By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.
Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.
1. Protocol
2. Representative Results
A successful PoRTS cranial window surgery allows the cranial window to remain clear for weeks to months. Figure 1 shows the vasculature beneath a PoRTS cranial window as visualized using backscattered light. These vasculature images were used as landmarks for locating the same brain areas for repetitive imaging. To visualize both the vasculature endothelial cells and GBM cells, we crossed the B6.Cg-Tg(Tek-cre)1Ywa/J mouse, which labels vascular endothelial cells with the B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J mouse, and generated mice with vascular endothelial cells labeled with the red fluorescent protein tdTomato. We then performed the PoRTS craniotomy procedure on these mice, and injected GFP-labeled GBM cells into their brains through a small opening on the side of a cranial window. We then performed in vivo two-photon imaging longitudinally on the injected mice. The laser excitation wavelength was set at 910 nm, which resulted in excitation of both GFP and tdTomato. Two-photon imaging was performed on a custom-built two-photon laser-scanning microscope with two detectors for simultaneous green/red fluorescence detection capability. Figure 2 shows an in vivo example of glioma initiation from injected GBM cells near the vasculature. The PoRTS cranial window is also an excellent choice for chronic time-lapse in vivo imaging. Figure 3 shows GFAP staining of cortical sections from a mouse without PoRTS surgery, a mouse 7 days following PoRTS surgery, and a mouse 7 days following PoRTS surgery and saline injection. It is evident from the images that the mouse without surgery and the mouse with PoRTS surgery do not have gliosis, whereas following saline injection, gliosis is observed in the brain tissue of the mouse, due to pipette penetration and saline injection in the brain. This result demonstrates that gliosis does not occur under the PoRTS cranial window, providing independent validation that the PoRTS cranial window described here does not lead to the gliosis that is typically associated with an “open skull” chronic cranial window. Figure 4 shows high-resolution imaging of dendritic spines from the same mouse on the day of surgery (Day 0) and 32 days after the PoRTS window creation (Day 32), demonstrating that the PoRTS window is also an excellent choice for high-resolution in vivo imaging.
Figure 1. Visualization of vasculature under a polished and reinforced thinned skull cranial window using backscattered light. Days represent the number of days after surgery; images were taken starting on the day of surgery immediately after completion of GBM cell injection. Scale bar: 0.5 mm. Click here to view larger figure.
Figure 2. Visualization of injected GBM cells and vasculature in vivo. Mice carrying both Tek-Cre and ROSA26 CAG-tdTomato were used for GBM cell injection. Vascular endothelial cells were labeled with tdTomato (red), and GBM cells were labeled with GFP (green). Images were acquired starting 24 hr after GBM cell injection. A total of 10-12 adjacent fields of view were taken and were assembled adjacent to one another, with the identified injected GBM cells in the center of the total field, to produce the final images. Days represent the number of days after surgery. Scale bar: 100 μm. Click here to view larger figure.
Figure 3. Gliosis does not occur following PoRTS cranial window surgery. Upper panels: Mouse brain coronal section under lower magnification. Left panel: a mouse without any cranial window surgery; no GFAP activation is observed in the brain tissue. Middle panel: a mouse with a PoRTS cranial window generated on the right side of the brain; no GFAP activation is observed in the brain tissue. Right panel: a mouse with a PoRTS cranial window and saline injection from the side of the PoRTS window. GFAP activation is observed on the side of the PoRTS window, but not on the side of intact brain tissue. Scale bar: 1 mm. Lower panel: higher-power images of brain tissues in areas as indicated in the white box of the upper-panel images. Scale bar: 100 μm. Click here to view larger figure.
Figure 4. High-resolution imaging of dendritic spines from Thy-1 YFPH mice. Images were acquired on the day of PoRTS surgery, and 32 days after PoRTS surgery. It is apparent from the images that most spines present on day 0 are clearly visible 32 days following PoRTS surgery. Scale bar: 2 μm.
The key to a successful PoRTS cranial window is the thinning and polishing. While the initial thinning can be performed quickly, care should be taken to ensure homogenous thinning of the skull over a large area. We typically apply a thin layer of saline to the skull, and then thin the skull one pass at a time, such that the saline solution evaporates shortly after the microdrill has passed over the skull once. This allows us to slowly but homogenously further thin the skull. A steady hand under the microscope is also key. Once skull-thinning nears the intended thickness, we normally use diamond paste to polish the thinned skull for 10-15 min, with subsequent polishing using tin oxide. We find that polishing the thinned skull first with diamond paste yields a better polishing result in general, and also allows us to further thin a large area of skull area without applying much pressure onto the thinned skull area.
We chose the PoRTS cranial window rather than other commonly used cranial windows, such as the “open skull” window and the “thinned skull” window, for the following reasons: With the open skull window, the cranial window usually becomes cloudy shortly after creation and then clears up on its own within the first two weeks, thus requiring a waiting period of approximately two weeks before any imaging experiments can be performed. This two-week waiting period would prevent visualization of glioma initiation during the initial two weeks following GBM cell injection. The thinned skull cranial window requires repetitive thinning before each imaging session, which renders it unsuitable for GBM cell injection and visualization of glioma initiation on a daily basis. In contrast, the PoRTS window allows us to perform GBM cell injection from the side of the window, which minimizes damage to the brain tissue, and also allows for performance of high-resolution imaging immediately following creation of the window. Information on glioma initiation can be collected within the first few weeks following GBM cell injection, which is a key advantage over the open skull technique for this type of studies.
The authors have nothing to disclose.
This work is supported by The Jackson Laboratory Cancer Center Pilot Grant and The Maine Cancer Foundation.
Name of reagent | Company | Catalogue number | Comments (optional) |
DMEM/F12(1:1) with Sodium Pyruvate | Thermo Sci Hyclone | SH3026101 | |
B-27 Serum Free Supplement (50x) | Invitrogen | 17504-044 | |
GlutaMax-1 Supplement | Invitrogen | 35050061 | |
Penicillin-Streptomycin Solution | Thermo Sci Hyclone | SV30010 | |
Accutase-Enzyme Cell Detachment Medium | eBiosciences | 00-4555-56 | |
T25 flasks-vent cap green | SARSTEDT | 83.1810.502 | |
70% alcohol | JAX LAHS | ||
10% povidone-iodine topical solution | JAX LAHS | ||
Ketamine HCl | Butler Animal Health Supply | NDC# 11695-0550-1 | |
Xylazine | Akorn, Inc. | NADA# 139-236 | |
Carprofen | JAX LAHS | ||
Ophthalmic ointment | Dechra Veterinary Products | 17033-211-38 | |
0.5% Lidocaine HCl | REGENT, Inc. | NDC 0517-0625-25 | |
Cyanoacrylate glue | Henkel Corp. | 46551 | |
Sterile Swabs | Fisher Scientific | 23-400-114 | |
Diamond paste | Widget Supply | BBE60 | |
Tin oxide | LORTONE, Inc. | 591-038 | |
Liner Bond 2V | KURARAY Medical Inc. | 1921-KA | |
Clearfil AP-X | KURARAY Medical Inc. | 1721-KA | |
Saline | JAX LAHS | ||
Cover glass | Warner Instruments | 64-0720 | |
Hex Nut | Small Parts, Inc. | HNX-0090-C | |
Syringe Pump | Syringepump.com | NE-1000 | |
Two-Photon imaging system | Custom built (Any commercial system would work) |