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Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.
Cells for human brain tumor xenografts can be sourced either from tumors propagated as subcutaneous growths in athymic mice, or from cell culture. Utilization of both cell sources is discussed below, along with demonstration of a method for cell implantation.
To prepare cells from subcutaneous tumors for transfer to the intracranial compartment, excised flank tumors are placed in culture dishes, where the tissue is initially minced with a scalpel and then mechanically disrupted by repetitive pipetting to create a cell aggregate suspension1. The cell aggregate suspension is then passed through a 70 μM nylon mesh filter to produce a single cell suspension suitable for intracranial injection. The cell suspension is centrifuged at 1000 rpm for 10 minutes at 4°C, and the supernatant aspirated before resuspending the cell pellet in an appropriate volume of serum-free media to obtain a final working concentration (see below). For preparing established cell lines for intracranial implantation, cells are harvested by trypsinizing monolayers, or by collecting neurosphere suspension cultures, then centrifuging and resuspending the cells as indicated above 2. The number of cells injected is variable dependent on neuroanatomical location of injection. For supratentorial injections we routinely inject 3-5 x 105 cells in 3 μL of serum-free media (DMEM), whereas for brainstem injections 3, as few as 5 x 104 cells are injected in 0.5 μL. Injecting larger volumes than recommended can result in tumor cell reflux through the needle tract, with resultant exophytic (Figure 1), rather than intracranial tumor growth. After withdrawing sample for intracranial injection, the remaining cell suspension should be placed in ice, with contents mixed frequently to maintain appropriate concentration while completing intracranial tumor establishment among the members of an injection series.
Note, all procedures described below have been reviewed and approved by the Institutional Animal Use and Care Committee at University of California San Francisco.
In the example shown in Figure 3A, mice receiving intracranial tumor cell injection were monitored for intracranial luminescence until successive mean luminescence values indicated progressive tumor growth, and at which time therapy was initiated (gray arrow beginning at day 34: erlotinib administered daily at 150 mg/kg until required euthanasia). Luminescence values for each mouse are set to a normalized value of 1 at time of initiating therapy, with subsequent luminescence readings for each mouse normalized to its final pretreatment imaging value. As an example, a mouse with a final pre-treatment luminescence reading of 2.0 x 107 photons/sec at day 34, whose luminescence had increased to 6.0 x 107 photons/sec at day 38, would have a day 38 normalized luminescence value of 3.0. Mean normalized bioluminescence and corresponding standard error for control and treatment groups are plotted for each imaging time point. In this example, a significant difference in mean normalized luminescence is apparent at the first imaging time point subsequent to the initiation of therapy (day 38), with the difference in mean group luminescence showing further increase at subsequent time points. In most instances, anti-tumor activity of therapy, as indicated by qBLI, is accompanied by a corresponding significant difference in survival (i.e., p < 0.05), as is the case here (Figure 3B). Panels 3C and 3D show adjacent hematoxylin & eosin and anti-EGFR stained sections of mouse brain obtained at time of euthanasia, following placement of resected brain in formalin and subsequent paraffin-embedding for sectioning.
Figure 1. Indications of intracranial injection errors. A) Exophytic (extracranial) tumor growth (red circle) can be caused by too large an injection volume, residual cell suspension attached to the syringe, or from withdrawing the syringe too quickly after injecting the tumor cells. B) Injecting tumor cells into the ventricles can cause spinal dissemination of tumor (mouse to the right), in contrast to properly injected tumor cells, the signal for which stays localized to the injection site (mouse to the left).
Figure 2. Heat map image representations of bioluminescence intensity for representative mice from control (left) and treatment (right) groups of a therapy response experiment. The Living Image software can be set to define regions of interest (red circles), or instrument operators can define regions of interest manually. For using images such as these for figure construction, we recommend that the instrument operator shows heat map images using the same bioluminescence heat map range (upper portion of figure), to provide visual representation of extent of bioluminescence difference between animal subjects.
Figure 3. Bioluminescence, survival, and tumor tissue analysis from an experiment in which therapeutic response is evident. A) Plot of mean bioluminescence readings for control and treatment group mice, with standard error indicated for each imaging point. B) Survival plot for same mice; p-value determined through use of log-rank test 7. C) H&E stained section of mouse brain with tumor. D) EGFR stained section. E and F) Magnifications of indicated areas from panels C and D, respectively, with panel E showing negative staining for tumor suppressor protein PTEN.
Orthotopic (intracranial) brain tumor xenograft establishment provides an appropriate microenvironment10 for modeling CNS cancer to be tested for therapeutic response. This type of modeling additionally provides information regarding therapeutic access to brain and brain tumor, which is critically important to determining whether an experimental agent should be advanced to clinical trial evaluation in patients. Because the amount of intracranial xenograft tumor can not be directly measured, such as by calipers, longitudinal monitoring of intracranial tumor growth and response to therapy requires non-invasive imaging, with our experience indicating bioluminescence imaging as the most practical approach for experiments whose primary objective is assessing extent of tumor response to therapy. When the results of bioluminescence imaging are combined with animal subject survival analysis, the two data sets provide a powerful and reliable approach for evaluating experimental therapeutic efficacy.
Finally, it is critically important that intracranial brain tumor xenografts are harvested from euthanized animal subjects in order to assess morphologic and molecular effects of therapy, and for this we prefer whole brain resection at time of euthanasia, with preservation of resected brain for subsequent analysis.
Whereas the preceding presentation has been made specific to brain tumor research, the concepts are certainly generalizeable to other human cancers that are amenable to orthotopic modeling in rodents.
No conflicts of interest declared.
NINDS grants NS49720 and NS65819, and NCI grant CA97257