Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.
The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two μl of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150μg luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in >10% loss of weight. Tumors were evident in all of the animals on necropsy.
1. Cell Culture
2. Surgery Setup 2
3. Intracranial Implantation 2
4. In vivo Bioluminescence Imaging 3
5. 3D Imaging 3
6. Data Analysis 3
7. Representative Results:
Successful cell implantation is evident when implanted cells are detectable using the IVIS spectrum on the day of surgery. Both GL261-luc and GL261-luc2 cells are detectable, however, the luc2 gene will provide a higher level of bioluminescence [Figure 5]. Images taken soon after implantation may have non-specific signaling on the animal’s paws and nose which should be disregarded as background. Signal located at the site of implantation is real and the signal will increase with time [Figure 6]. The decline in signal intensity on day 6 is reproducible and most likely due to loss of tumor take by some of the implanted cells. Quantitative measurements of tumor burden are reproducible in that they should rise steadily until the animal ultimately succumbs to the disease. However, growth curves will largely depend on the state of implanted cells, and or unavoidable minor variability in the implantation procedure. Our laboratory has elected to image implanted animals every three days.
Figure 1. After the mouse is properly anesthetized, it is placed in the stereotactic frame. The mouse head is secured using the mouth clamp.
Figure 2. After the skin is opened the main anatomical landmarks are identified including include the bregma, the coronal and sagittal sutures. The burrhole is made 2.3mm to the right of the bregma by slowly twisting a 16 gauge 1½ inch needle with a small amount of pressure until the skull is penetrated and the brain is exposed.
Figure 3. A kinetic comparison of subcutaneous () versus intraperitoneal () luciferin injection was performed to demonstrate the utility of a subcutaneous luciferin injection and to identify the optimal time to image following luciferin administration. Three minutes after the luciferin was injected the mouse was sedated, placed in the IVIS Spectrum instrument and imaged every 3 minutes for up to an hour and every 6 minutes after that to generate a kinetic curve of bioluminescence. This demonstrated that a subcutaneous route of luciferin administration was superior to an intraperitoneal injection in our hands, and that the optimal time to image the animals was approximately 25 minutes following the luciferin injection when GL261-luc cells were used.
Figure 4. Multiple views of a 3-Dimensional reconstruction of the intracranial implantation of GL261-luc2 cells co-registered with the mouse skeleton and brain.
Figure 5. Photon counts obtained from tumors resulting from GL261-luc cells vs GL261-luc2 cells. Results are an average of 5 animals.
Figure 6. Graph of GL261-luc tumor cell growth in an albino C57BL/6 mouse. Bioluminescence was measured every 3 days and plotted as in vivo photon count versus days post-implantation. Photographs show bioluminescence at various time points. Coloration is an indication of bioluminescence (pixel intensity) which is relative to tumor cell number (color bar is shown to the right). After the animal succumbed to the disease the brain was dissected and luciferin was added topically to obtain the ex vivo image shown in the figure inset.
The cell inoculum is infused at a depth of 2.6 mm from the surface of the brain after creating a 0.4 mm pocket. To ensure proper placement and depth of needle an X-ray can be taken using a C-Arm or similar X-ray image intensifying device; however, this is optional. Complications of the surgery may arise if the animal is not properly sedated, at which point the animal may move during cell infusion. This can cause leakage of cell mix or bleeding from needle track. Leakage of cells causes ectopic growth of tumor cells. It is also important not to puncture the ventricle which can be done if the burrhole is made medial to the 2.3 mm described in the protocol 4. Proper placement of the needle and cell spread was tested by infusing a mouse with 2 µl methylene blue dye and dissecting the brain tissue to check the location of the infused dye.
In this protocol we have used the IVIS Spectrum in vivo imaging system and the Living Image software (v 4.0) designed for use with this instrument. (Caliper Life Sciences). Any comparable in vivo imaging system and image analysis tools can be used to obtain similar results. These systems provide some advantages over traditional magnetic resonance imaging (MRI) to follow the growth of an experimental intracranial tumor. The most obvious is the relative costs of the 2 instruments – animal MRI machines are far more expensive and typically require the services of a skilled MRI technician. In vivo imaging such as what was described here can be done by the end user. The bioluminescence data is quantitative, while quantitation of MRI data is time-consuming and somewhat inexact. Furthermore, MRI images show edema and inflammation in addition to tumor cells and it can be difficult to separate tumor from treatment effect. For these reasons obtaining accurate volumetric measurements of growing tumor can be a challenge. Bioluminescence requires ATP, therefore only living tumor cells contribute to the tumor size data. Despite this, there are some advantages to MRI if a machine is available. Cells do not have to be labeled with a bioluminescent marker to be visualized by MRI. The ability to visualize peri-tumoral edema may be an advantage for some experimental protocols. A strength of both technologies is that using one does not preclude the use of the other, so data can be obtained on the growth of the tumor as well as the presence of peri-tumoral edema and inflammation from the same animal when both technologies are available to the researcher.
The authors have nothing to disclose.
We would like to acknowledge Dr. Joshua B. Rubin for the generous gift of the plasmids for the lentivirus system as well as Mahil Rao for helpful suggestion on the preparation of the GL261-luc cells.
We thank Students Supporting Brain Tumor Research (SSBTR), The Barrow Neurological Foundation and The Wallace Foundation for their generous support.
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee of St. Joseph’s Hospital and Medical Center.
Name of the reagent or supply | Company | Catalogue number | Yorumlar |
GL261-luc2 Bioware Ultra | Caliper Life Sciences | GL261-luc2 | |
Dulbecco’s Modified Eagle Medium (DMEM) | Invitrogen | 10313039 | |
Geneticin (G418) | Gibco (Invitrogen) | 11811-023 | |
Fetal Calf Serum (FCS) | Invitrogen | 26140079 | |
Phospate Buffered Saline (PBS) | Invitrogen | 70011044 | |
C57BL/6-cBrd/cBrd/Cr Mice | NCI-Frederick | ||
AKWA Tears Lubricant Opthalmic Ointment | Akorn Inc | 17478-062-35 | |
Ketaset (ketamine hydrochloride) | Wyeth | 11570775 | |
Sedazine (xylazine hydrochloride) | Wyeth | 10031894 | |
Small Animal Stereotaxic Instrument | Kopf Instruments | 900 | |
UltraMicroPump with SYS-Micro4 Controller | World Precision Instruments | UMP3-1 | If not available, it is possible to infuse manually |
10μl syringe with 26 gauge beveled needle | World Precision Instruments | SGE010RNS | |
Adison Forceps | World Precision Instruments | 500092 | |
Penfield Dissector | Codman | 65-1015 | |
16g 1½ Precision Glide Needle | Beckton, Dickinson and Company (BD) | 305198 | |
Surgical Blade Handle | BD | 371030 | |
Size 15 Blade | BD | 371315 | |
4-0 Vicryl Suture | Ethicon | VCP496G | |
Bone Wax | Medline | DYNJBW25 | |
Povidine-Iodine Swab Sticks | Medline | MD93901 | |
D-Luciferin Potassium Salt | Caliper Life Sciences | 122796 | |
Forane (Isoflurane) | Baxter | 1001936060 | |
OPMI Pentero Microscope | Carl Zeiss, Inc. | Any surgical microscope will suffice | |
Xenogen IVIS Spectrum with optional anesthesia system | Caliper Life Sciences |