We have developed a methodology for assessing whether nervous system neoplasms in genetically engineered mice accurately recapitulate the pathology of their human counterparts. Here, we apply these histologic techniques, defined pathologic criteria, and culture methodologies to neurofibromas and malignant peripheral nerve sheath tumors arising in the P0-GGFβ3 mouse model.
Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that subsequently transform into highly aggressive malignant peripheral nerve sheath tumors (MPNSTs). Understanding the process by which a PN transforms into an MPNST would be facilitated by the availability of genetically engineered mouse (GEM) models that accurately replicate the PN-MPNST progression seen in humans with NF1. Unfortunately, GEM models with Nf1 ablation do not fully recapitulate this process. This led us to develop P0-GGFβ3 mice, a GEM model in which overexpression of the Schwann cell mitogen neuregulin-1 (NRG1) in Schwann cells results in the development of PNs that progress to become MPNSTs with high frequency. However, to determine whether tumorigenesis and neoplastic progression in P0-GGFβ3 mice accurately model the processes seen in NF1 patients, we had to first prove that the pathology of P0-GGFβ3 peripheral nerve sheath tumors recapitulates the pathology of their human counterparts.
Here, we describe the specialized methodologies used to accurately diagnose and grade peripheral nervous system neoplasms in GEM models, using P0-GGFβ3 and P0-GGFβ3;Trp53+/- mice as an example. We describe the histologic, immunohistochemical, and histochemical methods used to diagnose PNs and MPNSTs, how to distinguish these neoplasms from other tumor types that mimic their pathology, and how to grade these neoplasms. We discuss the establishment of early-passage cultures from GEM MPNSTs, how to characterize these cultures using immunocytochemistry, and how to verify their tumorigenicity by establishing allografts. Collectively, these techniques characterize the pathology of PNs and MPNSTs that arise in GEM models and critically compare the pathology of these murine tumors to their human counterparts.
Over the last three decades, numerous laboratories have attempted to create mouse models of human cancers by introducing human cancer-associated mutations into the mouse genome or by overexpressing a gene product that is overexpressed in human cancers. The resulting genetically engineered mouse (GEM) models can be used for a variety of purposes such as establishing that the newly introduced genomic modification initiates tumorigenesis, identifying other subsequently occurring genetic or epigenetic changes that contribute to tumor progression, and defining the key signaling pathways that drive tumor initiation and progression. Unlike orthotopic xenograft models, which rely on the use of immunodeficient mice, GEM cancer models have a fully functional immune system and so more accurately model responses to candidate therapeutic agents. However, when using GEM cancer models for purposes such as these, it is essential that investigators confirm that observations made with GEM neoplasms are relevant to their human counterparts. This validation should include a thorough assessment of the pathology of the GEM neoplasms and a determination as to whether the pathologic features of the GEM neoplasms recapitulate the pathology of the corresponding human tumor type.
The tumor susceptibility syndrome neurofibromatosis type 1 (NF1) is the most common genetic disease affecting the human nervous system, occurring in approximately 1 in every 3,000-3,500 live births1,2,3. Individuals afflicted with NF1 develop multiple benign peripheral nerve sheath tumors known as neurofibromas in their skin (dermal neurofibromas) and in large nerves and nerve plexuses (plexiform neurofibromas). While both dermal and plexiform neurofibromas worsen the patient's quality of life by producing physical, behavioral, and/or social impairment, plexiform neurofibromas (PNs) are particularly dangerous4,5. This is because PNs frequently transform into malignant peripheral nerve sheath tumors (MPNSTs), which are aggressive spindle cell neoplasms with an exceptionally low survival rate1,2. In large part, this low survival rate is because the radio- and chemotherapeutic regimens that are currently used to treat MPNSTs are ineffective. However, developing new, more effective therapies has been challenging. This is because, despite how commonly MPNSTs occur in NF1 patients, they are still rare neoplasms. As a result, it is very difficult to obtain large numbers of human tumors for study; it is also challenging to recruit enough patients with MPNSTs for clinical trials. To overcome these limitations, several GEM models have been generated with the goal of gaining further insights into the abnormalities driving neurofibroma pathogenesis and PN-MPNST progression and to facilitate preclinical trials with candidate therapeutic agents.
NF1 patients have inactivating mutations in one copy of the NF1 gene. Neurofibroma pathogenesis is triggered when an inactivating mutation in the remaining functional NF1 gene occurs in a cell in the Schwann cell lineage. Surprisingly, however, when mice were generated with germline inactivating Nf1 mutations, they did not develop neurofibromas6,7. The subsequent demonstration that mice with Nf1-null Schwann cells and Nf1 haploinsufficiency in all other cell types (Krox20-Cre;Nf1flox/- mice) developed plexiform neurofibromas suggested that reduced Nf1 gene dosage in additional cell types was required for neurofibroma pathogenesis8. Even then, the plexiform neurofibromas in Krox20-Cre;Nf1flox/- mice did not progress to become MPNSTs and so only partially mimicked the biology of their human counterparts. MPNST pathogenesis did occur when Nf1 mutations were partnered with mutations in additional tumor suppressor genes such as Trp539 or Cdkn2a10, but MPNSTs in these GEM models developed de novo or from atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBPs)11,12, rather than from preexisting benign plexiform neurofibromas (see13,14 for excellent reviews of these models as well as other models introducing additional MPNST-associated loss of function mutations in genes such as Suz12 and Pten15).
These mouse models have been invaluable for establishing the role that genes such as NF1, TP53, and CDKN2A play in the pathogenesis of NF1-associated peripheral nervous system neoplasms and for preclinical trials testing candidate therapeutic agents. However, we still have an incomplete understanding of the process by which plexiform neurofibromas progress to become atypical neurofibromatous neoplasms of uncertain biologic potential (ANNUBPs16) and then MPNSTs. Some progress has recently been made in understanding this process with the recent report that mice with deletions in Nf1 and Arf develop ANNUBPs that progress to become MPNSTs11. However, Nf1 mutation-based mouse models that fully recapitulate the process of plexiform neurofibroma-MPNST progression seen in humans do not yet exist. In addition, it is not clear whether there are multiple distinct pathways that lead to the development of MPNSTs. Given this, it is possible that the GEMs described above only model a subset of several different pathways that lead to neurofibroma-MPNST progression and MPNST pathogenesis. This point is emphasized by the fact that MPNSTs also occur sporadically and that some sporadic MPNSTs apparently do not have NF1 mutations17,18.
Although this latter point has been challenged by Magollon-Lorenz et al.'s recent suggestion that at least some sporadic MPNSTs lacking NF1 mutations are melanomas or a different type of sarcoma19, we have recently reported a sporadic MPNST and a cell line derived from this tumor (2XSB cells) that was NF1 wild-type20. During the characterization of the parent tumor and the 2XSB cell line, we systematically ruled out alternative diagnostic possibilities, including melanoma and the multiple other sarcoma types that are routinely considered in the differential diagnosis of a sporadic malignant peripheral nerve sheath tumor20. In addition, we note that Magollon-Lorenz et al. acknowledged that their findings in the three sporadic MPNST cell lines that they studied could not be generalized to indicate that all tumors identified as sporadic MPNSTs are not MPNSTs.
To construct a GEM model in which neurofibroma and MPNST pathogenesis were not necessarily dependent upon specific tumor suppressor gene mutations, we generated transgenic mice in which overexpression of the potent Schwann cell mitogen neuregulin-1 (NRG1) was driven by the Schwann cell-specific myelin protein zero (P0) promoter (P0-GGFβ3 mice)21. We have previously shown that human neurofibromas, MPNSTs, and MPNST cell lines express several NRG1 isoforms together with the erbB receptor tyrosine kinases (erbB2, erbB3, and erbB4) that mediate NRG1 signaling and that these erbB receptors are constitutively activated22. We have also demonstrated that pharmacologic inhibitors of the erbB kinases potently inhibit MPNST proliferation22, survival23, and migration24. In keeping with our observations in humans, P0-GGFβ3 mice develop plexiform neurofibromas25 that progress to become MPNSTs at a high frequency21,25. We have shown that P0-GGFβ3 MPNSTs, like their human counterparts, commonly develop mutations of Trp53 and Cdkn2a, as well as numerous other genomic abnormalities that potentially contribute to tumorigenesis25. MPNSTs arising in P0-GGFβ3 mice do not have inactivating Nf1 mutations. However, using genetic complementation, we showed that NRG1 promotes tumorigenesis in P0-GGFβ3 mice predominantly through the same signaling cascades that are altered by Nf1 loss26; this conclusion is based on our finding that substituting NRG1 overexpression for Nf1 loss in the presence of Trp53 haploinsufficiency (P0-GGFβ3;Trp53+/- mice) produces animals in which MPNSTs develop de novo, as is seen in cis-Nf1+/-;Trp53+/- mice27.
To obtain this and other information demonstrating that P0-GGFβ3 mice accurately model the processes of neurofibroma pathogenesis and neurofibroma-MPNST progression seen in humans with NF1, we have developed specialized methodologies for processing tissues from these animals, accurately diagnosing their tumors, grading the MPNSTs arising in these mice, establishing and characterizing early-passage P0-GGFβ3 and P0-GGFβ3;Trp53+/- MPNST cultures and critically comparing the pathology of P0-GGFβ3 PNs and MPNSTs and P0-GGFβ3;Trp53+/- MPNSTs to that of their human counterparts. Many of these methodologies are generalizable to other GEM models of nervous system neoplasia. Additionally, several of these methodologies are more broadly applicable to GEM models in which neoplasms arise in other organ sites. Consequently, here we present a detailed description of these methodologies.
The procedures described here were approved by the Medical University of South Carolina's IACUC and were performed by properly trained personnel in accordance with the NIH Guide for the Care and Use of Laboratory Animals and MUSC's institutional animal care guidelines.
1. Determining tumor penetrance and survival in P0-GGFβ3 mice and identifying tumors in these animals for further characterization
2. Paraffin-embedding of grossly visible tumors and preparation of hematoxylin and eosin-stained sections for initial diagnostic assessment
3. Identify potential plexiform neurofibromas and perform special stains to confirm diagnoses
NOTE: We strongly recommend including an experienced human or veterinary pathologist in the evaluation of H&E and special stains of GEM tumor sections.
4. Special stains to diagnose MPNSTs and rule out alternative diagnoses
5. Grading of the MPNSTs
6. Preparation of cultures of early passage P0-GGFβ3 MPNST cells
7. Verifying the identity of early-passage MPNST cells by immunocytochemistry
8. Allograft of early-passage tumor cells to demonstrate tumorigenicity
Figure 2 illustrates examples of grossly evident neoplasms arising in P0-GGFβ3 mice. Tumors that are easily identifiable with the naked eye may be seen as masses distending body regions as shown in Figure 2A (arrow). When determining whether the neoplasm is potentially a peripheral nerve sheath tumor, it is essential to establish that the tumor is associated with a peripheral nerve. In this instance, an MRI scan (Figure 2B) demonstrates that the tumor is associated with the sciatic nerve (arrowhead); this association was confirmed after euthanizing the mouse and dissecting the tumor. It should be noted that a large size does not necessarily indicate that the tumor is malignant. In this case, a histologic examination of the tumor (Figure 2C) demonstrated that it was a neurofibroma. Most commonly, however, grossly evident tumors are not identified until performing the necropsy of the mouse. Figure 2D illustrates a large, fleshy MPNST that arose within the brachial plexus of this animal.
Figure 3 illustrates representative examples of MPNSTs and neurofibromas from P0-GGFβ3 mice prepared according to the procedures outlined in protocol section 1. Figure 3A–J illustrates ten examples of independently arising MPNSTs from our P0-GGFβ3 mouse colony. Note that the histologic appearance of MPNSTs can be highly variable, even between MPNSTs arising independently in the same animal. This histologic variability illustrates why we routinely confirm the diagnosis of P0-GGFβ3 MPNSTs with immunohistochemical stains. We would also point out that other, apparently unrelated tumor types occur sporadically at a low frequency in some inbred mouse strains (e.g., we have several times encountered lymphomas in P0-GGFβ3 mice carrying the transgene on a C57BL/6J background), further emphasizing the importance of using immunohistochemistry to confirm tumor diagnoses. Despite the variability of their histologic appearance, all ten of the tumors illustrated in Figure 3A–J were immunoreactive for S100β and nestin and negative for markers of other tumor types. Figure 3K illustrates a representative image of a properly decalcified vertebral column and associated tissues. Note that the spinal cord, nerve roots, vertebral body, and skeletal muscle all maintain their proper anatomic relationship with each other. Figure 3L is a higher-power image of the vertebral body and overlying spinal cord. Since this tissue is properly decalcified, the bone has cut easily without shredding or folding, and bone marrow is readily identifiable in the marrow spaces. If the tissue had not been decalcified properly, it would have caught on the microtome blade and been torn out of the section, doing significant damage to adjacent tissues (spinal cord, spinal nerve roots, and skeletal muscle. Figure 3M presents a representative image of a neurofibroma arising within a dorsal nerve root in a P0-GGFβ3 mouse. Note that this tumor is less cellular than the MPNSTs shown in Figure 3A–J. The key diagnostic for neurofibromas is that they are composed of a complex mixture of neoplastic Schwann cells and non-neoplastic mast cells, macrophages, fibroblasts, and perineurial-like elements that infiltrate the nerve and spread axons apart.
Figure 4 illustrates examples of the stains that are most useful for the initial identification of a plexiform neurofibroma and distinguishing between a plexiform neurofibroma and an MPNST. Figure 4A illustrates S100β immunoreactivity in a P0-GGFβ3 plexiform neurofibroma. Note that S100β immunoreactivity is only evident in a subpopulation of cells, which is in keeping with the fact that neurofibromas are composed of a mixture of neoplastic Schwann cells and other non-neoplastic elements (fibroblasts, mast cells, macrophages, perineurial-like cells, a poorly defined CD34-immunoreactive cellular population, and vasculature). Unfortunately, patchy S100β staining does not distinguish between plexiform neurofibromas and MPNSTs as S100β staining can be patchy in MPNSTs (H&E staining is useful for this purpose; however, since MPNSTs are typically more cellular than plexiform neurofibromas [see Figure 3]). Neoplastic Schwann cells are also immunoreactive for the intermediate filament nestin as demonstrated in the P0-GGFβ3 MPNST presented in Figure 4B. Neoplastic Schwann cells also often demonstrate nuclear immunoreactivity for the transcription factor Sox10, as shown in an MPNST in Figure 4C. Features useful for distinguishing between plexiform neurofibromas and MPNSTs are the presence of mast cells and prominent immunoreactivity for the Ki67 proliferation marker. Figure 4D illustrates an Unna stain performed on a P0-GGFβ3 plexiform neurofibroma to highlight the presence of mast cells, which are easily identifiable by the prominent metachromatic violet staining of their cytoplasmic granules. Mast cells are not present in MPNSTs. In contrast, Ki67 immunoreactivity is virtually non-existent in plexiform neurofibromas as seen in the P0-GGFβ3 tumor shown in Figure 4E. Nuclear Ki67 labeling is typically present in a very high fraction of tumor cells as seen in the microscopic MPNST arising in the trigeminal ganglion of a P0-GGFβ3 mouse (Figure 4F).
Figure 5 illustrates examples of the stains that we perform to fully characterize the cellular composition of neurofibromas in a newly developed GEM model. Although the stains shown in this figure were obtained in human dermal neurofibromas, they are identical in appearance to what we have seen in GEM tumors. Immunoreactivity for CD117 (c-Kit) is present in mast cells within neurofibromas and thus, has a distribution highly similar to what is seen with Unna stains (see Figure 3A). Macrophages are also present scattered throughout neurofibromas, as seen with the pan-macrophage marker Iba1 (see Figure 5D); this includes subclasses of macrophages that are immunoreactive for CD163 and CD86 (see Figure 5B and Figure 5C, respectively). A fraction of the Schwannian element in neurofibromas also demonstrates nuclear immunoreactivity for Sox10. Fibroblasts can be highlighted by their immunoreactivity for TCF4. In Figure 5G, CD31 labels the vascular elements within the neurofibroma, while CD34, demonstrated in Figure 5H, labels an enigmatic dendritic population of cells that have been suggested to be either a subpopulation of resident tissue macrophages30 or a novel population of nerve sheath cells that are neither Schwann cells nor fibroblasts31.
Figure 6 is included to enable the comparison of human plexiform neurofibromas and MPNSTs to the tumors seen in P0-GGFβ3 mice and to provide representative examples of some of the human tumor types that can be confused with MPNSTs. Figure 6A illustrates a plexiform neurofibroma that arose in the brachial plexus of an NF1 patient, while Figure 6B presents the WHO grade IV MPNST that arose within this same plexiform neurofibroma. Figure 6B shows some of the characteristic features of a WHO grade IV MPNST, including marked hypercellularity and cellular atypia, brisk mitotic activity, and tumor necrosis. For comparison, Figure 6C shows a WHO grade II MPNST that has significant cellular atypia but is less hypercellular than the grade IV MPNST and, although mitoses are present, demonstrates less mitotic activity. Figure 6D illustrates a fibrosarcoma with its characteristic "herringbone" pattern of interweaving sheaths of tumor cells. This pattern does not necessarily distinguish between fibrosarcomas and MPNSTs, however, because some MPNSTs will have a similar pattern. Further, the higher-power view presented in Figure 6E shows cellular morphology that is similar to that seen in the WHO grade IV MPNST presented in Figure 6B. Figure 6F illustrates a leiomyosarcoma. Unlike most MPNSTs, leiomyosarcomas are immunoreactive for muscle markers such as smooth muscle actin and desmin. Smooth muscle actin immunoreactivity can be variable from tumor to tumor, though, with some tumors demonstrating intense uniform immunoreactivity (Figure 6G) and others showing immunoreactivity that shows cellular variability within the tumor (Figure 6H). Desmin immunoreactivity can also be patchy in leiomyosarcomas (Figure 6I). Melanomas are highly variable in morphology, with some tumors being composed of polygonal cells (Figure 6J) and others being composed of spindled cells that can mimic the morphology of MPNST cells. Melanomas can be distinguished from MPNSTs by immunoreactivity for melanosome markers such as MART1 (Figure 6K). However, melanomas, like MPNSTs, frequently are positive for S100β and Sox10 (Figure 6L).
Figure 7 illustrates the pathologic features of WHO grade II, III, and IV MPNSTs isolated from P0-GGFβ3 mice. Figure 8 shows representative images of early-passage P0-GGFβ3 MPNST cells at low (Figure 7A) and high (Figure 7B) power. The tumorigenicity of these cells is demonstrated both by their ability to form colonies when suspended in soft agar (Figure 7C) and to form grafts when allografted subcutaneously in immunodeficient mice (Figure 7D).
Figure 1: Workflow used to process tumor and other tissues from P0-GGFβ3 mice. (A) Grossly visible tumors are harvested and segmented into three portions for 1) fixation in 4% paraformaldehyde followed by immunohistochemistry and histochemistry, 2) establishment of early passage tumor cell culture and/or genomic analyses, and 3) snap-freezing using liquid nitrogen for protein, DNA or RNA isolation. (B) After the excision of the tumor, the body of the mouse is fixed in 4% paraformaldehyde and the internal organs are removed. These organs are sampled for histologic examination performed to identify microscopic evidence of neoplasm and other pathologic processes. (C) Following removal of internal organs, the extremities (head, limbs, tail) and skin are removed from the carcass. The vertebral column, adjacent ribs, and adjacent skeletal muscle are decalcified using 0.3 M EDTA/4% paraformaldehyde (pH 8.0). The decalcified tissues are then paraffin-embedded and sections of the tissues prepared for immunohistochemical and histochemical examination. Please click here to view a larger version of this figure.
Figure 2: Representative images of grossly evident neurofibromas and MPNSTs in P0-GGFβ3 mice. (A) P0-GGFβ3 mouse with a large grossly evident tumor on the right flank (arrow). (B) An MRI scan of this mouse shows that the tumor is connected to the sciatic nerve (arrowhead) and that it has grown through the overlying fascia to expand within the subcutaneous (arrow, bulk tumor mass). (C) Microscopic examination of this tumor shows that, despite its large size, the tumor is a neurofibroma. (D) Large fleshy MPNST that arose in the brachial plexus of a P0-GGFβ3 mouse. Scale bar = 100 µm. (C). Abbreviations: MPNSTs = malignant peripheral nerve sheath tumors; MRI = magnetic resonance imaging. Please click here to view a larger version of this figure.
Figure 3: Representative images of MPNSTs, decalcified vertebral column and neurofibromas prepared as described in protocol section 1. (A-J) Excised and H&E-stained P0-GGFβ3 MPNSTs show histologic variability. All images are independently arising MPNSTs. Despite this histologic variability, these tumors all showed appropriate labeling for the MPNST markers indicated in Table 1. Scale bars = 200 µm. (K) Representative image of an H&E-stained cross-section of the decalcified vertebral column. In this image the following structures are easily visualized: the spinal cord; vertebral bone; the dorsal root ganglia on the dorsal spinal nerve root; and paravertebral skeletal muscle. Magnification 4x. (L) A higher-power image of the spinal cord and vertebra shown in K demonstrates the proper appearance of bone following decalcification with this methodology. Magnification 10x. (M) Representative image of a dorsal nerve root neurofibroma in a P0-GGFβ3 mouse. Magnification 40x. Scale bars = 100 µm. Abbreviations: MPNSTs = malignant peripheral nerve sheath tumors; H&E = hematoxylin and eosin. Please click here to view a larger version of this figure.
Figure 4: Diagnostic stain used for the initial identification of plexiform neurofibromas and their distinction from MPNSTs. (A) Immunostains for S100β in a P0-GGFβ3 plexiform neurofibroma. Note that intense brown staining for this antigen is only present in a subset of cells in this tumor, consistent with the fact that neurofibromas are composed of neoplastic Schwann cells and multiple other non-neoplastic cell types. (B) Immunofluorescence image of a P0-GGFβ3 MPNST stained for the intermediate filament nestin (red) and counterstained with bisbenzimide (blue, nuclear stain). (C) MPNST stained for the transcription factor Sox10. Intense immunoreactivity (brown) is evident in the nuclei of a subset of tumor cells. (D) High-power view of a P0-GGFβ3 plexiform neurofibroma after an Unna stain. This stain produces metachromatic (violet) staining of the granules in mast cells. Plexiform neurofibromas can be distinguished from MPNSTs because the latter lack mast cells. (E,F) Ki67 immunohistochemistry in (E) a P0-GGFβ3 plexiform neurofibroma and (F) a P0-GGFβ3 MPNST. Both sections have been counterstained with hematoxylin (blue nuclear staining), with Ki67 immunoreactivity being evident as brown nuclear staining in these immunoperoxidase stains. Note that no brown nuclear staining is evident in the plexiform neurofibroma, whereas most of the tumor cell nuclei are positive in the MPNST. Scale bars = 100 µm. Abbreviation: MPNST = malignant peripheral nerve sheath tumor. Please click here to view a larger version of this figure.
Figure 5: Representative images of immunostains used to identify the subpopulations of cells that compose neurofibromas. These immunofluorescent images from a human dermal neurofibroma have been stained for (A) CD117 (c-Kit; a marker of mast cells), (B) CD163 (M2 macrophages), (C) CD86 (M1 macrophages), (D) Iba1 (pan-macrophage marker), (E) Sox10 (Schwann cell marker), (F) TCF4 (fibroblast marker), (G) CD31 (marker of vasculature), and (H) CD34 (marks a distinct, poorly understood subpopulation of cells in neurofibromas). Magnification 60x, scale bars = 200 µm. Please click here to view a larger version of this figure.
Figure 6: Representative images of plexiform neurofibromas, MPNSTs, and some other human tumor types that are considered in the differential diagnosis of an MPNST. (A) Plexiform neurofibroma arising in the brachial plexus of an NF1 patient, showing the overall lower cellularity and benign appearance of this neoplasm. Although not easily visualized in H&E-stained sections, a complex mixture of cell types is present. Mitoses are not seen. Magnification: 40x. (B) A WHO grade IV MPNST that arose from the plexiform neurofibroma illustrated in A. Note the much higher degree of cellularity. The arrow indicates a mitotic figure, and the asterisk denotes a region of tumor necrosis in the upper right portion of this microscopic field. Magnification: 40x. (C) A WHO grade II MPNST. This tumor has a lower degree of cellularity than the WHO grade IV MPNST illustrated in B. However, there is more nuclear atypia and hyperchromasia than is evident in the plexiform neurofibroma illustrated in A. The arrow indicates one of the occasional mitotic figures that were encountered in this neoplasm. Magnification: 63x. (D) A low-power view of an adult-type fibrosarcoma illustrating the "herringbone" pattern (the interweaving sheaths of tumor cells) typically seen in this tumor type. Unfortunately, herringbone architecture is also encountered in some human MPNSTs and so cannot be used to distinguish between fibrosarcomas and MPNSTs. Magnification: 20x. (E) A higher-power view of the fibrosarcoma illustrated in D. Note the similarity between the cellular morphology in this fibrosarcoma and the cellular morphology evident in the WHO grade IV MPNST shown in B. Magnification: 40x. (F) High-power image of a leiomyosarcoma, demonstrating cellular morphology that falls within the range of variation seen in MPNSTs. Magnification: 40x. (G) Immunostains for smooth muscle actin in the leiomyosarcoma illustrated in F. Note that the tumor cells show uniform intense immunoreactivity for this antigen. Magnification: 40x. (H) Immunostains for smooth muscle actin in a different leiomyosarcoma. In this tumor, there is greater variability in the degree of immunoreactivity, with some cells staining more intensely than others. It is not uncommon for immunoreactivity for the same antigen to be uniformly present in one tumor and to be present only in a subset of tumor cells in a different neoplasm. Magnification: 40x. (I) Immunostain for desmin in a third leiomyosarcoma. In this tumor, only a subset of tumor cells is intensely immunoreactive for this antigen. (J) Metastatic melanoma. Melanomas are notorious for having highly variable morphology that can range from cuboidal to spindled; tumors with the latter morphology are most likely to be confused with MPNSTs, particularly given that both melanomas and MPNSTs can demonstrate S100β and Sox10 immunoreactivity. Magnification: 40x. (K) Immunoreactivity for the melanoma marker MART1 in the tumor illustrated in J. Magnification: 40x. (L) Nuclear immunoreactivity for the transcription factor Sox10 in the melanoma shown in J. Magnification: 40x, scale bars = 100 µm. Abbreviation: MPNST = malignant peripheral nerve sheath tumor. Please click here to view a larger version of this figure.
Figure 7: Representative images of WHO grade II-IV P0-GGFβ3 MPNSTs. (A) Low- and (B) high-power photomicrographs of a WHO grade II MPNST. Note that the cellularity is lower than the WHO grade III MPNST illustrated in panel C and that the nuclei of the tumor cells in D are more hyperchromatic (darker) than those seen in panel B. (C) Low- and (D) high-power photomicrographs of a WHO grade III MPNST. This tumor demonstrated >4 mitotic figures per 10 high-power fields, with cells that were more densely packed and more hyperchromatic, atypical nuclei. (E) Low- and (F) high-power views of a WHO grade IV MPNST. Note the focus of necrosis in the bottom center portion of panel F. Low power = 20x. High Power = 40x, scale bars = 100 µm. Please click here to view a larger version of this figure.
Figure 8: Representative images of early-passage P0-GGFβ3 MPNST cells and their tumorigenicity as demonstrated by growth in soft agar and their ability to grow as allografts. (A) Low- and (B) high-power phase contrast images of early-passage P0-GGFβ3 MPNST cells. (C) P0-GGFβ3 MPNST cells grown in soft agar and stained with Sudan black; the colonies are evident as black puncta in the agar. (D) Hematoxylin and eosin-stained image of a tumor that formed after P0-GGFβ3 MPNST cells were allografted subcutaneously in an immunodeficient mouse as described in the protocol. (E) Proliferation of early-passage P0-GGFβ3 MPNST cells over a 5-day period as determined by a Celigo Image Cytometer. Low power = 10x, high power = 40x; scale bar = 100 µm for all panels Please click here to view a larger version of this figure.
Name | Usage | Species Reactivity/Class |
CD117 | Prediluted | rabbit monoclonal |
CD163 | 1:200 | mouse monoclonl |
CD31 | 1:50 | rabbit polyclonal |
CD34 | 1:2000 | rabbit monoclonal |
CD86 | 1:1000 | rabbit monoclonal |
Cytokeratin | 1 µg/mL | mouse monoclonal |
Desmin | 1:50 | mouse monoclonal |
Iba1 | 1:500 | polyclonal rabbit |
Ki-67 | 1:50 | rabbit monoclonal |
MART1 | 1 µg/mL | mouse monoclonal |
Nestin | 1:1,000 | mouse monoclonal |
PMEL | 1:100 | rabbot monoclonal |
S100B | 1:200 | rabbit polyclonal |
SMA | 1:100 | mouse monoclonal |
Sox10 | 1:10 | mouse monoclonal |
TCF4/TCFL2 | 1:100 | rabbit monoclonal |
Table 1: Antibodies used for the diagnosis of plexiform neurofibroma and malignant peripheral nerve sheath tumors. Antibodies used for routine identification of GEM plexiform neurofibromas (S100β, Sox10, CD117, Ki67), the diagnosis of MPNSTs, and a complete assessment of other cell types present in neurofibromas. Abbreviation: MPNST = malignant peripheral nerve sheath tumor.
S100β | Nestin | Sox10 | MART1 | PMEL | Desmin | Smooth muscle actin | Cytokeratin | SS18-SSX Fusion | ||
MPNST | 50-90%, usually focal | Positive1 | ~30% | Negative | Negative | Negative | Negative | Negative | Negative | |
Fibrosarcoma | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | |
Leiomyosarcoma | Rare | Negative | Negative | Negative | Negative | 50-100% | Positive | ~40% | Negative | |
Epitheloid sarcoma | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Positive | Negative | |
Melanoma | Positive | Positive | 85% | Positive | Positive | Negative | Negative | Negative | Negative | |
Monophasic synovial sarcoma | ~30% | Negative | Negative | Negative | Negative | Negative | Negative | Positive | Positive |
Table 2: Markers used to establish tumor identity in humans. These immunohistochemical and histochemical markers, together with an assessment of tumor microscopic morphology, are used to distinguish MPNSTs from other neoplasms that mimic them. The differential diagnosis typically considered for human MPNSTs includes adult-type fibrosarcoma, epitheloid sarcoma, leiomyosarcoma, monophasic synovial sarcoma, and melanoma. Adult-type fibrosarcomas have a herringbone pattern microscopically and stain for vimentin, but not S100β. Leiomyosarcomas, but not MPNSTs, are immunoreactive for desmin; leiomyosarcomas also have nuclei with a notably blunt-ended morphology. Epitheloid sarcomas, but not MPNSTs, are immunoreactive for cytokeratin. Melanomas, like MPNSTs, may be S100β-positive, but are also immunoreactive for MART-1 and PMEL. Abbreviation: MPNST = malignant peripheral nerve sheath tumor. 1Combination of S100β and nestin highly predictive of MPNST.
The histological and biochemical methods presented here provide a framework for diagnosing and characterizing GEM models of neurofibroma and MPNST pathogenesis. Over the years, we have found these methodologies to be quite useful for assessing the pathology of peripheral nerve sheath tumors arising in GEM models21,25,26. However, while the protocols outlined here are useful for determining how accurately tumors in the GEM models recapitulate the pathology of their human counterparts, there are some limitations to these strategies that should be appreciated. To begin with, P0-GGFβ3 mice show almost complete penetrance of their tumor phenotype, which makes it relatively easy to obtain large numbers of mice with neurofibromas and MPNSTs21,25,26 that can be used for genomic studies and genome-scale shRNA screens. The high penetrance of the phenotype in this model also makes P0-GGFβ3 mice quite useful for preclinical trials. It should be recognized, though, that this will not be the case for all GEM cancer models. This is why we have emphasized the importance of determining the fraction of animals that can be predicted to develop tumors. When working with an animal model that less commonly develops tumors, we have found it advantageous to use imaging modalities such as magnetic resonance imaging or positron emission tomography to definitively identify animals carrying tumors that can be entered into preclinical trials or other studies. This approach can be cost-prohibitive, however, if repeated scans are required. This is why we also emphasized the importance of establishing the average survival time of the GEM model of interest26-understanding when an animal can be expected to develop a tumor reduces the number of scans that are required to identify animals with the desired phenotype and associated costs.
It is also important to recognize that a large tumor in a mouse is still actually a rather small amount of tissue. In this protocol, we recommend a process in which the tumor tissue is divided into thirds, with portions devoted to histology/immunohistochemistry, establishing early passage cell cultures, and isolation of biomolecules (proteins, RNA, DNA) of interest. However, tumor size will vary and if the tumor is quite small, this can preclude having enough tissue to divide in this fashion. In that case, the investigator must determine whether tumor diagnosis or another use of tumor tissue is prioritized32. Under those circumstances, our bias is that we must have a proper diagnosis to understand what we are working with before embarking on other experiments. We have found that tumor diagnoses can usually be readily established using less than a third of the neoplasm. When dealing with small tumors, we instead will typically remove a small fragment of tumor tissue, wrap it in histology tissue and place it in a tissue cassette to ensure that the tissue is not lost during processing. The disadvantage of this approach is that it may cause the investigator to undergrade the neoplasm if WHO grading is desired; this is because lower and higher-grade regions commonly coexist in cancers and, if a very small sample is taken, the grade obtained will depend on where the tumor sample was taken from.
The protocols we describe for establishing peripheral nerve sheath tumor identity rely heavily on immunohistochemistry. While there are occasional alternative approaches that can be used for some cell types (e.g., performing Unna stains to identify mast cells), that is not uniformly true for most of the cell types present in neurofibromas and MPNSTs. Consequently, great care must be taken to establish that the immunohistochemical procedures that will be used have been properly optimized. In our experience, several issues can compromise diagnostic immunohistochemistry. It is critically important that the investigator perform a dilutional series with a primary antibody that they have not used before; in addition, a dilutional series should be performed when using a new batch of an antibody that has been previously optimized33. Care must be taken as well to ensure that fresh batches of reagents are on hand. In our experience, when they age, hydrogen peroxide and DAB are particularly prone to not performing properly, which may lead the investigator to inappropriately decide that their stains are negative.
The immunohistochemical profiles that we describe for plexiform neurofibromas, MPNSTs, and the diverse types of neoplasms that are considered in the differential diagnosis of MPNSTs are those that we have most commonly encountered21,25,26,34. However, as divergent differentiation is not uncommon in MPNSTs and these other malignancies, the investigator may encounter staining profiles that differ somewhat from what we describe here. These nuances are why we strongly recommend that the team characterizing a new GEM model of tumorigenesis include an experienced human or veterinary pathologist. In this protocol, we have applied WHO grading to GEM and human MPNSTs. We prefer this grading system because the grading criteria are relatively well defined and are easy to compare between human and mouse MPNSTs2,28. We would note, though, that WHO grading criteria are not uniformly accepted by pathologists. This is because it is not clear that the three WHO grades that are applied to MPNSTs are predictive of clinical outcomes in patients. Because of that, many pathologists prefer to classify MPNSTs simply as low-grade or high-grade malignancies. Using this approach, approximately 85% of MPNSTs are considered high-grade malignancies characterized by brisk mitotic activity, marked cellular atypia, and hyperchromasia that may be accompanied by tumor necrosis. Low-grade MPNSTs show less cellularity, hyperchromasia, and mitotic activity.
We have found that allografting early passage MPNST cells is a straightforward means of verifying the tumorigenicity of these cultures. However, some issues should be considered when the cells do not establish an allograft32. In our experience, while the overwhelming majority of early-passage MPNST cells will establish an allograft, we have occasionally encountered early passage cultures that do not. Despite this failure, array comparative genomic hybridization (aCGH) and whole exome sequencing demonstrated that these early-passage cultures had multiple genomic abnormalities consistent with them being tumor cells25,26. We have also found early-passage cultures that are not healthy, usually because the cultures were allowed to become confluent before harvesting them for grafting. Cells damaged by rough handling (e.g., incubated overly long with non-enzymatic cell dissociation solution pipetted up and down an excessive number of times) also perform poorly when grafted. We would also note that the times we have indicated for graft establishment are an estimate based on our experience. On occasion, we have encountered early-passage cultures that will successfully establish allografts but take a longer time to do so.
The approaches outlined above provide a comprehensive means of characterizing key aspects of a newly developed GEM model of peripheral nervous system neoplasia and properly diagnosing any neurofibromas and MPNSTs that these animals develop. The methods that we outline for establishing early passage MPNST cultures and using these cultures for allografting also provide clear evidence for the tumorigenicity of neoplasms identified in GEM models. We would emphasize that we are not performing the selection of individual clones when establishing early passage cultures. Although we recognize that some selection is unavoidable when placing tumor cells in cultures, these initial findings suggest that the mutations identified in early-passage GEM MPNST cultures remain highly like those present in the parent tumor. However, using aCGH, we have also found that continuing to carry these early passage cultures for more extended passages results in genomic changes that begin to diverge from what is seen in the earlier passages of these tumor cells.
The authors have nothing to disclose.
This work was supported by grants from the National Institute of Neurological Diseases and Stroke (R01 NS048353 and R01 NS109655 to S.L.C.; R01 NS109655-03S1 to D.P.J.), the National Cancer Institute (R01 CA122804 to S.L.C.) and the Department of Defense (X81XWH-09-1-0086 and W81XWH-12-1-0164 to S.L.C.).
100 mm Tissue Culture Plates | Corning Falcon | 353003 | |
3, 3'- Diaminobensidine (DAB) | Vector Laboratories | SK-400 | |
6- well plates | Corning Costar | 3516 | |
Acetic Acid | Fisher Scientific | A38-212 | |
Alexa Fluor 488 Secondary (Goat Anti-Mouse) | Invitrogen | A11029 | |
Alexa Fluor 568 Secondary (Goat Anti-Mouse) | Invitrogen | A21043 or A11004 | |
Alexa Fluor 568 Secondary (Goat Anti-Rabbit) | Invitrogen | A11036 | |
Ammonium Chloride (NH4Cl) | Fisher Scientific | A661-500 | |
BCA Protein Assay Kit | Thermo Scientific | 23225 | |
Bovine Serum Albumin | Fisher Scientific | BP1600-100 | |
Caldesmon | ABCAM | E89, ab32330 | |
CD117 | Cell Marque | 117R-18-ASR | |
CD163 | Leica | NCL-L-CD163 | |
CD31 | ABCAM | ab29364 | |
CD34 | ABCAM | ab81289 | |
CD86 | ABCAM | ab53004 | |
Cell Scraper | Sarstedt | 83.183 | |
Cell Stripper | Corning | 25-056-CI | |
Circle Coverslip | Fisher Scientific | 12-545-100 | |
Citrisolve Hybrid (d-limonene-based solvent) | Decon Laboratories | 5989-27-5 | |
Critic Acid | Fisher Scientific | A104-500 | |
Cytokeratin | ABCAM | C-11, ab7753 | |
Desmin | Agilent Dako | clone D33 (M0760) | |
Diaminobensizdine (DAB) Solution | Vector Laboratories | SK-4100 | |
DMEM | Corning | 15-013-CV | |
Eosin Y | Thermo Scientific | 7111 | |
Ethanol (200 Proof) | Decon Laboratories | 2716 | |
Fetal Calf Serum | Omega Scientific | FB-01 | |
Forksolin | Sigma-Aldrich | F6886 | |
Glycerol | Sigma-Aldrich | G6279 | |
Hank's Balanced Salt Solution (HBSS) | Corning | 21-022-CV | |
Harris Hematoxylin | Fisherbrand | 245-677 | |
Hemacytometer | Brightline-Hauser Scientific | 1490 | |
Hydrochloric Acid | Fisher Scientific | A144-212 | |
Hydrogen Peroxide | Fisher Scientific | 327-500 | |
Iba1 | Wako Chemicals | 019-19741 | |
ImmPRESS HRP (Peroxidase) Polymer Kit ,Mouse on Mouse | Vector Laboratories | MP-2400 | |
ImmPRESS HRP (Peroxidase) Polymer Kit, Horse Anti-Rabbit | Vector Laboratories | MP-7401 | |
Incubator | Thermo Scientific | Heracell 240i CO2 incubator | |
Isoflurane | Piramal | NDC 66794-017-25 | |
Isopropanol | Fisher Scientific | A415 | |
Ki-67 | Cell Signaling | 12202 | |
Laminin | Thermo Fisher Scientific | 23017015 | |
Liquid Nitrogen | |||
MART1 | ABCAM | M2-9E3, ab187369 | |
Microtome | |||
Nestin | Millipore | Human: MAD5236 (10C2), Human:MAB353 (Rat-401) | |
Neuregulin 1 beta | In house | Made by S.L.C. (also available as 396-HB-050/CF from R&D Systems) | |
Neurofibromin | Santa Cruz Biotechnology | sc-67 | |
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice | Jackson Laboratory | 5557 | |
Nonfat Dry Milk | Walmart | Great Value Brand | |
P0-GGFβ3 mice | In house | ||
Paraffin Wax | Leica | Paraplast 39601006 | |
Parafilm M | Sigma-Aldrich | PM-999 | |
Paraformaldehyde (4%) | Thermo Scientific | J19943-K2 | |
Permount (Xylene Mounting Medium) | Fisher Scientific | SP15-100 | |
pH Meter | Mettler Toldedo | Seven Excellence, 8603 | |
Phosphate Buffered Saline (Dulbecco's) | Corning | 20-031-CV | |
PMEL | ABCAM | EP4863(2), ab137078 | |
Poly-L-Lysine Hydrobromide | Sigma-Aldrich | P5899-5MG | |
Portable Isoflurance Machine | VetEquip Inhalation Anesthesia Systems | ||
PVA-DABCO (Aqueous Mounting Medium) | Millipore Sigma | 10981100ML | |
Rice Cooker | Beech Hamilton | ||
S100B | Agilent Dako | Z0311 (now GA504) | |
SMA | Ventana Medical Systems | clone 1A4 | |
Sodium Chloride | Fisher Scientific | S640 | |
Sodium Citrate (Dihydrate) | Fisher Scientific | BP327-1 | |
Sox10 | ABCAM | ab212843 | |
Steel histology mold | |||
Superfrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
TCF4/TCFL2 | Cell Signaling | (CH48H11) #2569 | |
Tissue Cassette | |||
Toluidine Blue | ACROS Organics | 348600050 | |
Triton X-100 | Fisher Scientific | BP151-500 | |
TRIzol | Invitrogen | 15596026 | |
Trypsin | Corning | 25-051-31 |