 | OD=1.00 mm, ID=0.78 mm, Length =10 cm Cell injection |
| Cell culture dish -35 mm diameter | Thermofisher NZ | NUN153066 | Fish husbandry |
| Cell culture dish -100 mm diameter | Sigma-Aldrich | CLS430167-500EA | Fish husbandry |
| Cell culture flask 75 cm2 | In Vitro Technologies | COR430641 | Cell culture |
| CellTracker Green | Invitrogen | C2925 | Cell labelling, Stock concentration (10 mM in DMSO), working concentration (0.2 μM in serum-free media) |
| Dimethyl sulfoxide | Sigma-Aldrich | D8418 | Drug treatment, Cell labelling |
| E3 Media (60x in 2 L of water) 34.8 g NaCl 1.6 g KCl 5.8 g CaCl2·2H2O 9.78 g MgCl2·6H2O adjust to pH 7.2 with NaOH |
In house [1] | Fish husbandry | |
| Ethyl-3-aminobenzoate methanesulfonate (Tricaine) | Sigma-Aldrich | E10521 | Xenotransplantation, Imaging |
| Filter tip 1000 μL | VWR | 732-1491 | Used during multiple steps |
| Filter tip 200 μL | VWR | 732-1489 | Used during multiple steps |
| Filter tip 10 μL | VWR | 732-1487 | Used during multiple steps |
| Fluorescence microscope | Leica | MZ16FA | Preparation of embryos |
| FBS (NZ origin) | Thermofisher Scientific | 10091148 | Cell culture |
| Gloves | Any commercial brand | Used during multiple steps | |
| Haemocytometer cell counting chamber Improved Neubauer | HawksleyVet | AC1000 | Xenotransplantation |
| Heraeus Multifuge X3R Centrifuge | Thermofisher Scientific | 75004500 | Cell culture, Cell labelling |
| Hoechst 33342 | Thermofisher Scientific | 62249 | Cell labelling, Stock concentration (1 mg/ml in DMSO), working concentration (6 μg/ml in serum-free media) |
| Low Melting Point, UltraPure Agarose | Thermofisher Scientific | 16520050 | Imaging |
| Methycellulose | Sigma-Aldrich | 9004 67 5 | Xenotransplantation |
| Methylene blue | sigma-Aldrich | M9140 | Fish husbandry |
| Microloader 0.5-20 μL pipette tip for loading microcapillaries | Eppendorf | 5242956003 | Xenotransplantation |
| Micropipettes | Any commercial brand | Used during multiple steps | |
| Micropipette puller P 87 | Sutter Instruments | Xenotransplantation | |
| Microscope cage incubator | Okolab | Time-lapse imaging | |
| Microwave | Any commercial brand | Imaging | |
| Mineral oil | Sigma-Aldrich | M3516 | Xenotransplantation |
| Minimal Essential Media (MEM) – alpha | Thermofisher Scientfic | 12561056 | Cell Culture |
| MPPI-2 Pressure Injector | Applied Scientific Instrumentation | Xenotransplantation | |
| Narishige micromanipulator | Narishige Group | Xenotransplantation | |
| Nikon D Eclipse C1 Confocal Microscope | Nikon | Imaging | |
| N-Phenylthiourea (PTU) | Sigma-Aldrich | P7629 | Fish husbandry |
| PBS | Gibco | 10010023 | Cell culture |
| Penicillin Streptomycin | Life Technologies | 15140122 | Cell culture |
| S1 pipet filler | Thermoscientific | 9501 | Cell culture |
| Serological stripette 10 mL | Corning | 4488 | Cell culture |
| Serological stripette 25 mL | Corning | 4489 | Cell culture |
| Serological stripette 5 mL | Corning | 4485 | Cell culture |
| Serological stripette 2 mL | Corning | 4486 | Cell culture |
| Terumo Needle 22 gauge | Amtech | SH 182 | Fish husbandry |
| Tissue culture incubator | Thermofisher Scientfic | HeraCell 150i | Cell culture |
| Tivozanib (AV951) | AVEO Pharmaceuticals | Drug treatment | |
| Transfer pipette 3 mL | Mediray | RL200C | Fish husbandry |
| Trypsin/EDTA (0.25% ) | Life Technologies | T4049 | Cell culture |
| Tweezers | Fine Science Tools | 11295-10 | Fish husbandry |
| Volocity Software (v6.3) | Improvision/Perkin Elmer | Image analysis |
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