A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.
Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug’s ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.
1. Initial Preparation of Non-nude Immunodeficient Mice (not Necessary for Nude Strains):
2. Preparation of MPNST Cells for Injection on the Day of Grafting
3. Grafting Protocol
4. Assessment of Graft Success and Randomization Into Study Groups
5. Representative Results:
Figure 1 illustrates the typical progressive increase in bioluminescence observed 1 to 18 days post-grafting in a properly established orthotopic xenograft in a nude (NIH III) mouse (A-C). Quantification of the bioluminescence signals observed 1-24 days after grafting shows that, although these signals progressively increase, tumor growth markedly accelerates in the later stages of the study period (Figure 1D). To rigorously demonstrate that tumor growth has occurred in grafted mice, we routinely collect the sciatic nerve and, if it appears that tumor has ruptured the epineurium and invaded adjacent tissue, surrounding soft tissue and skeletal muscle. Given the aggressive growth of MPNSTs, it is not surprising that we often find that the grafted tumor cells have breached the normal barriers of the nerve and invaded adjacent tissues (Figure 1E). We also often find that grafted MPNST cells migrate aggressively into nerve proximal and distal to the graft site.
Figure 1. Bioluminescence imaging of NIH III mouse orthotopically grafted with luciferase-tagged MPNST cells 1 day post-grafting (A). Note that the signals detected within the region of interest (ROI) in different mice are all within one order of magnitude of each other. Reimaging 10 days (B) and 18 days (C) post-grafting shows that bioluminescent signals progressively increase at the graft site in individual mice. (D) Graph illustrating the progressive increase in the relative photon counts/second detected over the graft site 1-24 days post-grafting. (E) Photomicrograph of the graft site from this mouse demonstrating tumor growth and focal invasion into adjacent skeletal muscle.
The detailed orthotopic xenografting method presented here is one that we developed using ST88-14 MPNST cells, a line which is widely used in studies of these NF1-associated peripheral nerve sheath tumors. However, this methodology is easily adaptable for preclinical studies with other MPNST cell lines. For instance, we have also performed orthotopic xenografting with STS-26T10cells, a line derived from a sporadically occurring MPNST and T265-2c11cells, which are derived from a NF1-associated MPNST, with success similar to that we observed with ST88-14 cells.
Having said that, we would note that the exact conditions we describe here for orthotopic xenografting of ST88-14 cells cannot be directly adopted for the grafting of other MPNST lines. Instead, key parameters of the methodology must be empirically determined for each cell line. These parameters include the number of MPNST cells initially grafted, the time allowed for graft development and, to a lesser extent, the volume in which the tumor cells are injected. To establish these parameters for a new line, we graft groups of mice (5 mice per group) with 103 to 5 x 106 tumor cells, checking two concentrations of cells for each order of magnitude (i.e., 103 cells, 5 x 103 cells, 104 cells, 5 x 104 cells, etc.). Larger numbers of tumor cells (>106) must be injected in a larger volume; we have found that up to 5 mL can be injected without loss of cells from the grafted nerve. We have also found that no more than 5 x 106 tumor cells per 5 mL volume can be injected as denser suspensions become increasingly prone to shear which kills the tumor cells. We follow these mice until at least three animals within each group have palpable tumors, at which point we terminate that group and examine the nerve histologically to confirm graft growth. Obviously, the time required to reach this point will differ, depending on the number of cells injected; in general, we are seeking a concentration of cells that will reach maximal allowable tumor growth within 30-60 days. More rapid growth can make it difficult to achieve effective therapeutic concentrations prior to termination of the experiment and/or prevent the collection of sufficient bioluminescence imaging datapoints over the course of the experiments. A time course longer than 60 days makes adjusting experimental parameters unwieldy and unnecessarily prolongs the duration of the planned experiments.
Finally, we would also note that we have used this orthotopic xenografting methodology with ST88-14 cells grafted into NIH III mice to demonstrate the therapeutic effectiveness of tamoxifen6. A detailed description of the methods we routinely use to assess the effects of candidate therapeutic agents on orthotopic xenografts can be found in that manuscript. It has also been demonstrated that neurofibroma cells can be successfully grafted into peripheral nerve12, suggesting that, with modifications, the procedures outlined in this protocol can be used to perform preclinical trials with candidate therapeutic agents directed against neurofibromas.
The authors have nothing to disclose.
This work was supported by the National Institute of Neurological Diseases and Stroke (R01 NS048353 to S.L.C.), the National Cancer Institute (R01 CA122804 to S.L.C., R01 CA134773 to Kevin A. Roth and S.L.C. and CA13148-35 to K.R.Z.) and the Department of Defense (X81XWH-09-1-0086 to S.L.C.). Funds supporting the operation of the UAB Comprehensive Cancer Center Small Animal Imaging Shared facility were provided by a NCI Core Support grant (P30 CA13148-35; E. Partridge, P.I.). We thank the Alabama Neuroscience Blueprint Core Center (P30 NS57098) and the UAB Neuroscience Core Center (P30 NS47466) for their assistance. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Department of Defense.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
Foxchase outbred SCID mice | Charles River | #236 | ||
NIH III mice | Taconic | #NIHBNX | ||
NOD-SCIDγ mice | Jackson Laboratories | #005557 | ||
Cell Stripper | Fisher Scientific | 25-056-cl | ||
Vetbond surgical glue | 3M | 1469SB | ||
Hamilton syringe | Hamilton Company | #80030 | 10 μL volume | |
Hamilton syringe needles | Hamilton Company | #7803-05 | 33 Ga., 0.5 inch, PT4 (custom made) | |
Ear tags | National Band and Ear Tag Co. | 1005-1 | ||
Ophthalmic ointment | Dechra | NDC 17033-211-38 |