Brain Tumor Pathol DOI 10.1007/s10014-014-0190-4
CASE REPORT
Anaplastic meningioma with rapid growth after omental flap transposition: a case report and experimental study Kenichiro Iwami • Hiroyuki Momota • Masazumi Fujii Atsushi Natsume • Shunjiro Yagi • Kazuhiro Toriyama Yuzuru Kamei • Toshihiko Wakabayashi
• •
Received: 24 March 2014 / Accepted: 18 April 2014 Ó The Japan Society of Brain Tumor Pathology 2014
Abstract Meningiomas occasionally display aggressive behavior, but the mechanisms of malignant transformation remain unclear. We encountered the case of a 65-year-old man with a 10-year history of recurrent meningioma. The patient had undergone multiple tumor resections, radiotherapy treatments, and reconstructive surgeries due to wound infection. After the third resection of the tumor and reconstruction with an omental flap, the tumor demonstrated rapid growth and lung metastasis. The final pathological diagnosis was anaplastic meningioma. Because the drastic change of the tumor was observed after omental flap transposition, we investigated the effect of the omentum on tumor cells and performed histopathological analyses of meningiomas using a mouse model. We found that meningioma cells have a high affinity to the omentum and show a growth advantage when co-cultured with adipocytes. Immunohistochemical staining revealed that meningioma cells adjacent to the omentum strongly expressed fatty acid-binding protein 4, a lipid transfer protein, in both mouse and human. Our results suggest that
K. Iwami H. Momota (&) M. Fujii A. Natsume T. Wakabayashi Department of Neurosurgery, Nagoya University, Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan e-mail: [email protected] Present Address: K. Iwami Department of Neurosurgery, Japanese Red Cross Nagoya Daiichi Hospital, Nagoya, Japan S. Yagi K. Toriyama Y. Kamei Department of Plastic and Reconstructive Surgery, Nagoya University, Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
tumor cells can receive lipid supply from omental adipocytes, and the surrounding tissues may induce tumor progression. We conclude that although omental tissue is an ideal material for reconstruction surgery, close follow-up is recommended in meningioma patients when used for cranioplasty. Keywords Meningioma Omentum Adipocyte FABP4 Mouse model
Introduction Meningiomas are the most commonly diagnosed primary intracranial neoplasms, constituting approximately 36 % of all primary tumors in this location [1]. Approximately 90 % of meningiomas are benign [grade I of the World Health Organization (WHO) grading scheme], 5–7 % are atypical (WHO grade II), and 1–3 % are anaplastic/ malignant (WHO grade III) [2]. Despite substantial advances in modern therapies, surgical resection remains the best treatment option for most patients with meningioma [3–5]. However, even histologically benign meningiomas occasionally recur and can result in incurable disease [6, 7]. WHO grade II and III meningiomas have a high recurrence rate after both surgical and radiosurgical managements [8–10], and repeated surgery may be required in patients with recurrent meningioma. Craniotomy can lead to cranial infection, and repeated surgery is a known risk factor for craniotomy infection [11]. The standard management of cranial bone infection includes wound debridement, bone removal, and delayed cranioplasty with artificial bone or other materials [12, 13]. Infection can moreover be reactivated after cranioplasty as a result of the epidural cavity becoming filled with serous
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fluid, resulting in abscess formation. We have previously reported on the successful treatment of cranial bone infection using a reconstructive technique that fills the dead space with a titanium mesh plate enclosed in an omental flap [14]. The omentum is a highly vascularized peritoneal fold comprising mesothelial cell layers enveloping the adipose tissue [15]. Its rich blood supply and large lymphatic network prevent infection, and its absorptive capacity resorbs exudates [14]. However, a recent study indicated that omental adipocytes attract cancer cells and promote migration and invasion, and showed that fatty acid-binding protein 4 (FABP4) plays a key role as a lipid transporter through this cell–cell interaction [16]. We here report a case of meningioma with malignant transformation in the course of multiple recurrences. Shortly after a cranioplasty using an omental flap, the tumor became invasive and metastasized to the lungs. Because lipid transfer from adjacent adipose tissue has been implicated in the progression of certain cancers, and since it may have played a role in the late progression of the high-grade meningioma in this case, we moreover
conducted in vitro and in vivo experiments to further investigate its potential role in meningioma tumor progression.
Fig. 1 Magnetic resonance imaging (MRI) scans (a–e) and macroscopic appearance (f) of the meningioma in our patient. a Axial T1weighted image with gadolinium contrast (T1-Gd) in 2001 showed an en plaque left frontal tumor (red arrow). b T1-Gd MRI in 2006 showed a recurrent tumor in the left sphenoid ridge (red arrow). c T1Gd MRI in 2010 demonstrated multiple recurrences in the parasagittal and left frontal convexities (red arrows). d T1-Gd in July 2011 showing the omental flap (black arrowheads) and titanium mesh plate
(red arrowhead) in the left cranium. e T1-weighted MRI without gadolinium contrast in 2011 showed a large extra-axial tumor in the left cranium compressing the brain and protruding toward the skin. MRI with contrast enhancement was not performed in the emergency setting. Red arrowheads delineate the tumor border. f At the fourth operation, the tumor presented as an irregularly shaped mass protruding under the skin on the left side of the head (red arrow)
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Case report Clinical presentation A 55-year-old man presented with a mild headache in 2001. Cranial magnetic resonance imaging (MRI) revealed an en plaque left frontal tumor invading the brain parenchyma (Fig. 1a). Two months after the initial presentation, he underwent the first surgery of gross total tumor removal by left frontal craniotomy. The patient developed a cranial bone infection at the craniotomy site, and the bone flap was removed. A secondary cranioplasty using hydroxyapatite was performed 6 months later. In 2005, a small local recurrent lesion was found in the left frontal convexity, and the patient underwent single-fraction gamma knife radiosurgery at marginal (12 Gy) and maximum (20 Gy) doses.
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In 2006, cranial MRI revealed a small tumor at the left sphenoid ridge (Fig. 1b), and the second surgical removal of the tumor was performed. In 2008, recurrent tumors were again identified: this time located in the parasagittal and left frontal convexities (Fig. 1c). Because the left frontal tumor had already been treated by gamma knife radiosurgery, only the parasagittal tumor was involved in the field of following stereotactic radiotherapy of 40 Gy in 10 fractions. The left frontal convexity tumor then continued to grow, and the third surgical resection of the tumor was conducted in 2010. The left frontal tumor was macroscopically removed and the dural defect was replaced with a synthetic dura mater. Follow-up MRI showed complete removal of the left frontal convexity tumor and a stable residual parasagittal tumor. In April 2011, the patient had a surgical wound infection and received antibiotic therapy. Because artificial bone can be a breeding ground for pathogens, he underwent a cranioplasty using an omental flap and titanium mesh plate in the left cranium in June 2011. Follow-up MRI revealed no evidence of tumor recurrence or growth of the residual tumor (Fig. 1d). In
September 2011, the patient reported severe headache and gait disturbance. MRI showed a huge tumor mass invading the omental flap both inside and outside the titanium mesh (Fig. 1e), and an emergency operation of the fourth tumor resection was performed. Macroscopically, the tumor presented as an irregular mass under the scalp (Fig. 1f), and upon examination of the partially resected tumor, the omental flap was found to be replaced by the solid tumor mass. Total tumor removal could not be achieved as the tumor invaded wide areas of the surrounding normal tissue. Post-surgery, the residual tumor showed further progression, and computed tomography scans demonstrated multiple lung and bone metastases. In November 2011, the patient died of elevated intracranial pressure and multiorgan failure.
Fig. 2 Histopathological findings of meningioma specimens stained with hematoxylin and eosin (H&E) (a–e), FABP4 (f, h), and Ki-67 (g). a The tumor specimen from the first operation (specimen 1) showed meningothelial meningioma with whorl formation (black arrows) and some clear cells (inset). b The tumor sample from the second operation (specimen 2) demonstrated an increased clear cell component. c The tumor resected in the third operation (specimen 3) showed an abundant clear cell population and mitoses. d The tumor tissue from the fourth operation (specimen 4) showed an invasive
tumor with fat cells (red arrows) and rhabdoid cells (inset). e The recurrent meningioma showed an adipocyte–tumor cell interface. f FABP4 staining of the adjacent section of panel e revealed strong expression of FABP4 in the cytoplasm of both adipocytes (black arrows) and tumor cells (red arrows). g Ki-67 staining of the adjacent section of panel e indicated that the cells around the adipocytes were proliferating tumor cells. h FABP4 staining of the meningioma cells distant from adipocytes demonstrated the absence of FABP4 expression. Scale bars indicate 50 lm
Pathological findings Histologic analysis of the resected tumor from the first operation (specimen 1) displayed the classic features of meningothelial meningioma (WHO grade I), including
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whorls and psammoma body formation (Fig. 2a). Clear cells existed focally in the tumor (Fig. 2a, inset). The tumor from the second operation (specimen 2) revealed an atypical meningioma (WHO grade II) with a more abundant clear cell population (Fig. 2b). Similarly, the tumor excised in the third operation (specimen 3) was also diagnosed as an atypical meningioma (Fig. 2c). Upon examination of the resected tumor from the fourth operation (specimen 4), we found that tumor cells had spread throughout the omentum tissue with the areas of geographical necrosis, converting the soft omental flap into a solid tumor mass (Fig. 2d). Rhabdoid cells were focally observed, whereas clear cells were sparse (Fig. 2d, inset). The pathological diagnosis of specimen 4 was an anaplastic meningioma (WHO grade III). The lobular proliferation pattern of the tumor cells separated by collagenous septa was commonly observed among all 4 specimens. Clear cell components in specimens 1–3 and rhabdoid cells in specimen 4 were focally positive to periodic acid–Schiff (PAS) staining, and INI1 staining was positive in specimen 4 (data not shown). The Ki-67 staining index was \1, 6, 12, and 32 % in specimens 1–4, respectively, indicating an increased proliferation rate of recurrent tumors (Table 1). We performed immunohistochemical analyses to investigate the common pathway abnormalities in meningioma. EGFR and VEGF protein levels gradually increased with recurrences but decreased in specimen 4. Expression of p16 was high in specimens 1 and 2 but absent in specimens 3 and 4. The p53 staining index was 23, 11, 3, and \1 % in specimens 1–4, respectively (Table 1). These results suggest that some molecular events occurred at each stage, but the mechanisms responsible for the rapid tumor growth observed after the cranioplasty with an omental flap remain unknown. Because FABP4 had been reported to have a key role in cancer progression at the adipocyte– tumor cell interface [16], we conducted further immunohistochemical analyses of FABP4 in our patient samples. In accordance with the study mentioned above, we found that FABP4 was strongly expressed in the tumor cells of specimen 4 at the adipocyte–tumor cell interface (Fig. 2e– Table 1 WHO grade and immunohistochemical expression profile in the meningioma specimens Specimen 1 Specimen 2
Specimen 3
Specimen 4
WHO grade
I
I
II
III
Ki-67 (%)
\1
6
12
32
p53 (%)
23
11
3
\1
p16
?
?
-
-
EGFR
?
?
??
±
VEGF
?
?
??
-
- negative, ± occasional or weakly positive, ? positive, ?? strongly positive
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g), whereas FABP4 expression was not detected in specimens 1–3 (data not shown) or in tumor cells distant from the adipocytes in specimen 4 (Fig. 2h).
Experimental study Materials and methods Cell culture The human meningioma cell lines IOMM-Lee, HKBMM, and KT21MG1 were kindly provided by Dr. Shinichi Miyatake (Osaka Medical University, Osaka, Japan). The human embryonic kidney cell line GP2-293 was obtained from the American Type Culture Collection (Manassas, VA). All cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10 % fetal bovine serum and penicillin/streptomycin. Cell lines were grown at 37 °C in a humidified atmosphere of 5 % CO2. Human meningioma cell lines expressing green fluorescent protein (GFP) were constructed using the Retro-X Universal Packaging System (Clontech, Mountain View, CA) and RetroNectin-coated plates (Takara-Bio Inc., Otsu, Japan) according to the manufacturer’s instructions. Histopathological analysis Histopathological examination was performed on formalinfixed, paraffin-embedded tissue sections. Five-micrometerthick paraffin sections were routinely stained with hematoxylin and eosin (H&E) and PAS. Next, the slides were stained with primary antibodies against epidermal growth factor receptor (EGFR) (2-18C9, Dako, Glostrup, Denmark; prediluted), FABP4 (ab13979, Abcam, Cambridge, MA; 1:400), INI1 (25, BD Biosciences, San Jose, CA; 1:100), Ki-67 (MIB-1, Dako; 1:100), p16 (E6H4, mtm Laboratories, Heidelberg, Germany; prediluted), p53 (DO1, Santa Cruz Biotechnology, Santa Cruz, CA; 1:100), and vascular endothelial growth factor (VEGF) (A-20, Santa Cruz Biotechnology; 1:100). The peroxidase signal was developed using the ABC Elite and DAB Substrate Kit (Vector Laboratories, Burlingame, CA) or using the provided reagents for EGFR and p16. BODIPY 493/503, Hoechst 33342, and collagenase type I were purchased from Invitrogen (Carlsbad, CA). Xenograft mouse model All animal experiments were approved by the Nagoya University Animal Ethics Committee. Eight-week-old female BALB/c Slc-nu/nu mice (purchased from Chubu
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Adipocyte extraction and proliferation assay
Kagaku Shizai Co. Ltd., Nagoya, Japan) were used for the xenograft model. For intracranial implantation, 3 lL of 1.5 9 105 freshly dissociated GFP-expressing IOMM-Lee cells were administered to 5 mice using the postglenoid foramen injection technique as previously described [17]. For peritoneal implantation, 3 lL of 1.5 9 105 freshly dissociated GFP-expressing IOMM-Lee cells were administered to 10 mice. For subcutaneous implantation, 3 lL of 1.5 9 105 IOMM-Lee cells alone or 100 lL packed cell volume of IOMM-Lee cells and adipocytes were administered to 10 mice. Tumors were allowed to grow for either 2 or 4 weeks. All mice were monitored daily for signs or symptoms of tumor development. Mice were humanely euthanized with CO2 and examined histologically. GFP fluorescence in tumor cells was analyzed using a fluorescence imaging system (IVIS Spectrum; Caliper Life Sciences, Alameda, CA). HKBMM and KT21MG1 cells did not grow as a xenograft in nude mice.
Adipocytes were prepared by collagenase digestion from visceral adipose tissues of 20-week-old female BALB/c Slc-nu/nu mice as described elsewhere [16, 18]. The separated adipocytes were used either as cell aggregates or as dissociated cells, which were counted by a hemocytometer and maintained in serum-free DMEM/F12 medium (Gibco, Basel, Switzerland) supplemented with 0.1 % bovine serum albumin. Meningioma cells were co-cultured with adipocytes for 24 h, and the adipocytes were then removed by washing. Tumor cells were fixed in 10 % formalin, stained with Hoechst 33342, and lipids in the tumor cells were visualized with BODIPY 493/503. Tumor cells (3 9 103) were cultured with or without adipocyte aggregates in serum-free DMEM/F12 (1:5, adipocytes: DMEM/ F12). After centrifugation, the number of cells was counted using a hemocytometer over 3 days.
Fig. 3 Microscopic images of lipid accumulation and proliferation assay in meningioma cell lines. Three meningioma cell lines (a, d IOMM-Lee; b, e HKBMM; and c, f KT21MG1) were cultured alone (a–c) or co-cultured with adipocytes (d–f). BODIPY staining revealed cytoplasmic lipid accumulation (green) in meningioma cells only when co-cultured with adipocytes. Nuclear counterstaining was
performed with Hoechst 33342 (blue). g In vitro proliferation assay in meningioma cells alone (control) or co-cultured with adipocytes (adipocyte) indicated an increased proliferative effect exerted by adipocytes on meningioma cells. A significant difference in cell proliferation was observed in IOMM-Lee (*P = .013) and HKBMM cells (**P = .022). Scale bars indicate 50 lm
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Fig. 4 Xenograft tumor models and ex vivo adhesion assay in meningioma cell lines. a Representative fluorescence images of the intracranial xenograft model on day 14 after implantation of green fluorescent protein (GFP)-labeled IOMM-Lee cells. A tumor developed along the skull base dura mater. b Intraperitoneal xenograft model on day 14 after implantation of GFP-labeled IOMM-Lee cells. Large tumor masses developed on the omentum (white arrow), and small tumors grew in the visceral peritoneal surfaces (white arrowheads). c The bar graph shows the average tumor weight per mouse and standard deviation on day 14 for the intracranial xenograft (IC) and intraperitoneal xenograft (IP) groups. d Ex vivo adhesion assay.
The bar graph represents the average number of meningioma cells adhering to each tissue and the standard deviation. All 3 meningioma cell lines (IOMM-Lee, HKBMM, and KT21MG1) showed higher binding affinities to the dura mater and omentum than the brain and parietal peritoneum (*P \ .001). e The bar graph shows the average weight of the subcutaneous tumor per mouse and the standard deviation for each group. Mice in the control group received a subcutaneous injection of IOMM-Lee cells alone, while mice in the adipocyte group received a subcutaneous injection of IOMM-Lee cells with adipocytes. Representative tumor images from the same mice are included. Scale bars indicate 5 mm
Ex vivo adhesion assay
Statistical analysis
The ex vivo adhesion assay was performed as previously described [19]. Twenty-week-old female BALB/c Slcnu/nu mice were euthanized under general anesthesia with ether. The brain, dura mater, visceral peritoneum, and omentum specimens were extracted using an operative microscope [20–22]. Each resected tissue sample was washed with PBS and added to a suspension of 5 9 104 GFP-labeled meningioma cells in 1 mL DMEM. Subsequently, each mixture was incubated in a 2.0 mL round-bottom micro-test tube at 37 °C for 2 h at 10 rpm. After incubation, the tissues were washed twice with PBS and analyzed under a microscope (IX71; Olympus, Tokyo, Japan). The number of tumor cells adhering to the tissue surface was counted in 5 randomly selected microscopic fields (1.0 9 1.0 mm in size) at 1009 magnification.
Statistical analysis was performed using paired t test or Mann–Whitney exact test where appropriate. One-way repeated-measures ANOVA was used for the proliferation assay. All statistical analyses were performed using Statcel2 software (OMS Publishing Co, Saitama, Japan). Differences were considered significant at P \ .05.
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Results In vitro lipid transfer and proliferation assay To determine whether tumor cells can receive lipid supply from adipocytes, as previously reported [16], murine visceral adipocytes were isolated without contamination from other cell types and co-cultured with 3 human meningioma cell lines. BODIPY staining revealed cytoplasmic lipid
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accumulation in meningioma cells only when co-cultured with adipocytes, suggesting direct transfer of lipids from adipocytes to meningioma cells (Fig. 3a–f). Subsequently, we investigated the effect of adipocytes on cell proliferation in meningioma cell lines. By co-cultivation with adipocytes, IOMM-Lee and HKBMM cells demonstrated significantly increased cell growth (P = .013 and P = .022), while a trend was observed in KT21MG1 cells (P = .065) (Fig. 3g).
was seen in the intracranial model (data not shown). These results are likely relevant to humans as well and support our hypothesis that adipocytes in the omental flap conferred a growth advantage to the meningioma cells in our patient.
Xenograft models and ex vivo adhesion assay
Nieman et al. [16] showed in their study that omental adipocytes attract cancer cells and promote migration and invasion by providing fatty acids via FABP4, and in our study, we found that this mechanism was likely behind the meningioma growth observed after reconstructive surgery with an omental flap. Because the meningioma cell lines used in this study were derived from high-grade meningiomas, our experimental results might not be applicable to benign meningiomas, but rather high-grade meningiomas. Although our case was diagnosed as meningothelial meningioma (WHO grade I) at onset, the tumor had an invasive nature and multiple recurrences with a clear cell component histology at the beginning, indicating malignant features for meningioma. Because clear cell meningioma is an aggressive meningioma variant classified as WHO grade II [24], the clear cell component of this patient may represent a possible cause of tumor recurrence and malignant transformation other than omental adipocytes. Other possible causes for tumor progression include multiple radiation therapy and repeated infection. Accordingly, our patient had received gamma knife radiosurgery and stereotactic radiotherapy in his left cranium, indicating a role for radiation-induced transformation in this case. Although the present case suggested that an implanted omentum promotes tumor progression, the use of an omental flap has several advantages in reconstructive surgery [14, 25, 26]. After reviewing the medical records of 31 consecutive patients who had undergone reconstruction of head and neck defects using an omental flap at our institution, including 2 cases of meningioma with a residual tumor at the time of reconstructive surgery, no tumor invasion into the omental flap was observed. To investigate whether similar cases of tumor invasion have been reported, we performed a literature review, in which we identified 1 case of drop metastasis of meningioma (WHO grade I) in an omental flap 50 months after the reconstruction surgery [27]. Although this case showed no malignant transformation, it suggests that meningioma cells have a binding affinity to omental tissue. Further investigation will be needed in a subset of patients with omental flap transposition to verify if omentum promotes tumor progression. In conclusion, we report on a case of malignant meningioma with rapid growth after cranioplasty with an
To further investigate the effects of adipocytes on tumor cell proliferation in vivo, GFP-labeled IOMM-Lee cells were constructed and used in xenograft mouse models. The tumor cells were implanted into the intracranial or intraperitoneal space in mice, and the tumor weight was measured 2 weeks later. In the intracranial xenograft model, the tumor developed along the skull base dura mater and did not invade the brain (Fig. 4a), whereas in the intraperitoneal xenograft model, relatively large tumor masses developed on the omentum and small tumors grew on the visceral peritoneal surfaces, but did not invade the digestive tract (Fig. 4b). The intraperitoneal tumors were approximately 4 times heavier than the intracranial tumors (mean weight 83.2 vs. 318.2 mg; P \ .001) (Fig. 4c). Both the omentum and mesenterium comprise visceral peritoneum layers containing adipose tissues and a vascular network. The tissue tropism of tumors has been recognized, and tumor-cell adhesion to the tissue has been demonstrated to be an important step in tumor invasion [19, 23]. Next, we conducted an ex vivo adhesion assay to compare the tissue affinity of meningioma cells. Brain, dura mater, visceral peritoneum, and omentum specimens were obtained from the mice and co-cultured with GFPlabeled meningioma cell lines. Interestingly, the meningioma cell lines had a higher affinity for the dura mater and omentum compared with the brain and parietal peritoneum specimens (Fig. 4d). To investigate whether adipocytes could confer a growth advantage to meningioma cells in vivo, subcutaneous implantation was performed in mice. As making a pedicled omental flap in the subcutaneous xenograft mouse model was technically difficult, we instead used subcutaneous injection of tumor cells with adipocytes. Co-injection of IOMM-Lee cells with adipocytes resulted in the formation of significantly larger tumor masses in comparison to the tumors developed from IOMM-Lee cells alone (mean weight 311.6 vs. 738.0 mg; P \ .001) (Fig. 4e). Finally, we investigated the FABP4 protein expression by immunohistochemistry in the xenograft mouse models. FABP4 expression was occasionally observed in the meningioma cells adjacent to the omentum in the intraperitoneal xenograft model, while no expression of FABP4
Discussion
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omental flap. We found that meningioma cells can receive a lipid supply and growth advantage from omental adipocytes, and that FABP4 would play a key role in this phenomenon. Although this study has several limitations, including only comprising a single-case evaluation and relatively simple animal experiments, we believe that, based on our results, there is a possibility that omental adipocytes promote malignant progression of meningioma, and close interval imaging follow-up is thus recommended in meningioma patients receiving reconstructive surgery with omental grafts. Acknowledgments The authors would like to thank the members of the Division of Experimental Animals and the Department of Pathology (Nagoya University) and Akira Kato for histology assistance and mouse maintenance. This work was supported by Grant-inAid for Young Scientists (B) (21790398) to Hiroyuki Momota, by Grant-in-Aid for Scientific Research (B) (21390408) to Atsushi Natsume, and by Grant-in-Aid for Scientific Research (C) (21591867) to Masazumi Fujii from the Japan Society for the Promotion of Science.
11.
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13. 14.
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17. Conflict of interest The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper. 18.
References 1. Dolecek TA, Propp JM, Stroup NE, Kruchko C (2012) CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2005–2009. Neuro Oncol 14(Suppl 5):v1–v49 2. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109 3. Black P, Kathiresan S, Chung W (1998) Meningioma surgery in the elderly: a case-control study assessing morbidity and mortality. Acta Neurochir (Wien) 140:1013–1016 discussion 1016–1017 4. Black PM, Morokoff AP, Zauberman J (2008) Surgery for extraaxial tumors of the cerebral convexity and midline. Neurosurgery 62:1115–1121 discussion 1121–1113 5. Sanai N, Sughrue ME, Shangari G, Chung K, Berger MS, McDermott MW (2010) Risk profile associated with convexity meningioma resection in the modern neurosurgical era. J Neurosurg 112:913–919 6. Monleo´n D, Morales JM, Gonzalez-Segura A, Gonzalez-Darder JM, Gil-Benso R, Cerda´-Nicola´s M, Lo´pez-Gine´s C (2010) Metabolic aggressiveness in benign meningiomas with chromosomal instabilities. Cancer Res 70:8426–8434 7. Perry A, Scheithauer BW, Stafford SL, Lohse CM, Wollan PC (1999) ‘‘Malignancy’’ in meningiomas: a clinicopathologic study of 116 patients, with grading implications. Cancer 85:2046–2056 8. Harris AE, Lee JY, Omalu B, Flickinger JC, Kondziolka D, Lunsford LD (2003) The effect of radiosurgery during management of aggressive meningiomas. Surg Neurol 60:298–305 discussion 305 9. Huffmann BC, Reinacher PC, Gilsbach JM (2005) Gamma knife surgery for atypical meningiomas. J Neurosurg 102(Suppl):283–286 10. Stafford SL, Pollock BE, Foote RL, Link MJ, Gorman DA, Schomberg PJ, Leavitt JA (2001) Meningioma radiosurgery: tumor
123
19.
20.
21.
22.
23.
24.
25.
26.
27.
control, outcomes, and complications among 190 consecutive patients. Neurosurgery 49:1029–1037 discussion 1037–1028 Delgado-Lopez PD, Martin-Velasco V, Castilla-Diez JM, Galacho-Harriero AM, Rodriguez-Salazar A (2009) Preservation of bone flap after craniotomy infection. Neurocirugia (Astur). Spain, p 124–131 Auguste KI, McDermott MW (2006) Salvage of infected craniotomy bone flaps with the wash-in, wash-out indwelling antibiotic irrigation system. Technical note and case series of 12 patients. J Neurosurg 105:640–644 Bruce JN, Bruce SS (2003) Preservation of bone flaps in patients with postcraniotomy infections. J Neurosurg 98:1203–1207 Asai S, Kamei Y, Torii S (2004) One-stage reconstruction of infected cranial defects using a titanium mesh plate enclosed in an omental flap. Ann Plast Surg 52:144–147 Wilkosz S, Ireland G, Khwaja N, Walker M, Butt R, de GiorgioMiller A, Herrick SE (2005) A comparative study of the structure of human and murine greater omentum. Anat Embryol (Berl) 209:251–261 Nieman KM, Kenny HA, Penicka CV, Ladanyi A, Buell-Gutbrod R, Zillhardt MR, Romero IL, Carey MS, Mills GB, Hotamisligil GS, Yamada SD, Peter ME, Gwin K, Lengyel E (2011) Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat Med 17:1498–1503 Iwami K, Momota H, Natsume A, Kinjo S, Nagatani T, Wakabayashi T (2012) A novel method of intracranial injection via the postglenoid foramen for brain tumor mouse models. J Neurosurg 116:630–635 Rodbell M (1964) Metabolism of isolated fat cells. I. effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375–380 Okamura A, Yazawa S, Nishimura T, Tanaka S, Takai I, Kudo S, Asao T, Kuwano H, Matta KL, Akamatsu S, Kochibe N (2000) A new method for assaying adhesion of cancer cells to the greater omentum and its application for evaluating anti-adhesion activities of chemically synthesized oligosaccharides. Clin Exp Metastasis 18:37–43 Baillie LD, Hagen V, Gardner KM, Mulligan SJ (2011) Functional imaging within individual pain fibres ex vivo with optical microscopy. J Neurosci Methods 198:274–279 Cavallotti C, Frati A, Cavallotti D, Tranquilli Leali FM (2004) Dopaminergic receptors in rat dura mater: pharmacological characteristics. Clin Exp Pharmacol Physiol 31:190–194 Khan SM, Funk HM, Thiolloy S, Lotan TL, Hickson J, Prins GS, Drew AF, Rinker-Schaeffer CW (2010) In vitro metastatic colonization of human ovarian cancer cells to the omentum. Clin Exp Metastasis 27:185–196 Nguyen DX, Bos PD, Massague´ J (2009) Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer 9:274–284 Jain D, Sharma MC, Sarkar C, Suri V, Garg A, Singh M, Sharma BS, Mahapatra AK (2007) Clear cell meningioma, an uncommon variant of meningioma: a clinicopathologic study of nine cases. J Neurooncol 81:315–321 Yagi S, Kamei Y, Fujimoto Y, Torii S (2007) Use of the internal mammary vessels as recipient vessels for an omental flap in head and neck reconstruction. Ann Plast Surg 58:531–535 Kamei Y, Takada T, Yagi S, Fujimoto Y, Saito K, Takahashi M, Torii S (2005) Use of an omental flap for middle skull base reconstruction. Head Neck Cancer 31:498–502 Ichikawa M, Saito K, Ito E, Kawai K, Fujimoto Y, Kamei Y (2010) Iatrogenic drop metastasis of recurrent meningioma in a vascularized free omental flap—case report. Neurol Med Chir (Tokyo) 50:70–73
CASE REPORT
Anaplastic meningioma with rapid growth after omental flap transposition: a case report and experimental study Kenichiro Iwami • Hiroyuki Momota • Masazumi Fujii Atsushi Natsume • Shunjiro Yagi • Kazuhiro Toriyama Yuzuru Kamei • Toshihiko Wakabayashi
• •
Received: 24 March 2014 / Accepted: 18 April 2014 Ó The Japan Society of Brain Tumor Pathology 2014
Abstract Meningiomas occasionally display aggressive behavior, but the mechanisms of malignant transformation remain unclear. We encountered the case of a 65-year-old man with a 10-year history of recurrent meningioma. The patient had undergone multiple tumor resections, radiotherapy treatments, and reconstructive surgeries due to wound infection. After the third resection of the tumor and reconstruction with an omental flap, the tumor demonstrated rapid growth and lung metastasis. The final pathological diagnosis was anaplastic meningioma. Because the drastic change of the tumor was observed after omental flap transposition, we investigated the effect of the omentum on tumor cells and performed histopathological analyses of meningiomas using a mouse model. We found that meningioma cells have a high affinity to the omentum and show a growth advantage when co-cultured with adipocytes. Immunohistochemical staining revealed that meningioma cells adjacent to the omentum strongly expressed fatty acid-binding protein 4, a lipid transfer protein, in both mouse and human. Our results suggest that
K. Iwami H. Momota (&) M. Fujii A. Natsume T. Wakabayashi Department of Neurosurgery, Nagoya University, Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan e-mail: [email protected] Present Address: K. Iwami Department of Neurosurgery, Japanese Red Cross Nagoya Daiichi Hospital, Nagoya, Japan S. Yagi K. Toriyama Y. Kamei Department of Plastic and Reconstructive Surgery, Nagoya University, Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan
tumor cells can receive lipid supply from omental adipocytes, and the surrounding tissues may induce tumor progression. We conclude that although omental tissue is an ideal material for reconstruction surgery, close follow-up is recommended in meningioma patients when used for cranioplasty. Keywords Meningioma Omentum Adipocyte FABP4 Mouse model
Introduction Meningiomas are the most commonly diagnosed primary intracranial neoplasms, constituting approximately 36 % of all primary tumors in this location [1]. Approximately 90 % of meningiomas are benign [grade I of the World Health Organization (WHO) grading scheme], 5–7 % are atypical (WHO grade II), and 1–3 % are anaplastic/ malignant (WHO grade III) [2]. Despite substantial advances in modern therapies, surgical resection remains the best treatment option for most patients with meningioma [3–5]. However, even histologically benign meningiomas occasionally recur and can result in incurable disease [6, 7]. WHO grade II and III meningiomas have a high recurrence rate after both surgical and radiosurgical managements [8–10], and repeated surgery may be required in patients with recurrent meningioma. Craniotomy can lead to cranial infection, and repeated surgery is a known risk factor for craniotomy infection [11]. The standard management of cranial bone infection includes wound debridement, bone removal, and delayed cranioplasty with artificial bone or other materials [12, 13]. Infection can moreover be reactivated after cranioplasty as a result of the epidural cavity becoming filled with serous
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fluid, resulting in abscess formation. We have previously reported on the successful treatment of cranial bone infection using a reconstructive technique that fills the dead space with a titanium mesh plate enclosed in an omental flap [14]. The omentum is a highly vascularized peritoneal fold comprising mesothelial cell layers enveloping the adipose tissue [15]. Its rich blood supply and large lymphatic network prevent infection, and its absorptive capacity resorbs exudates [14]. However, a recent study indicated that omental adipocytes attract cancer cells and promote migration and invasion, and showed that fatty acid-binding protein 4 (FABP4) plays a key role as a lipid transporter through this cell–cell interaction [16]. We here report a case of meningioma with malignant transformation in the course of multiple recurrences. Shortly after a cranioplasty using an omental flap, the tumor became invasive and metastasized to the lungs. Because lipid transfer from adjacent adipose tissue has been implicated in the progression of certain cancers, and since it may have played a role in the late progression of the high-grade meningioma in this case, we moreover
conducted in vitro and in vivo experiments to further investigate its potential role in meningioma tumor progression.
Fig. 1 Magnetic resonance imaging (MRI) scans (a–e) and macroscopic appearance (f) of the meningioma in our patient. a Axial T1weighted image with gadolinium contrast (T1-Gd) in 2001 showed an en plaque left frontal tumor (red arrow). b T1-Gd MRI in 2006 showed a recurrent tumor in the left sphenoid ridge (red arrow). c T1Gd MRI in 2010 demonstrated multiple recurrences in the parasagittal and left frontal convexities (red arrows). d T1-Gd in July 2011 showing the omental flap (black arrowheads) and titanium mesh plate
(red arrowhead) in the left cranium. e T1-weighted MRI without gadolinium contrast in 2011 showed a large extra-axial tumor in the left cranium compressing the brain and protruding toward the skin. MRI with contrast enhancement was not performed in the emergency setting. Red arrowheads delineate the tumor border. f At the fourth operation, the tumor presented as an irregularly shaped mass protruding under the skin on the left side of the head (red arrow)
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Case report Clinical presentation A 55-year-old man presented with a mild headache in 2001. Cranial magnetic resonance imaging (MRI) revealed an en plaque left frontal tumor invading the brain parenchyma (Fig. 1a). Two months after the initial presentation, he underwent the first surgery of gross total tumor removal by left frontal craniotomy. The patient developed a cranial bone infection at the craniotomy site, and the bone flap was removed. A secondary cranioplasty using hydroxyapatite was performed 6 months later. In 2005, a small local recurrent lesion was found in the left frontal convexity, and the patient underwent single-fraction gamma knife radiosurgery at marginal (12 Gy) and maximum (20 Gy) doses.
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In 2006, cranial MRI revealed a small tumor at the left sphenoid ridge (Fig. 1b), and the second surgical removal of the tumor was performed. In 2008, recurrent tumors were again identified: this time located in the parasagittal and left frontal convexities (Fig. 1c). Because the left frontal tumor had already been treated by gamma knife radiosurgery, only the parasagittal tumor was involved in the field of following stereotactic radiotherapy of 40 Gy in 10 fractions. The left frontal convexity tumor then continued to grow, and the third surgical resection of the tumor was conducted in 2010. The left frontal tumor was macroscopically removed and the dural defect was replaced with a synthetic dura mater. Follow-up MRI showed complete removal of the left frontal convexity tumor and a stable residual parasagittal tumor. In April 2011, the patient had a surgical wound infection and received antibiotic therapy. Because artificial bone can be a breeding ground for pathogens, he underwent a cranioplasty using an omental flap and titanium mesh plate in the left cranium in June 2011. Follow-up MRI revealed no evidence of tumor recurrence or growth of the residual tumor (Fig. 1d). In
September 2011, the patient reported severe headache and gait disturbance. MRI showed a huge tumor mass invading the omental flap both inside and outside the titanium mesh (Fig. 1e), and an emergency operation of the fourth tumor resection was performed. Macroscopically, the tumor presented as an irregular mass under the scalp (Fig. 1f), and upon examination of the partially resected tumor, the omental flap was found to be replaced by the solid tumor mass. Total tumor removal could not be achieved as the tumor invaded wide areas of the surrounding normal tissue. Post-surgery, the residual tumor showed further progression, and computed tomography scans demonstrated multiple lung and bone metastases. In November 2011, the patient died of elevated intracranial pressure and multiorgan failure.
Fig. 2 Histopathological findings of meningioma specimens stained with hematoxylin and eosin (H&E) (a–e), FABP4 (f, h), and Ki-67 (g). a The tumor specimen from the first operation (specimen 1) showed meningothelial meningioma with whorl formation (black arrows) and some clear cells (inset). b The tumor sample from the second operation (specimen 2) demonstrated an increased clear cell component. c The tumor resected in the third operation (specimen 3) showed an abundant clear cell population and mitoses. d The tumor tissue from the fourth operation (specimen 4) showed an invasive
tumor with fat cells (red arrows) and rhabdoid cells (inset). e The recurrent meningioma showed an adipocyte–tumor cell interface. f FABP4 staining of the adjacent section of panel e revealed strong expression of FABP4 in the cytoplasm of both adipocytes (black arrows) and tumor cells (red arrows). g Ki-67 staining of the adjacent section of panel e indicated that the cells around the adipocytes were proliferating tumor cells. h FABP4 staining of the meningioma cells distant from adipocytes demonstrated the absence of FABP4 expression. Scale bars indicate 50 lm
Pathological findings Histologic analysis of the resected tumor from the first operation (specimen 1) displayed the classic features of meningothelial meningioma (WHO grade I), including
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whorls and psammoma body formation (Fig. 2a). Clear cells existed focally in the tumor (Fig. 2a, inset). The tumor from the second operation (specimen 2) revealed an atypical meningioma (WHO grade II) with a more abundant clear cell population (Fig. 2b). Similarly, the tumor excised in the third operation (specimen 3) was also diagnosed as an atypical meningioma (Fig. 2c). Upon examination of the resected tumor from the fourth operation (specimen 4), we found that tumor cells had spread throughout the omentum tissue with the areas of geographical necrosis, converting the soft omental flap into a solid tumor mass (Fig. 2d). Rhabdoid cells were focally observed, whereas clear cells were sparse (Fig. 2d, inset). The pathological diagnosis of specimen 4 was an anaplastic meningioma (WHO grade III). The lobular proliferation pattern of the tumor cells separated by collagenous septa was commonly observed among all 4 specimens. Clear cell components in specimens 1–3 and rhabdoid cells in specimen 4 were focally positive to periodic acid–Schiff (PAS) staining, and INI1 staining was positive in specimen 4 (data not shown). The Ki-67 staining index was \1, 6, 12, and 32 % in specimens 1–4, respectively, indicating an increased proliferation rate of recurrent tumors (Table 1). We performed immunohistochemical analyses to investigate the common pathway abnormalities in meningioma. EGFR and VEGF protein levels gradually increased with recurrences but decreased in specimen 4. Expression of p16 was high in specimens 1 and 2 but absent in specimens 3 and 4. The p53 staining index was 23, 11, 3, and \1 % in specimens 1–4, respectively (Table 1). These results suggest that some molecular events occurred at each stage, but the mechanisms responsible for the rapid tumor growth observed after the cranioplasty with an omental flap remain unknown. Because FABP4 had been reported to have a key role in cancer progression at the adipocyte– tumor cell interface [16], we conducted further immunohistochemical analyses of FABP4 in our patient samples. In accordance with the study mentioned above, we found that FABP4 was strongly expressed in the tumor cells of specimen 4 at the adipocyte–tumor cell interface (Fig. 2e– Table 1 WHO grade and immunohistochemical expression profile in the meningioma specimens Specimen 1 Specimen 2
Specimen 3
Specimen 4
WHO grade
I
I
II
III
Ki-67 (%)
\1
6
12
32
p53 (%)
23
11
3
\1
p16
?
?
-
-
EGFR
?
?
??
±
VEGF
?
?
??
-
- negative, ± occasional or weakly positive, ? positive, ?? strongly positive
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g), whereas FABP4 expression was not detected in specimens 1–3 (data not shown) or in tumor cells distant from the adipocytes in specimen 4 (Fig. 2h).
Experimental study Materials and methods Cell culture The human meningioma cell lines IOMM-Lee, HKBMM, and KT21MG1 were kindly provided by Dr. Shinichi Miyatake (Osaka Medical University, Osaka, Japan). The human embryonic kidney cell line GP2-293 was obtained from the American Type Culture Collection (Manassas, VA). All cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10 % fetal bovine serum and penicillin/streptomycin. Cell lines were grown at 37 °C in a humidified atmosphere of 5 % CO2. Human meningioma cell lines expressing green fluorescent protein (GFP) were constructed using the Retro-X Universal Packaging System (Clontech, Mountain View, CA) and RetroNectin-coated plates (Takara-Bio Inc., Otsu, Japan) according to the manufacturer’s instructions. Histopathological analysis Histopathological examination was performed on formalinfixed, paraffin-embedded tissue sections. Five-micrometerthick paraffin sections were routinely stained with hematoxylin and eosin (H&E) and PAS. Next, the slides were stained with primary antibodies against epidermal growth factor receptor (EGFR) (2-18C9, Dako, Glostrup, Denmark; prediluted), FABP4 (ab13979, Abcam, Cambridge, MA; 1:400), INI1 (25, BD Biosciences, San Jose, CA; 1:100), Ki-67 (MIB-1, Dako; 1:100), p16 (E6H4, mtm Laboratories, Heidelberg, Germany; prediluted), p53 (DO1, Santa Cruz Biotechnology, Santa Cruz, CA; 1:100), and vascular endothelial growth factor (VEGF) (A-20, Santa Cruz Biotechnology; 1:100). The peroxidase signal was developed using the ABC Elite and DAB Substrate Kit (Vector Laboratories, Burlingame, CA) or using the provided reagents for EGFR and p16. BODIPY 493/503, Hoechst 33342, and collagenase type I were purchased from Invitrogen (Carlsbad, CA). Xenograft mouse model All animal experiments were approved by the Nagoya University Animal Ethics Committee. Eight-week-old female BALB/c Slc-nu/nu mice (purchased from Chubu
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Adipocyte extraction and proliferation assay
Kagaku Shizai Co. Ltd., Nagoya, Japan) were used for the xenograft model. For intracranial implantation, 3 lL of 1.5 9 105 freshly dissociated GFP-expressing IOMM-Lee cells were administered to 5 mice using the postglenoid foramen injection technique as previously described [17]. For peritoneal implantation, 3 lL of 1.5 9 105 freshly dissociated GFP-expressing IOMM-Lee cells were administered to 10 mice. For subcutaneous implantation, 3 lL of 1.5 9 105 IOMM-Lee cells alone or 100 lL packed cell volume of IOMM-Lee cells and adipocytes were administered to 10 mice. Tumors were allowed to grow for either 2 or 4 weeks. All mice were monitored daily for signs or symptoms of tumor development. Mice were humanely euthanized with CO2 and examined histologically. GFP fluorescence in tumor cells was analyzed using a fluorescence imaging system (IVIS Spectrum; Caliper Life Sciences, Alameda, CA). HKBMM and KT21MG1 cells did not grow as a xenograft in nude mice.
Adipocytes were prepared by collagenase digestion from visceral adipose tissues of 20-week-old female BALB/c Slc-nu/nu mice as described elsewhere [16, 18]. The separated adipocytes were used either as cell aggregates or as dissociated cells, which were counted by a hemocytometer and maintained in serum-free DMEM/F12 medium (Gibco, Basel, Switzerland) supplemented with 0.1 % bovine serum albumin. Meningioma cells were co-cultured with adipocytes for 24 h, and the adipocytes were then removed by washing. Tumor cells were fixed in 10 % formalin, stained with Hoechst 33342, and lipids in the tumor cells were visualized with BODIPY 493/503. Tumor cells (3 9 103) were cultured with or without adipocyte aggregates in serum-free DMEM/F12 (1:5, adipocytes: DMEM/ F12). After centrifugation, the number of cells was counted using a hemocytometer over 3 days.
Fig. 3 Microscopic images of lipid accumulation and proliferation assay in meningioma cell lines. Three meningioma cell lines (a, d IOMM-Lee; b, e HKBMM; and c, f KT21MG1) were cultured alone (a–c) or co-cultured with adipocytes (d–f). BODIPY staining revealed cytoplasmic lipid accumulation (green) in meningioma cells only when co-cultured with adipocytes. Nuclear counterstaining was
performed with Hoechst 33342 (blue). g In vitro proliferation assay in meningioma cells alone (control) or co-cultured with adipocytes (adipocyte) indicated an increased proliferative effect exerted by adipocytes on meningioma cells. A significant difference in cell proliferation was observed in IOMM-Lee (*P = .013) and HKBMM cells (**P = .022). Scale bars indicate 50 lm
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Fig. 4 Xenograft tumor models and ex vivo adhesion assay in meningioma cell lines. a Representative fluorescence images of the intracranial xenograft model on day 14 after implantation of green fluorescent protein (GFP)-labeled IOMM-Lee cells. A tumor developed along the skull base dura mater. b Intraperitoneal xenograft model on day 14 after implantation of GFP-labeled IOMM-Lee cells. Large tumor masses developed on the omentum (white arrow), and small tumors grew in the visceral peritoneal surfaces (white arrowheads). c The bar graph shows the average tumor weight per mouse and standard deviation on day 14 for the intracranial xenograft (IC) and intraperitoneal xenograft (IP) groups. d Ex vivo adhesion assay.
The bar graph represents the average number of meningioma cells adhering to each tissue and the standard deviation. All 3 meningioma cell lines (IOMM-Lee, HKBMM, and KT21MG1) showed higher binding affinities to the dura mater and omentum than the brain and parietal peritoneum (*P \ .001). e The bar graph shows the average weight of the subcutaneous tumor per mouse and the standard deviation for each group. Mice in the control group received a subcutaneous injection of IOMM-Lee cells alone, while mice in the adipocyte group received a subcutaneous injection of IOMM-Lee cells with adipocytes. Representative tumor images from the same mice are included. Scale bars indicate 5 mm
Ex vivo adhesion assay
Statistical analysis
The ex vivo adhesion assay was performed as previously described [19]. Twenty-week-old female BALB/c Slcnu/nu mice were euthanized under general anesthesia with ether. The brain, dura mater, visceral peritoneum, and omentum specimens were extracted using an operative microscope [20–22]. Each resected tissue sample was washed with PBS and added to a suspension of 5 9 104 GFP-labeled meningioma cells in 1 mL DMEM. Subsequently, each mixture was incubated in a 2.0 mL round-bottom micro-test tube at 37 °C for 2 h at 10 rpm. After incubation, the tissues were washed twice with PBS and analyzed under a microscope (IX71; Olympus, Tokyo, Japan). The number of tumor cells adhering to the tissue surface was counted in 5 randomly selected microscopic fields (1.0 9 1.0 mm in size) at 1009 magnification.
Statistical analysis was performed using paired t test or Mann–Whitney exact test where appropriate. One-way repeated-measures ANOVA was used for the proliferation assay. All statistical analyses were performed using Statcel2 software (OMS Publishing Co, Saitama, Japan). Differences were considered significant at P \ .05.
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Results In vitro lipid transfer and proliferation assay To determine whether tumor cells can receive lipid supply from adipocytes, as previously reported [16], murine visceral adipocytes were isolated without contamination from other cell types and co-cultured with 3 human meningioma cell lines. BODIPY staining revealed cytoplasmic lipid
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accumulation in meningioma cells only when co-cultured with adipocytes, suggesting direct transfer of lipids from adipocytes to meningioma cells (Fig. 3a–f). Subsequently, we investigated the effect of adipocytes on cell proliferation in meningioma cell lines. By co-cultivation with adipocytes, IOMM-Lee and HKBMM cells demonstrated significantly increased cell growth (P = .013 and P = .022), while a trend was observed in KT21MG1 cells (P = .065) (Fig. 3g).
was seen in the intracranial model (data not shown). These results are likely relevant to humans as well and support our hypothesis that adipocytes in the omental flap conferred a growth advantage to the meningioma cells in our patient.
Xenograft models and ex vivo adhesion assay
Nieman et al. [16] showed in their study that omental adipocytes attract cancer cells and promote migration and invasion by providing fatty acids via FABP4, and in our study, we found that this mechanism was likely behind the meningioma growth observed after reconstructive surgery with an omental flap. Because the meningioma cell lines used in this study were derived from high-grade meningiomas, our experimental results might not be applicable to benign meningiomas, but rather high-grade meningiomas. Although our case was diagnosed as meningothelial meningioma (WHO grade I) at onset, the tumor had an invasive nature and multiple recurrences with a clear cell component histology at the beginning, indicating malignant features for meningioma. Because clear cell meningioma is an aggressive meningioma variant classified as WHO grade II [24], the clear cell component of this patient may represent a possible cause of tumor recurrence and malignant transformation other than omental adipocytes. Other possible causes for tumor progression include multiple radiation therapy and repeated infection. Accordingly, our patient had received gamma knife radiosurgery and stereotactic radiotherapy in his left cranium, indicating a role for radiation-induced transformation in this case. Although the present case suggested that an implanted omentum promotes tumor progression, the use of an omental flap has several advantages in reconstructive surgery [14, 25, 26]. After reviewing the medical records of 31 consecutive patients who had undergone reconstruction of head and neck defects using an omental flap at our institution, including 2 cases of meningioma with a residual tumor at the time of reconstructive surgery, no tumor invasion into the omental flap was observed. To investigate whether similar cases of tumor invasion have been reported, we performed a literature review, in which we identified 1 case of drop metastasis of meningioma (WHO grade I) in an omental flap 50 months after the reconstruction surgery [27]. Although this case showed no malignant transformation, it suggests that meningioma cells have a binding affinity to omental tissue. Further investigation will be needed in a subset of patients with omental flap transposition to verify if omentum promotes tumor progression. In conclusion, we report on a case of malignant meningioma with rapid growth after cranioplasty with an
To further investigate the effects of adipocytes on tumor cell proliferation in vivo, GFP-labeled IOMM-Lee cells were constructed and used in xenograft mouse models. The tumor cells were implanted into the intracranial or intraperitoneal space in mice, and the tumor weight was measured 2 weeks later. In the intracranial xenograft model, the tumor developed along the skull base dura mater and did not invade the brain (Fig. 4a), whereas in the intraperitoneal xenograft model, relatively large tumor masses developed on the omentum and small tumors grew on the visceral peritoneal surfaces, but did not invade the digestive tract (Fig. 4b). The intraperitoneal tumors were approximately 4 times heavier than the intracranial tumors (mean weight 83.2 vs. 318.2 mg; P \ .001) (Fig. 4c). Both the omentum and mesenterium comprise visceral peritoneum layers containing adipose tissues and a vascular network. The tissue tropism of tumors has been recognized, and tumor-cell adhesion to the tissue has been demonstrated to be an important step in tumor invasion [19, 23]. Next, we conducted an ex vivo adhesion assay to compare the tissue affinity of meningioma cells. Brain, dura mater, visceral peritoneum, and omentum specimens were obtained from the mice and co-cultured with GFPlabeled meningioma cell lines. Interestingly, the meningioma cell lines had a higher affinity for the dura mater and omentum compared with the brain and parietal peritoneum specimens (Fig. 4d). To investigate whether adipocytes could confer a growth advantage to meningioma cells in vivo, subcutaneous implantation was performed in mice. As making a pedicled omental flap in the subcutaneous xenograft mouse model was technically difficult, we instead used subcutaneous injection of tumor cells with adipocytes. Co-injection of IOMM-Lee cells with adipocytes resulted in the formation of significantly larger tumor masses in comparison to the tumors developed from IOMM-Lee cells alone (mean weight 311.6 vs. 738.0 mg; P \ .001) (Fig. 4e). Finally, we investigated the FABP4 protein expression by immunohistochemistry in the xenograft mouse models. FABP4 expression was occasionally observed in the meningioma cells adjacent to the omentum in the intraperitoneal xenograft model, while no expression of FABP4
Discussion
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omental flap. We found that meningioma cells can receive a lipid supply and growth advantage from omental adipocytes, and that FABP4 would play a key role in this phenomenon. Although this study has several limitations, including only comprising a single-case evaluation and relatively simple animal experiments, we believe that, based on our results, there is a possibility that omental adipocytes promote malignant progression of meningioma, and close interval imaging follow-up is thus recommended in meningioma patients receiving reconstructive surgery with omental grafts. Acknowledgments The authors would like to thank the members of the Division of Experimental Animals and the Department of Pathology (Nagoya University) and Akira Kato for histology assistance and mouse maintenance. This work was supported by Grant-inAid for Young Scientists (B) (21790398) to Hiroyuki Momota, by Grant-in-Aid for Scientific Research (B) (21390408) to Atsushi Natsume, and by Grant-in-Aid for Scientific Research (C) (21591867) to Masazumi Fujii from the Japan Society for the Promotion of Science.
11.
12.
13. 14.
15.
16.
17. Conflict of interest The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper. 18.
References 1. Dolecek TA, Propp JM, Stroup NE, Kruchko C (2012) CBTRUS statistical report: primary brain and central nervous system tumors diagnosed in the United States in 2005–2009. Neuro Oncol 14(Suppl 5):v1–v49 2. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109 3. Black P, Kathiresan S, Chung W (1998) Meningioma surgery in the elderly: a case-control study assessing morbidity and mortality. Acta Neurochir (Wien) 140:1013–1016 discussion 1016–1017 4. Black PM, Morokoff AP, Zauberman J (2008) Surgery for extraaxial tumors of the cerebral convexity and midline. Neurosurgery 62:1115–1121 discussion 1121–1113 5. Sanai N, Sughrue ME, Shangari G, Chung K, Berger MS, McDermott MW (2010) Risk profile associated with convexity meningioma resection in the modern neurosurgical era. J Neurosurg 112:913–919 6. Monleo´n D, Morales JM, Gonzalez-Segura A, Gonzalez-Darder JM, Gil-Benso R, Cerda´-Nicola´s M, Lo´pez-Gine´s C (2010) Metabolic aggressiveness in benign meningiomas with chromosomal instabilities. Cancer Res 70:8426–8434 7. Perry A, Scheithauer BW, Stafford SL, Lohse CM, Wollan PC (1999) ‘‘Malignancy’’ in meningiomas: a clinicopathologic study of 116 patients, with grading implications. Cancer 85:2046–2056 8. Harris AE, Lee JY, Omalu B, Flickinger JC, Kondziolka D, Lunsford LD (2003) The effect of radiosurgery during management of aggressive meningiomas. Surg Neurol 60:298–305 discussion 305 9. Huffmann BC, Reinacher PC, Gilsbach JM (2005) Gamma knife surgery for atypical meningiomas. J Neurosurg 102(Suppl):283–286 10. Stafford SL, Pollock BE, Foote RL, Link MJ, Gorman DA, Schomberg PJ, Leavitt JA (2001) Meningioma radiosurgery: tumor
123
19.
20.
21.
22.
23.
24.
25.
26.
27.
control, outcomes, and complications among 190 consecutive patients. Neurosurgery 49:1029–1037 discussion 1037–1028 Delgado-Lopez PD, Martin-Velasco V, Castilla-Diez JM, Galacho-Harriero AM, Rodriguez-Salazar A (2009) Preservation of bone flap after craniotomy infection. Neurocirugia (Astur). Spain, p 124–131 Auguste KI, McDermott MW (2006) Salvage of infected craniotomy bone flaps with the wash-in, wash-out indwelling antibiotic irrigation system. Technical note and case series of 12 patients. J Neurosurg 105:640–644 Bruce JN, Bruce SS (2003) Preservation of bone flaps in patients with postcraniotomy infections. J Neurosurg 98:1203–1207 Asai S, Kamei Y, Torii S (2004) One-stage reconstruction of infected cranial defects using a titanium mesh plate enclosed in an omental flap. Ann Plast Surg 52:144–147 Wilkosz S, Ireland G, Khwaja N, Walker M, Butt R, de GiorgioMiller A, Herrick SE (2005) A comparative study of the structure of human and murine greater omentum. Anat Embryol (Berl) 209:251–261 Nieman KM, Kenny HA, Penicka CV, Ladanyi A, Buell-Gutbrod R, Zillhardt MR, Romero IL, Carey MS, Mills GB, Hotamisligil GS, Yamada SD, Peter ME, Gwin K, Lengyel E (2011) Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat Med 17:1498–1503 Iwami K, Momota H, Natsume A, Kinjo S, Nagatani T, Wakabayashi T (2012) A novel method of intracranial injection via the postglenoid foramen for brain tumor mouse models. J Neurosurg 116:630–635 Rodbell M (1964) Metabolism of isolated fat cells. I. effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375–380 Okamura A, Yazawa S, Nishimura T, Tanaka S, Takai I, Kudo S, Asao T, Kuwano H, Matta KL, Akamatsu S, Kochibe N (2000) A new method for assaying adhesion of cancer cells to the greater omentum and its application for evaluating anti-adhesion activities of chemically synthesized oligosaccharides. Clin Exp Metastasis 18:37–43 Baillie LD, Hagen V, Gardner KM, Mulligan SJ (2011) Functional imaging within individual pain fibres ex vivo with optical microscopy. J Neurosci Methods 198:274–279 Cavallotti C, Frati A, Cavallotti D, Tranquilli Leali FM (2004) Dopaminergic receptors in rat dura mater: pharmacological characteristics. Clin Exp Pharmacol Physiol 31:190–194 Khan SM, Funk HM, Thiolloy S, Lotan TL, Hickson J, Prins GS, Drew AF, Rinker-Schaeffer CW (2010) In vitro metastatic colonization of human ovarian cancer cells to the omentum. Clin Exp Metastasis 27:185–196 Nguyen DX, Bos PD, Massague´ J (2009) Metastasis: from dissemination to organ-specific colonization. Nat Rev Cancer 9:274–284 Jain D, Sharma MC, Sarkar C, Suri V, Garg A, Singh M, Sharma BS, Mahapatra AK (2007) Clear cell meningioma, an uncommon variant of meningioma: a clinicopathologic study of nine cases. J Neurooncol 81:315–321 Yagi S, Kamei Y, Fujimoto Y, Torii S (2007) Use of the internal mammary vessels as recipient vessels for an omental flap in head and neck reconstruction. Ann Plast Surg 58:531–535 Kamei Y, Takada T, Yagi S, Fujimoto Y, Saito K, Takahashi M, Torii S (2005) Use of an omental flap for middle skull base reconstruction. Head Neck Cancer 31:498–502 Ichikawa M, Saito K, Ito E, Kawai K, Fujimoto Y, Kamei Y (2010) Iatrogenic drop metastasis of recurrent meningioma in a vascularized free omental flap—case report. Neurol Med Chir (Tokyo) 50:70–73
