Het hanteren van de Cotton Rat in Studies voor de Pre-klinische evaluatie van Oncolytic Virussen
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
Oncolytische virus (OV) selectief repliceren in tumorcellen door gebruik biochemische verschillen tussen normale en tumorcellen 1,2. Er zijn twee soorten OVS; die geen mutatie vereisen selectieve oncolyse bereiken, genoemd natuurlijk voorkomende wildtype virussen en die welke moeten worden gemanipuleerd om selectieve oncolyse bereiken. De collectie van mutaties binnen een bepaald tumortype bepaalt de aard van de selectieve groei voordeel boven normale cellen voor OV 2. De veiligheid en voordeel van OVS is aangetoond in klinische proeven 3-7. Ondanks vooruitgang op het gebied van oncolytische virotherapy bestaan spleten tussen preklinische en klinische resultaten suggereren dat betere modellen nodig zijn om de antitumor werkzaamheid van OVS evalueren.
Boviene herpesvirus type 1 (BHV-1) is een lid van de familie Herpesviridae en Alphaherpesviridae onderfamilie. BHV-1 initiates runder respiratoire ziekte bij runderen complex, manifesteren in een grote verscheidenheid van symptomen die lijken verkouden 8,9. BHV-1 bindt hechting en binnenkomst receptoren door HSV-1, zoals heparan sulfaat en nectin-1 10. Echter, het bindt CD155 in de plaats van nectin-2 10. BHV-1 is een zeer smalle gastheerbereik zodanig dat het niet efficiënt voeren en replicatie initiëren in normale en getransformeerde muizencellen 3,4,10. Dit maakt het gebruik van conventionele muizenmodellen problematisch. De oncolytische vermogen van BHV-1 is aangetoond in vitro 11,12. BHV-1 is aangetoond dat replicatie in te leiden en te doden humane tumorcellen van verschillende histologische oorsprongen, zoals borstkankercellen en borstkanker initiërende cellen 11,12. Echter, de antitumor capaciteit van BHV-1 in vivo worden beoordeeld in het kader van een immunocompetente gastheer.
Humaan adenovirus (Ad), waarvoorzijn er 57 geïdentificeerd serotypen, meestal veroorzaakt ziekte van de luchtwegen bij de mens. Oncolytisch Ad vectoren zijn geëvalueerd op hun antitumoreffectiviteit met diverse oprukkende in klinische studies 13-15. Ondanks de veelbelovende pre-klinische gegevens, hebben klinische resultaten de verwachtingen niet ingelost. Humane tumor xenograft modellen worden gewoonlijk gebruikt om de antitumor werkzaamheid van Ad vectoren bestuderen, hoewel ze vertonen verzwakte immuunrespons op het virus 16,17. Bovendien syngene muismodellen zijn niet tolerant Ad infectie, wat het beoordelen van immuunreacties van de gastheer met deze modellen onpraktisch 17,18.
De gastheer immuunsysteem is geïdentificeerd als de meest invloedrijke mechanisme waarmee OVS opwekken tumorceldood 19. Antitumor-responsen tussen tolerant en niet- tolerant tumor- geassocieerde antigen (TAA) modellen verschillen en een grote invloed heeft het succes van OV therapie. De HSV-1 OV KM100 (ICP0 N212VP16 in 1814 20) 20,21 opgewekt tumorregressie in 80% van tumordragende muizen in een muizen Polyoma Midden T antigen borstcarcinoom model 22. In HER-2 / neu-modellen, de antitumor werkzaamheid van KM100 varieerde tussen 20% volledige regressie in syngene muizen tumor stasis in transgene, HER2-tolerant muizen. Samen vormen deze gegevens benadrukken het belang van de volledige evaluatie van OVS gebruik van dierlijke modellen die het beste samen te vatten het menselijk immuunsysteem landschap om volledig te begrijpen welke functies bepalen therapeutisch succes.
Het katoen rat (Sigmodon hispidus), inheems in Noord-en Zuid-Amerika, wordt het meest gebruikt als een model van respiratoir syncytieel virus infectie (zoals beoordeeld in 5). Cotton ratten worden ook gebruikt in anti-BHV-1 vaccinonderzoek zij recapituleren de pathologie geassocieerd met bovine respiratoire aandoeningen complex 6,23. Bovendien BHV-1 infectie van katoenrattenimmunogeen induceren duurzame mucosale en systemische immuunresponsen 6,23-25. Cellijnen werden afgeleid van spontane fibrosarcoma en osteosarcoom van de borstklier (LCRT) en bot (CCRT en VCRT), respectievelijk 26. Cotton ratten zijn gebruikt om de in vivo effectiviteit van oncolytische Ad vectoren evalueren zij gevoelig Ad infectie en vertonen soortgelijke pathologie mens 27-29. Het gebruik van immuun modellen voor de pre-klinische evaluatie van OVS zijn niet alleen minder indicatief voor klinische respons op de behandeling, maar ze geen rekening met de rol van het immuunsysteem bij oncolytische virotherapy 30,31. Daarom is de syngene en tumor tolerant katoen ratmodellen mammacarcinoom en osteosarcoom relevant modellen om de preklinische effectiviteit van OVS, zoals BHV-1 en Ad die niet kunnen worden onderzocht met conventionele muizenmodellen evalueren.
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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