Håndtering af bomuld Rat i Undersøgelser for præklinisk evaluering af onkolytiske Vira
Summary
Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.
Abstract
Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.
Introduction
Onkolytisk vira (OV) selektivt replikerer i tumorceller ved at udnytte biokemiske forskelle mellem normale og tumorceller 1,2. Der er to typer af OvS: dem, der ikke kræver en mutation til at opnå selektiv onkolyse, benævnt naturligt forekommende vildtype-virus, og de, der skal manipuleres til at opnå selektiv onkolyse. Indsamlingen af mutationer inden for en given tumortype bestemmer arten af den selektive vækst fordel over normale celler til en OV 2. Sikkerheden og fordel ved at bruge OVS er påvist i kliniske forsøg 3-7. Trods fremskridt inden for onkolytiske virotherapy der findes mellemrum mellem præ-kliniske og kliniske resultater, tyder på, at der er behov for bedre modeller til at evaluere antitumor effekt af OvS.
Bovin herpesvirus type 1 (BHV-1) er et medlem af Herpesviridae-familien, og Alphaherpesviridae underfamilien. BHV-1 initiates bovint respiratorisk sygdom kompleks i kvæg, manifesterer sig i en lang række symptomer, der ligner en forkølet 8,9. BHV-1 binder vedhæftede og indrejse receptorer, der anvendes af HSV-1, såsom heparansulfat og nectin-1 10. Det binder imidlertid CD155 i stedet for nectin-2 10. BHV-1 har en meget smal værtsområde, således at det er i stand til effektivt at komme ind og initiere replikation i normale og transformerede murine celler 3,4,10. Dette gør anvendelsen af konventionelle murine modeller problematisk. Onkolytisk kapacitet BHV-1 er blevet påvist in vitro 11,12. BHV-1 er blevet vist at initiere replikation i og dræbe humane tumorceller fra en række histologiske oprindelse, herunder brystcancerceller og brystkræft indledning celler 11,12. Dog skal antitumor kapacitet BHV-1 vurderes in vivo i forbindelse med en immunkompetent vært.
Humant adenovirus (Ad), hvorDer er 57 identificerede serotyper, mest almindeligt forårsager respiratorisk sygdom hos mennesker. Onkolytisk Ad vektorer er blevet evalueret for deres antitumorvirkning med flere fremrykkende i kliniske forsøg 13-15. Trods lovende prækliniske data har kliniske resultater levet op til forventningerne. Human tumor xenograftmodeller typisk anvendt til at undersøge antitumorvirkningen af Ad-vektorer, selvom de udviser svækket immunrespons til virus 16,17. Endvidere syngene murine modeller er ikke-permissiv for Ad-infektion, hvorved evalueringen af værten immunresponser ved hjælp af disse modeller upraktiske 17,18.
Værten immunsystem er blevet identificeret som den mest indflydelsesrige mekanisme, hvormed OVS fremkalde tumorcelledød 19. Antitumor reaktioner mellem tolerante og ikke-tolerante tumor-associeret antigen (TAA) modeller er forskellige og kan i høj grad påvirke succes OV terapi. HSV-1 OV KM100 (ICP0 N212VP16 i 1814 20) 20,21 fremkaldte tumorregression i 80% af tumorbærende mus i en murin Polyoma Middle T antigen brystcancer model 22. Men i HER-2 / neu modeller antitumorvirkningen af KM100 varierede mellem 20% fuldstændig regression i syngene mus og tumorstase i transgene HER2-tolerante mus. Sammen disse data fremhæver betydningen af fuldt ud at evaluere OVS hjælp dyremodeller, der bedst rekapitulere menneskelige immunsystem landskab til fuldt ud at forstå, hvilke funktioner bestemme terapeutisk succes.
Bomulden rotte (Sigmodon hispidus), indfødte til Nord- og Sydamerika, er mest almindeligt anvendt som en model af respiratorisk syncytialvirus infektion (som revideret i 5). Bomuldsrotter bruges også i anti-BHV-1 vaccination forskning som de rekapitulere patologien forbundet med bovint respiratorisk sygdom kompleks 6,23. Endvidere BHV-1-infektion af bomuldsrotterer immunogen, inducere langvarig mucosal og systemiske immunresponser 6,23-25. Cellelinjer er udledt fra spontane fibrosarkom og osteosarkomer af mælkekirtlen (LCRT) og knogle (CCRT og VCRT), henholdsvis 26. Bomuldsrotter er blevet anvendt til at vurdere in vivo-effektivitet af onkolytiske Ad-vektorer, som de er modtagelige for Ad-infektion og udviser lignende patologi for mennesker 27-29. Anvendelsen af immunkompromitterede modeller for præklinisk evaluering af OvS er ikke kun mindre indikerer klinisk respons på behandlingen, men de undlader at tage hensyn til den rolle af immunsystemet i onkolytisk virotherapy 30,31. Derfor syngene og tumor-tolerante bomuld rottemodeller af brystcarcinom og osteosarkom er relevante modeller til at vurdere den præ-kliniske effekt af OvS, såsom BHV-1 og Ad, som ikke kan studeres ved anvendelse af traditionelle murine modeller.
Protocol
Representative Results
Discussion
Cotton rats are highly excitable and have a strong flight response. Therefore, special care should be taken to minimize any undue stress on the animal. The cage setup described will allow for safe and easy capture of the animals, with the placement of the enrichment tube being of the utmost importance. When setting up cages, ensure that the enrichment tubes meet the size and shape requirements, and are placed in proper orientation in the cage. It is also important to ensure that any technicians who might be aiding in ani…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Breanne Cuddington holds a fellowship from the Canadian Breast Cancer Foundation. This work was sponsored by operating grants from the Cancer Research Society and the Canadian Cancer Society Research Institute (formerly the Canadian Breast Cancer Research Alliance). We thank Ann Tollefson (Saint Louis University School of Medicine) for LCRT cells and Dr. Kathleen Delaney and Marion Corrick for technical assistance with cotton rat housing and sedation.
Materials
| Name of Material/Equipment | Company | Catalog # | Comments/Description |
| Dulbecco’s modified Eagle’s medium | Gibco | 11965-092 | May use any brand |
| 1X Phosphate Buffered Saline | Can prepare in lab, filter to sterilize | ||
| 200 mM L-glutamine | Gibco | 25030164 | May use any brand |
| 100x Antibiotic-Antimycotic | Gibco | 15240-062 | May use any brand |
| Fetal bovine serum | Quality Biological Inc. | 110-001-101HI | May use any brand |
| T-150cm2 tissue culture flask | Fisher Scientific | 14-826-80 | May use any brand |
| 1X TypLE Express | Life Technologies | 12604-013 | |
| 12-well cell culture plate, flat bottom | Fisher Scientific | 08-772-29 | May use any brand, must be tissue culture treated |
| alamarBlue | Life Technologies | DAL1025 | May use an alternative reagent for determination of cell viability |
| 8640 Teklad 22/5 Rodent diet | Harlan | 8640 | |
| 1/8” corncob rodent bedding | Harlan | 7092 | |
| Nestlets | Ancare | – | Made of pulped virgin cotton fiber, dust-free and autoclavable |
| 50 mL Conical tubes | Fisher Scientific | 14-432-22 | May use any brand, must be sterile |
| Isoflurane USP, 99.9 %, inhalation anesthetic | Pharmaceutical Partners of Canada Inc. | M60302 | |
| 70% Ethanol | Can prepare in lab | ||
| 10 % Neutral Buffered Formalin | Sigma-Aldrich | HT501128 | May use any brand |
| Name of Material/Equipment | Company | Catalog # | Comments/Description |
| NAPCO NapFlow 1200 Class II A/B3 Biosafety Microbiological Safety Cabinet (cell culture hood) | NAPCO | Model used not currently available | May use any brand |
| Thermo Fisher Scientific Precision Heated Water Bath | Fisher Scientific | Model used not currently available | May use any brand |
| Reichert Bright-line Hemacytometer | Sigma-Aldrich | Z359629 | May use any brand |
| Typhoon Trio BioAnalyzer | GE Healthcare Life Sciences | Model used not currently available | May use any fluorescence plate reader |
| Tecan Safire2 Multi-detection Microplate Reader | Tecan | Model used not currently available | May use any fluorescence plate reader |
| Allegra 6R benchtop centrifuge | Beckman Coulter | 366816 | May use any brand |
| Table Top Anaesthesia machine | VetEquip | Model used not currently available | May use any brand, must be portable |
| Wahl Peanut Mini Clippers | Wahl | May use any brand of small clippers | |
| Insulin syringes 29 G x 1/2', 0.3 mL | BD | 329464 | May use any brand. Insulin syringes are recommended as they make injections easier through the rat’s tough skin. |
| Cotton swabs | MedPro | 018-425 | May use any brand |
| Sharp-Pointed Dissecting Scissors | Fisher Scientific | 8940 | May use any brand |
| Dissecting Tissue Forceps | Fisher Scientific | 13-812-41 | May use any brand |
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