A Simple Guide Schroef Methode voor Intracraniële xenograft Studies in Muizen
Summary
Met het oog op nieuwe therapeutische paradigma's voor de behandeling van gliomen te evalueren, fysiologisch relevante modellen zijn essentieel. We maken gebruik van een implanteerbare gids schroef procedure voor de oprichting van intracraniale xenotransplantaatmodellen dat is sneller en veiliger dan stereotactische benaderingen.
Abstract
Het enten van menselijke tumorcellen in de hersenen van muizen immunosuppressie is een gevestigde methode voor de studie van de hersenen kanker inclusief glioblastoma (glioom) en medulloblastoom. De meest gebruikte stereotactische aanpak alleen toestaat voor de injectie van een enkel dier in een tijd, is arbeidsintensief en vereist zeer gespecialiseerde apparatuur. De gids schroef methode, oorspronkelijk ontwikkeld door Lal et al.., 1 werd ontwikkeld om omslachtige stereotactische procedures te elimineren. We hebben nu beschrijven een aangepaste gids schroef aanpak die is snel en uitzonderlijk veilig, die beide een kritische, ethische overwegingen. Met name onze werkwijze omvat nu een infusiepomp waarmee maximaal 10 dieren tegelijk worden geïnjecteerd met tumorcellen.
Om aan te tonen het nut van deze procedure, wij menselijke U87MG glioomcellen opgericht als intracraniële xenotransplantaten in muizen, die vervolgens werden behandeld met AMG102, een volledig humaan antilichaam gericht op HGF / verstrooien factor ondergaat momenteel klinische evaluatie 2-5. Systemische injectie van AMG102 verlengde significant de overleving van alle muizen met intracraniële U87MG xenografts en resulteerde in een aantal volledige genezing.
Deze studie toont aan dat de gids schroef-methode is een goedkope, zeer reproduceerbaar aanpak voor het vaststellen van intracraniële xenotransplantaten. Bovendien biedt een relevante fysiologische model voor de validatie van nieuwe therapeutische strategieën voor de behandeling van hersentumoren.
Protocol
Discussion
The intracranial guide screw method presented here enables the rapid and reproducible establishment of intracranial xenografts and in the hands of a trained animal technician, this procedure is very easily performed without the need for a stereotactic device. Our method combines the use of a guide screw, to accurately locate the region of brain most suitable for in vivo tumor development, and an automated infusion pump that allows for the simultaneous inoculation of up to 10 animals at a time. The guide screw augmentation procedure takes less than 5 minutes to perform. The automated infusion of cells at 30 μl/hour takes just 10 minutes for a 5 μl volume. This ensures accurate dosing at a constant rate and reduces the risk of cell expulsion into the extra-cranial area due to increased intracranial pressure. We have also performed this procedure with 10 μl volumes and have not observed any extra-cranial tumor growth due to cellular reflux. As the automated infusion pump also allows several animals to be inoculated simultaneously we can inject up to 60 animals per hour compared to a stereotactic procedure which is limited to approximately 15 animals per hour.
The most critical step in this procedure is the location of guide screw in the skull as its position determines the ultimate location of the tumor bulk. If the guide screw is too central, the cells will be injected into a region between the two brain hemispheres whereas if the guide is placed to far forward, the cells will not be injected into the brain at all. It is therefore essential to be as accurate as possible in locating the guide 2.5 mm lateral and 1 mm anterior to the bregma1. In this study we used a steel guide screw, although plastic guide screws are also available for those studies that require brain imaging such as MRI or PET6. Even if the automated infusion pump is cost prohibitive for some groups, cells can still be injected manually more quickly than stereotactic approaches. Overall, this rapid, accurate and reproducible method can be used to study glioma, medulloblastoma and brain stem tumors7. This procedure has been performed on over 500 mice between our two laboratories with deaths due to unexpected anesthetic reactions (<1%). Mice that were bolted with the guide screw and subsequently infused with cells did not die or suffer any neurological complications as a result of the procedure. Animals have survived past 100 days without any complications. Although we preferred to use a hairless mouse, an immuno-compromised mouse with hair like the SCID would be just as appropriate provided the skull hair was removed prior to the surgery to keep the site clean and to prevent hair interfering with guide screw.
There are several novel therapeutics currently being developed for the treatment of glioma8-10. Recently we showed that subcutaneous U87MG glioma xenografts can be inhibited by treatment with AMG1023; a humanized antibody directed to HGF currently undergoing clinical evaluation in glioma5. We used the method describe here to establish intracranial U87MG tumors in the brains of nude mice. Our study clearly shows the utility of the method and demonstrates that AMG102 significantly inhibits the growth of orthotopic U87MG xenografts. We will continue to use this model to determine what other therapeutics can be used in combination with AMG102 to obtain more effective inhibition of tumor growth.
Disclosures
The authors have nothing to disclose.
Acknowledgements
De auteurs willen graag Verlene Henry en Lindsay Holmes bedanken voor de hulp bij de ontwikkeling van dit model. Dit werk werd gedeeltelijk gefinancierd door de James S. McDonnell Foundation (# 220020173).
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| DMEM/F12 | Invitrogen | 12634010 | |
| FCS | Bovogen | SFBS | |
| Trypsin | Invitrogen | 15050065 | |
| EDTA | Sigma | E6758 | |
| Balb/c nu/nu mice | ARC Perth | ||
| Screw Holder | Plastics One | SD-1 | |
| Drill Holder | Plastics One | DH-1 | |
| Drill Bit | Plastics One | D#60 | |
| Screw guide (metal) | Plastics One | C212SG | |
| Screw Dummy (stylus) | Plastics One | C212SD | |
| Hamilton Syringe (10 μl 26g cemented needle) | Grace Division Discovery Science | 80075 | |
| PHD 22/2000 infusion pump | Harvard Apparatus | 70-2003 | Optional |
| Vetbond | 3M | 1469SB | |
| Thermo pad | Harvard Apparatus | 340390 |
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