The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.
Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal – particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.
In vitro studies of brain tumor cells are invaluable for dissecting molecular mechanisms driving growth, survival, migration, and invasion of cancer cells; cultured cell experiments can define signaling pathways, suggest potential therapeutic targets, and characterize cellular response to drug treatment. But in vitro systems are far too simplistic to predict organismal response to pharmaceuticals; they lack the physiological reactions, immune responses, cell microenvironment, and overall heterogeneity of living animal systems. Genetically engineered models can be invaluable, when available, but molecular differences exist between species and murine cells may not recapitulate events in human processes, resulting in significant discrepancies when comparing animal models to clinical observations1. Mouse xenograft models involving subcutaneous (SQ) injection of human brain tumor cell lines under the skin of the flank are easy to perform and measure; they can be used to address effects of gene modification and drug administration/delivery, metabolism and toxicity. Significant drawbacks, however, limit the utility of SQ models. The microenvironment does not recapitulate that of a naturally occurring brain tumor: the interactions of various cell types and tissues; the local vasculature, and myriad other factors unique to the brain cannot be replicated. To more accurately reproduce the unique milieu of a naturally occurring brain tumor and test the effects of pharmaceutical interventions, a mouse orthotopic model should be utilized. Furthermore, orthotopic techniques may be used as part of a genetically engineered approach in which human primary non-cancerous cells (differentiated or progenitor) are genetically modified and injected into the relevant site of a mouse, with or without human stroma cells, resulting in tumorigenesis similar to that seen in humans1.
This article describes a methodology to precisely and reproducibly create brain tumors in mice. Using this technique, the user can accurately inject a small aliquot of suspended cells into a specified location of the fronto-parieto-temporal region of the mouse cerebral cortex. Mouse mortality is extremely low; in our hands, no mice have died from surgical complications after 185 procedures. Characteristics of the resultant tumor can be compared with that of typical human clinical tumors; for example: rapidity of growth, degree of necrosis, extent of invasion, heterogeneity of cell type, presence of mitotic cells, markers of proliferation and apoptosis, etc. Cell lines or disaggregated human tissue or tumor samples can then be evaluated based on their ability to simulate actual clinical presentation. Pharmaceuticals, selected based on their performance in cell culture, can be tested in the context of a functioning metabolism, circulatory system, and blood-brain barrier as they exist in an animal burdened with a tumor, all in a relevant architectural context. Furthermore, the cells chosen for injection may be genetically modified to investigate the impact of specific knockdowns, deletions, knock-ins, mutations, etc. on tumor growth and survival.
A number of publications document tumors studies using a variety of intracranial techniques. Yamada et al. did a detailed study of the injection of dye and of U87 cells and found that minimizing volume and injection rate produced the best tumor2. Brooks et al. found superior reproducibility and efficiency using a microprocessor-controlled injector rather than a manual method to deliver viral vectors; their conclusions regarding optimal injection parameters are applicable to cell delivery3. Shankavaram et al. showed that glioblastoma multiforme (GBM) cell lines injected orthotopically (using a manual method) into the brain recapitulated the gene expression profile of the clinical tumors more closely than either in vitro or SQ xenografts, supporting the use of intracranial models for preclinical studies4. Giannini et al. injected cells from human surgical specimens that had been sustained in the flanks of nude mice by serial passaging into the brains of additional mice, and showed that this approach preserved patient tumor gene alterations in the model5. Similar results were reported by Yi et al6. Using a stereotaxic setup, carefully defined injection site, and a slow and steady injection rate, they obtained reproducible brain tumors with consistent growth rates and high (100%) engraftment rate. The validity of this technique has therefore been well established; a literature search suggests that the applications of this technique are extensive. Carty et al. used intracranial injections to successfully deliver viral vectors expressing therapeutic genes into the frontal cortex of transgenic model of Alzheimer’s disease7. Thaci et al. described the use of intracranial injections to deliver therapeutic oncolytic adenovirus in a neural stem cell based carrier into nude mice already carrying orthotopically injected GBM tumors8. Clearly, intracranial injections are a versatile and effective tool for preclinical research. Earlier publications in The Journal of Visualized Experiments describe fundamental approaches9-11, but we take the concept of intracranial tumor injection and orthotopic modeling to a higher level of precision using easy-to-master technology.
All described procedures were reviewed and approved by our institutional animal care and use committee.
1. Plan the Experiment
2. Assemble the Equipment
3. Prepare Cells for Injection
4. Anesthetize and Prepare Mouse for Surgery
5. Perform Injection
6. Finish and Monitor Recovery & Tumor Development
Reliable intracranial xenografts can be created with this described technique. Identifying the critical structures of the mouse skull (Figure 1) will allow for recognition of the bregma and guide the investigator to a precise and reproducible injection location. In these studies the U251 parental line, U251 cells transfected with luciferase (U251-Luc), or U87 immortalized human GBM tissue culture cells were suspended in 4 to 6 µl of SF-DMEM and injected 2.5 mm lateral (right), 1.5 mm anterior, and 3.5 mm ventral with respect to bregma (Figure 2).
The resulting tumors can be visualized and analyzed by magnetic resonance imaging (MRI, Figure 3), in vivo luminescent imaging (IVIS, Figure 4), or routine gross pathology techniques (H&E staining, Figure 5). Particular care must be taken and optimization performed to determine the appropriate cells and location of injection to represent timing, growth rate, and tumor model desired.
Figure 1. Skull anatomy. The anatomical features of the mouse head and skull are illustrated. The bregma, which is on the midline axis between the eyes and the ears at the intersection of the coronal and the sagittal sutures, is used to reproducibly locate the injection coordinates.
Figure 2. Incision and injection map. The features used to determine skin incision and precisely locate the injection site are illustrated. A diagonal incision is made to allow access to both bregma and the injection site. Application of 30% H2O2 to the surface of the skull helps to visualize skull sutures. Position a syringe with needle onto the micropump and maneuver the needle tip directly over the bregma. Set coordinates on the alignment console to zero; all skull measurements are then reproducibly made with respect to bregma.
Figure 3. Representative intracranial xenograft tumors: MRI images. MRI T2 weighted images of a tumor derived from (A) 2 x 105 U87 cells compared with a tumor derived from (B) 2 x 105 U251 cells. (C) Tumor volumes calculated from MRI images of three individual mice injected with U251 cells plotted over time shows a reproducible window of tumor development and growth that is consistent in all experiments.
Figure 4. Representative xenograft tumors: IVIS images. IVIS image of U251-Luciferase transduced GBM cells injected intracranially into nude mice (A). The mouse on the left was successfully injected as described with U251-Luc cells and shows a very strong focalized signal (photons/sec) in the desired location. The mouse on the right demonstrates an unsuccessful result from improper injection location. H&E examination of the spine revealed tumor cell growth in the spinal column resulting from injection too close to midline with ventricular dissemination. (B) Tumor size is estimated from luciferase activity (photons/second) in the brain region of interest, plotted over time. The blue line corresponds to the mouse with a successful intracranial injection, while the red line corresponds to the mouse with tumor cell displacement to the spine.
Figure 5. H&E of Intracranial xenograft tumors. Whole brains were collected from mice post sacrifice and fixed in formalin, mounted in paraffin, sectioned and stained with H&E. Tumor (A) was derived from U87 GBM cells and illustrates an area of dense tumor growth (left upper corner) and adjacent normal brain tissue with microscopic invasion of malignant cells (right lower corner). Tumor (B) was taken from a section of the center of a tumor derived from U251 GBM cells. The section very closely replicates the bizarre histopathology with multinucleated malignant cells seen in typical human GBM.
Orthotopic mouse models of human brain cancer can be an excellent tool for assessing the effectiveness of clinical therapies, but care must be taken to optimize the placement of cells in brain tissue. Studies have shown that excessive aliquot volumes, suboptimal injection technique and hasty injection rates can lead to leakiness and the appearance of tumor cells in undesirable locations (ventricles, spinal cord, extradural regions, etc.) and high variation in tumor size2 (personal observations). An analysis of microprocessor-driven delivery of noncellular samples found that the use of a micropump produced more focalized delivery, less sample reflux and less variability than manual methods of injection, attributed to the smooth, uniform delivery and consistent pressures3. While it may be challenging to condense the needed number of cells in a volume of only 2 or 4 µl, the use of a stereotaxic instrument and alignment console equipped with a programmable micro-pump can yield reproducible and reliable tumors with similar growth rates. A xenograft model can never truly duplicate the microenvironment, initiation and development of a naturally occurring tumor, particularly with immune deficient hosts, but a well designed and implemented orthotopic model is the best alternative and is far superior to an ectopic model.
The major advantage of this protocol is the establishment of detectable tumors within a consistent time frame. The timing of tumor appearance is dependent on cell type and the number of cells injected, but is fairly predictable, and most tumors are established within a narrow window of time relative to the duration of tumor growth (i.e., time until endpoint). This enables the researcher to identify time points for data collection (such as MRI or IVIS) or intervention (such as drug treatment). Tumor growth rates vary with cell type, number of cells injected, and from mouse to mouse (much as they do in human patients), but are consistent from experiment to experiment.
While this protocol employs a technique that may require more instrumentation and time than less exact manual methods of injection and may not be applicable to large scale investigations, techniques for intracranial injections that allow for throughput of large numbers of animals (such as that described by Iwami et al.12) by definition involve rapid, high pressure injection and often involve hand-held instrumentation and manual measurement, which are subject to unsteadiness and uncertainty. These factors may be associated with leakage and off-target delivery2,3. The precision, reproducibility, and low mortality of this procedure will allow the investigator to design treatment experiments using fewer mice to obtain statistically significant results – a net savings.
One step is critical to the success of tumor implantation: the precise location of the injection. Cells injected too close to the ventricles may lead to CSF spread of disease through the ventricular system or into extracranial regions. Cells injected too shallow may grow out through the needle track. Predetermined coordinates are of little value if needle placement is sloppy. Take time to position the mouse head securely in the stereotaxic unit. Use the stereotaxic adjustment options and the ear bars to fit the equipment to the mouse, and ensure that the head is stably positioned and will not rock or twist during the procedure. Changes or variation if the injection site may affect tumor characteristics, including tumor take, growth, invasive potential, and access to drug delivery and oxygen supply.
Other parameters that have a profound impact on the resulting tumor include the injection rate, cell number and volume; all must be determined empirically. Minimize the volume for the number of cells required for tumor growth; use the slowest injection rate practical. Cell number and passage number also have major effects on tumor take and growth rate. The specialized equipment used here offer superior control, but the concepts of minimal volume, precise targeting, minimal and consistent injection rate, and slow needle withdrawal may be applied to a variety of techniques (including manual injection) and a variety of instruments.
This procedure is open to a variety of modifications: the injection site may be customized to recapitulate particular types of tumors. Adherent tissue culture cells, genetically modified clones, neurospheres, disaggregated mouse tumors, or human tissue fragments may be engrafted into a mouse brain. This technique may even be adapted for noncellular studies, including viral gene transfer3. Once a method for reliably producing tumors of the desired characteristics is established, experiments comparing the efficacy of therapies, drug treatments and combinations, and other options such as gene transfer may be performed.
The authors have nothing to disclose.
Dr. Keating is funded by DOD grant CA100335 and is a St. Baldrick’s Foundation Scholar.
Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
Equipment | |||
Small Animal Stereotaxic Instrument with Digital Display Console. | Kopf | Model 940 | |
Mouse Gas Anesthesia Head Holder | Kopf | Model 923-B | |
Mouse Ear Bars | Kopf | Medel 922 | |
Fiber Optic Illuminator | Fisher | 12-562-36 | |
UltraMicroPump III | WPI | UMP3 | |
Micro4 microprocessor | WPI | UMC4 | |
Variable speed hand-held rotary drill | Dremel | Model 300 | |
Dental drill bit, 1.0 mm | Spoelting | 514554 | |
Adaptor for dental drill bit: 3/32 inch collet | Dremel | 481 | |
Heating pad | for mice | ||
Isoflurane vaporizer system | for mice | ||
Medical tubing and connectors | to connect isoflurane vaporizer with stereotaxic frame | ||
Instruments | |||
Precision 25 ul micro syringe | Hamilton | 7636-01 | Model 702, without needle |
Microsyringe needles, 26s gauge | Hamilton | 7804-04 | RN, 25 mm point style 2 |
Fine-tipped scissors (straight, sharp/sharp) | |||
Medium-sized standard scissors | |||
Standard serrated forceps | |||
Serrated hemostats (2) | |||
Fine-tipped forceps | |||
Supplies | |||
Sutures 5-0 vicryl P-3 13 mm (Ethicon) | MWI | J463G | |
Surgical blades #10, stainless (Feather) | Fisher | 296#10 | |
Isoflurane (Fluriso) | VetOne | NDC 13985-528-60 | Item #502017. Liquid inhalation anesthetic. federal law restricts this drug to use by or on the order of a licensed veterinarian. |
Carprofen (Rimadyl Injectable 50 mg/mL) | Pfizer | NDC 61106-8507-01 | dilute in saline |
Ophthalmic ointment (artificial tears) | Rugby | NDC 0536-6550-91 | |
Topical antibiotic (AK-Poly-Bac ) | Akorn | NDC 17478-238-35 | |
Povidone-iodine topical antiseptic, 10% (Betadine) | Betadine | NDC 67618-150-04 | |
Hydrogen Peroxide, 30% | Fisher | H325-100 | for visualizing skull landmarks |
Sterile saline | VetOne | NDC 13985-807-25 | for diluting solutions, cleaning tissue |
Bone wax | WPI | Item #501771 | |
Sterile drapes | McKesson | 25-517 | |
Sterile surgical gloves | McKesson | (to fit) | |
Sterile gauze pads, 2 x 2 | Fisherbrand | 22028556 | |
Sterile gauze pads, 4 x 4 | Fisherbrand | 22-415-469 | |
Alcohol prep pads (medium) | PDI | B603 | |
Sterile cotton-tipped applicators | Fisherbrand | 23-400-114 | |
Sterile 0.5 ml screw cap tube with caps for cells | USA Scientific | 1405-4700 | for cells |
Individually wrapped sterile dispo pipettes | Fisher | BD 357575 | for needle cleaning solutions |
BD insulin syringes with needles | Fisher | 329461 | for analgesic |
70% ethanol | for cleaning | ||
Sterile di H2O | for cleaning | ||
Microfuge tubes for cleaning solutions | for needle cleaning solutions | ||
Felt tip pen (dedicated) | for marking skull |