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Na-K-2Cl cotransporter and aquaporin 1 in arachnoid granulations, meningiomas, and meningiomas invading dura
Human Pathology (2013) 44, 1118–1124
www.elsevier.com/locate/humpath
Original contribution
Na-K-2Cl cotransporter and aquaporin 1 in arachnoid granulations, meningiomas, and meningiomas invading dura Mahlon D. Johnson MD, PhD ⁎, Mary O'Connell BS Department of Pathology, Division of Neuropathology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA Received 25 August 2012; revised 26 September 2012; accepted 28 September 2012
Keywords: NKCC1; Aquaporin 1; Arachnoid granulations; Meningioma; Invasion
Summary Meningioma invasion of the dura may contribute to the high rate of recurrence. Recently, ion channels that affect cell shape and movement have been implicated in cancer invasion. Combined Na-K2Cl cotransporter (NKCC1) and aquaporin 1 (AQP1) expression in arachnoid granulations and meningiomas with and without dural invasion has not been characterized. Arachnoid granulations associated with dura were collected from 10 adult formalin-fixed dura/leptomeninges. Thirty-four frozen meningiomas were evaluated by Western blot. An additional 58 formalin-fixed, paraffin-embedded meningiomas including 36 World Health Organization grade I, 15 grade II, and 7 grade III meningiomas were evaluated by immunohistochemistry. By Western blot, NKCC1 was found in 17 (100%) of 17 World Health Organization grade I, 13 (87%) of 15 grade II, and both grade III meningiomas. Distinct AQP1 was not detected in the meningioma lysates tested. By immunohistochemistry, extensive NKCC1 but no AQP1 immunoreactivity was detected in the arachnoid granulation cells. Extensive NKCC1 was detected in meningioma cells in 56 and in capillaries in 43 by immunohistochemistry. In those tumors with dural or bone/soft tissue invasion, NKCC1 immunoreactivity was seen in invading cells in all cases and in their capillaries in the majority. AQP1 was detected in meningioma cells in 29 and in capillaries in all. AQP1 was also detected in cells and capillaries invading the dura or bone in 10 and 18 of 18, respectively. This was extensive in all subtypes of meningiomas studied. These findings suggest that NKCC1 and AQP1 participate in meningioma biology and invasion. NKCC1 might be targeted by FDA-approved NKCC1 inhibitors. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Invasion of the dura by meningioma cells is thought to contribute to the high rate of meningioma recurrence [1–3]. ⁎ Corresponding author. Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Box 626, Rochester, NY 14623. E-mail address: [email protected] (M. D. Johnson). 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2012.09.020
The mechanisms underlying this invasion are poorly understood. Activation of collagenases and metaloproteases has been demonstrated in meningiomas and may contribute [4–7]. However, recently, ion and water channels that affect cell shape and movement have been implicated in neoplastic cell migration and metastasis [8–10]. Neutral Na-K-2Cl cotransporter (NKCC1) is a membrane-bound cation chloride cotransporter family membrane encoded by the SLC12 gene family. NKCC1 mediates
NKCC1 and AQP1 in meningiomas potassium and chloride movement across the plasmalemma in cells of several tissues including the brain. It may also be involved in choroid plexus formation of cerebrospinal fluid. Abnormalities in astrocyte and neuronal cation/chloride homeostasis may contribute to many pathophysiologic conditions including cerebral edema and seizure disorders [11,12]. However, NKCC1 expression and function in the leptomeninges and meningiomas have not been systematically studied, although NKCC1s have recently been identified in arachnoid cysts and thought to contribute to fluid accumulation in these lesions [13]. Aquaporins include a family of 19 water channels that allow the transfer of water and small molecules along the cell membrane. Of these, aquaporins 1, 4, and 9 have been identified in the mammalian brain. Aquaporin 1 (AQP1) has been identified on choroid plexus epithelium and tumors of the choroid plexus. Aquaporin 4 is located primarily on astrocyte end-feet lining capillaries [14,15]. Recently, AQP1 has been identified in cells of a cystic meningioma, the dural attachment and sites of invasion of meningiomas [16,17]. In the present study, we evaluated the expression of NKCC1 and AQP1 in capillaries and venous channels in the dura, meningiomas, and meningiomas with dural invasion.
2. Materials and methods 2.1. Tissue Frozen tissue from 34 meningiomas including 17 World Health Organization (WHO) grade I and 15 WHO grade II and 2 grade III meningiomas were collected at the University of Rochester Medical Center or obtained primarily from the Cooperative Human Tissue Network (Philadelphia, PA) with institutional review board approval (Table 1). Arachnoid granulations associated with dura were collected from 10 adult formalin-fixed dura/leptomeninges from consecutive autopsies between 2010 and 2012. Fifty-eight formalin-fixed, paraffin-embedded meningiomas including 36 WHO grade I meningiomas, 15 WHO grade II, and 7 WHO grade III including 9 with dural invasion and 9 cases with bone including 3 cases bone with soft tissue invasion were identified in the University of Rochester Medical Center archives from 2000 to 2011 and classified [18] (Table 2).
2.2. Western blots For Western blots, meningioma lysates were obtained by mechanical homogenization in RIPA Lysis Buffer (Upstate Biotechnology) with 1:100 Protease Inhibitor Cocktail (Sigma, St Louis, MO) and then frozen at –85°C. The tissue slurries were thawed on ice and vortexed vigorously. Solids were removed by centrifugation. Protein concentrations were quantified using a Bradford assay (BioRad Protein Assay reagent), and then 50 μg protein from each was loaded on
1119 Table 1 Summary of meningiomas used for NKCC1 and AQP1 Western blots Histologic type
Mean age (y)
Sex
n
WHO grade I Meningothelial Transitional Fibrous Total WHO grade II Meningothelial Transitional Fibrous Microcystic Total WHO grade III Fibrous Transitional Total
57
14F, 3M 7 7 3 17
54
11F, 4M 6 4 4 1 15
52
1F, 1M 1 1 2
Abbreviations: F, female; M, male.
7.5% acrylamide gel and transferred to 0.45-μm nitrocellulose membrane. The membrane was blocked 1 hour in 5% milk in Tris-Cl buffer with Tween 20 and then reacted with an affinity purified primary antibody overnight at 4°C. This was followed by horseradish peroxidase–conjugated secondary antibody treatment. Detection was achieved with Western Lightening (Perkin Elmer, Waltham, MA, USA) on Xomat film (Kodak, Rochester, NY, USA). Western blots were analyzed with a polyclonal antibody to NKCC1 (Santa Cruz Biotechnology Inc, Santa Cruz, CA). Loading was assessed with an antibody to actin (Cell Signaling, Beverly MA, USA).
Table 2 Summary of tissues used for NKCC1 and AQP1 immunohistochemistry Histologic type
Mean age (y) Sex
Dura with arachnoid 58 granulations WHO grade I 58 Meningothelial Transitional Fibrous Psammomatous Total WHO grade II 60 Meningothelial Transitional Fibrous Total WHO grade III 68 Meningothelial/Undifferentiated Papillary Total Abbreviations: F, female; M, male; U, unknown.
8F, 2M
n 10
31F, 5M 12 18 4 2 36 9F, 5M, 1U 8 5 2 15 3F, 4M 4 3 7
1120
M. D. Johnson, M. O'Connell
Fig. 1 NKCC1 expression in WHO grade I and II meningiomas. Representative Western blot analysis from 8 WHO grade I (M1-M8), 7 grade II, and 2 grade III (M26-M34) meningiomas showing 170-kd NKCC1 (top) and actin (bottom). NKCC1 immunoreactivity was found in 17 (100%) of 17 WHO grade I, 13 (87%) of 15 grade II, and 2 (100%) of 2 grade III meningiomas and was extensive in all common subtypes.
2.3. Immunohistochemistry Each case was analyzed with a polyclonal antibody to human AQP1 (Abcam Inc, Cambridge, MA) goat polyclonal antibody to NKCC1 (Santa Cruz Biotechnology) with MAC4 universal HRP-polymer (Biocare, Concord, CA) or Goat on Rodent HRP-polymer kit (Biocare) with diaminobenzidene chromogen and hematoxylin counterstain (Biocare). For antigen retrieval, tissue sections were incubated in a thermoresistant chamber with X Reveal Decloaker (Biocare Medical) at 120 to 123°C and pressure of 20 to 24 psi, according to the manufacturers' specifications. Immunoreactivity in tumor cells (excluding blood vessels) was assessed by 2 of us as “0” for no distinct immunoreactivity, “1+” if 1% to 30% of cells were immunoreactive, “2+” if 25% to 50%, and “3+” if greater than 50% of cells were positive. Percentage immunoreactivity in blood vessel endothelium was graded as “0” for no distinct immunoreactivity was found, “1+” if 1% to 30% of cells were immunoreactive, and “2+” if more than 30% of blood vessels had immunoreactivity.
3. Results
difference in grade or subtype. In 6 grade I cases analyzed by Western blot, where formalin-fixed tissue was available, all 6 cases also showed NKCC1 immunoreactivity. Immunoreactivity was membranous and also cytoplasmic. Meningioma cells invading the dura and dural channels also exhibited extensive NKCC1 (Table 3; Fig. 4A and C). Extensive NKCC1 was also seen in cells in all 9 cases with bone (Table 3; Fig. 5A and B) or bone with soft tissue extension. It was also seen in capillaries in 7 of 9 meningiomas invading bone without or with soft tissue. AQP1 immunoreactivity was seen in capillaries in all meningiomas (Table 3 and Fig. 3B) and in meningioma cells (Table 3 and Fig. 3D) of 19 meningiomas. In meningiomas invading dura, invasion of endothelium-lined vascular channels was found in 4 of 9 cases. In each case, the capillaries showed extensive AQP1 immunoreactivity. AQP1 was also detected in capillaries and some meningioma cells in a case invading the brain parenchyma. In meningioma cells invading bone or bone with soft tissue, AQP1 was seen in cells in 6 of 9 and was extensive in capillaries in 9 of 9 (Table 3 and Fig. 5C).
4. Discussion
3.1. Western blots As shown in Fig. 1, NKCC1 was found in 17 (100%) of 17 WHO grade I, 13 (87%) of 15 grade II, and 2 (100%) of 2 grade III meningiomas. This was extensive in all subtypes of meningiomas studied. In the 6 cases where formalin-fixed tissue was available, NKCC1 immunoreactivity was extensive. A distinct 30-kd AQP1 band was not detected by Western blot for these tissues.
3.2. Immunohistochemistry As summarized in Table 3, extensive NKCC1 but no AQP1 was detected in all arachnoid granulation cells (Table 3 and Fig. 2). NKCC1 immunoreactivity was detected in meningioma cells by immunohistochemistry in all 40 meningiomas without documented dural or bone invasion and was extensive (3+) in 38 of 40 (Table 3 and Fig. 3A). NKCC1 was found in capillaries in 29 of 40. Because the immunoreactivity was extensive, there was no apparent
The presence of NKCC1 channels has not been described in the arachnoid or meningiomas. Its widespread membranous localization in arachnoid granulations and meningiomas suggests that NKCC1 channels are important to the biology of normal and neoplastic leptomeningeal cells. Several studies suggest that NKCC1 channels participate in neoplastic processes including cell division, apoptosis, and migration [8–10,12]. The presence of NKCC1 in capillaries throughout meningiomas raises the possibility that NKCC1 channels facilitate movement of cerebrospinal fluid in meningiomas and may form an intercellular network for cerebrospinal fluid distribution throughout these tumors. Previously, we have shown that cerebrospinal fluid is a mitogen for meningiomas cells [19]. However, it is not known whether cerebrospinal fluid reaches central portions of tumors or merely stimulates cell proliferation at the margin of meningiomas. The extensive NKCC1 channel distribution reported here, along with variable AQP1, raises the possibility that growth regulatory cerebrospinal fluid components may reach much of the tumor and that distribution could be targeted by Federal
NKCC1 and AQP1 in meningiomas
1121
Drug Administration−approved NKCC1 inhibitors such as bumetamide [20]. Recent studies suggest that ion channels facilitate the migration and metastasis of some carcinomas and gliomas by altering cell volume and shape [8–10]. At the infiltrating edge of tumors, NKCC1 cotransporters facilitate intracellular Table 3
accumulation of potassium and chloride resulting in changes in the osmotic gradient across the cell membrane and attendant intracellular shift in fluids via AQP1 and other channels resulting in changes in tumor cell volume and shape. This results in displacement of the adjacent parenchyma. The subsequent efflux of ions and fluid mediated by voltage-gated
Summary of NKCC1 and AQP1 arachnoid granulations, meningiomas, and meningiomas with dural invasion NKCC1
Arachnoid granulation cells Grade I meningiomas
Grade II meningiomas
AQP1
cells
capillaries
10/10 3+ 1/27 1+ 1/27 2+ 25/27 3+ 10/10 3+
NA 10/27 0 17/27 1+
1/10 0 4/10 1+ 5/10 2+ 2/3 1+ 1/3 2+ 1/3 0 2/3 1+
Grade III meningiomas
3/3 3+
Grade I meningiomas invading dura
3/3 3+
Grade II meningiomas invading dura
4/4 3+
4/4 1+
Grade III meningiomas invading dura
2/2 3+
Grade I meningiomas invading bone
6/6 3+
Grade II meningiomas invading bone
1/1 3+ 2/2 3+
1/2 0 1/2 1+ 2/6 0 2/6 1+ 2/6 2+ 1/1 2+ 2/2 2+
Grade III meningiomas invading bone
Abbreviation: NA, not applicable. a Capillaries in meningiomas.
a
cells
capillaries a
10/10 0 14/27 0 4/27 1+ 9/27 2+ 5/10 0 3/10 1+ 2/10 2+ 2/3 0 1/3 1+ 1/3 0 1/3 1+ 1/3 2+ 2/4 0 1/4 1+ 1/4 2+ 2/2 0
NA
2/6 0 3/6 1+ 1/6 2+ 1/1 2+ 1/2 0 1/2 1+
27/27 2+
10/10 2+
3/3 2+
1/3 1+ 2/3 2+
1/4 1+ 3/4 2+
2/2 2+
6/6 2+
1/1 2+ 2/2 2+
1122
Fig. 2 NKCC1 immunoreactivity in arachnoid granulations. NKCC1 immunoreactivity, largely membranous, was detected in all arachnoid cap cells by immunohistochemistry (hematoxylin counterstain and diaminobenzidine chromogen, original magnification ×400).
M. D. Johnson, M. O'Connell chloride, calcium-activated potassium channels, and aquaporins subsequently “collapses” the volume/shape of the infiltrating cells, whereas actin polymerization alters cell shape allowing the advancing edge of a cell, that is, lamellipodium, to pull the advancing cell forward into the parenchyma [21–23]. The role of NKCC1 and AQP1 channels in meningioma cell invasion of the dura and brain is unknown. However, the apparent insinuation of NKCC1 and AQP1 expressing meningioma cells into unlined and lined vascular spaces raises the possibility that NKCC1/ AQP1-mediated changes in meningioma cell shape may contribute to meningioma invasion of the dura, skull, and soft tissue. Nonetheless, additional in vitro and in vivo studies evaluating meningioma invasion of the dura and brain after pharmacologic or genetic inhibition of NKCC1 and AQP1 activity are required to test this hypothesis. The dura contains a complex network of capillaries, unlined and venous channels that connect with the arachnoid granulations and superior sagittal sinus [24–28]. Their role
Fig. 3 NKCC1 and AQP1 immunoreactivity in meningiomas. NKCC1 immunoreactivity (A) was largely membranous and in meningioma cells, whereas AQP1 immunoreactivity (B) was primarily capillaries in this meningothelial meningioma. In some meningiomas with extensive NKCC1, immunoreactivity (C) also had AQP1 immunoreactivity (D) in meningioma cells as well as capillaries (hematoxylin counterstain and diaminibenzidine chromogen, original magnification ×400 [A and B] and ×200 [C and D]).
NKCC1 and AQP1 in meningiomas
Fig. 4 NKCC1 in meningioma cells invading dura. A, NKCC1 immunoreactivity was extensive in WHO grade I meningioma cells invading dura in endothelium-lined channels. B, In some cases, meningioma cells were also found in unlined dural spaces adjacent to a blood vessel (hematoxylin and eosin). C, NKCC1 was also seen in these meningioma cells (hematoxylin counterstain and diaminobenzidine chromogen, original magnification ×400 [A and C]).
1123
Fig. 5 NKCC1 and AQP1 in meningioma cells invading the skull. A, NKCC1 immunoreactivity in WHO grade I meningioma invading bone Haversian canal. B, NKCC1 immunoreactivity was extensive in WHO grade I meningioma cells invading the skull bone. C, AQP1 was found in variable numbers of meningioma cells and meningioma capillaries invading the bone (hematoxylin counterstain and diaminobenzidine chromogen, original magnification ×400).
1124 as a passageway for meningioma cell invasion or as a reservoir for recurring meningiomas cells is not established. These channels may also be a conduit for cerebrospinal fluid, water, and cytokines. Of note, electron microscopy has shown that these venules have pores [24]. Previously, we have shown that cerebrospinal fluid stimulates proliferation of human leptomeningeal and meningioma cells of all grades [19,29,30]. Growth factor/cytokines and their receptor tyrosine kinases likely participate in these effects [29,30]. However, other factors such as cation and chloride homeostasis may also participate. AQP1 was consistently found in endothelial cells of capillaries, but not larger blood vessels, and variably detected in meningioma cells. AQP1 has been reported in a cystic meningioma in a newborn and in meningioma cells at the point of dural invasion of the dura [16,17]. In the present study, AQP1 was also detected in capillaries in meningioma cells invading the dura. We also found aquaporin expression at sites of dural invasion, but expression was not confined to that site. By Western blot, a definite 30-kd band was not detected in the meningiomas analyzed. This likely reflects the limited amounts of AQP1 detected by immunohistochemistry in meningiomas. NKCC1 and AQP1 were detected in all subtypes and grades of meningiomas, suggesting a role in meningioma biology rather than in progression. Although Merlin was not tested in many cases, this also suggests their presence both in tumor with and in tumor without Merlin. Consequently, pharmacologic agents targeting these channels might have widespread applicability as an adjunct chemotherapy. In summary, these studies have shown that NKCC1 and AQP1 are widely distributed in meningiomas and meningioma cells invading the dura. Considering recent findings implicating ion and water channels in invasion and metastasis, additional studies evaluating their role in meningioma invasion of surrounding tissues are warranted.
References [1] Stafford SL, Perry A, Suman VJ, et al. Primarily resected meningiomas: outcomes and prognostic factors in 581 Mayo Clinic patients, 1978 through 1988. Mayo Clin Proc 1998;73:936-42. [2] Johnson MD, Sade B, Milano MT, Lee JH, Toms SA. New prospects for management and treatment of inoperable and recurrent skull base meningioma. J Neuro-Oncol 2008;86:109-22. [3] Alahmadi H, Croul SE. Pathology and genetics of meningiomas. Sem Diagn Pathol 2011;28:314-24. [4] Josefine A, von Randow U, Schindler S, Tews DS. Expression of extracellular matrix-degrading proteins in classic, atypical and anaplastic meningiomas. Pathol Res Pract 2006;202:365-72. [5] Kandenwein JA, Park-Simon T-J, Schramm J, Simon M. uPA, PAI-1 expression and uPA promoter methylation in meningiomas. J Neurooncol 2011;103:533-9. [6] Rooprai HK, Rucklidge GJ, Panou C, Pilkington GJ. The effects of exogenous growth factors on matrix metalloproteinase secretion by human brain tumors. Br J Cancer 2000;82:52-5.
M. D. Johnson, M. O'Connell [7] Kondraganti S, Gondi CS, McCutcheon I, et al. RNAi-mediated downregulation of plasminogen activator and its receptor in human meningioma cells inhibits tumor invasion and growth. Int J Oncol 2006;28:1353-60. [8] Cuddapah VA, Sontheimer H. Ion channels and transporters in cancer. 2. Ion channels and the controls of cancer cell migration. Am J Physiol 2011;301:C541-9. [9] Machida Y, Ueda Y, Shimasaki M, et al. Relationship of AQP1, 3, and 5 expression in lung cancer cells to cellular differentiation, invasive growth and metastasis potential. HUM PATHOL 2011;42:669-78. [10] Sontheimer H. An unexpected role of ion channels in brain tumor metastasis. Exp Biol Med 2008;233:779-91. [11] Russel JM. Sodium-potassium-chloride cotransport. Phys Rev 2000;80:212-70. [12] Kahle KT, Staley KJ, Nahed BV, et al. Roles of the cation-chloride cotransporters in neurological disease. Nat Clin Pract Neurol 2008;4: 490-503. [13] Helland CA, Aarhus M, Knappskog P, et al. Increased NKCC1 expression in arachnoid cysts supports secretory basis for cyst formation. Exp Neurol 2010;224:424-8. [14] Yool AJ. Aquaporins: multiple roles in the central nervous system. Neuroscientist 2007;13:470-80. [15] MacAulay N, Zeuthen T. Water transport between CNS compartments: contributions of aquaporins and cotransporters. Neuroscience 2010;168:941-56. [16] Marton E, Feletti A, Basaldella L, et al. Atypical cystic meningioma overexpressing AQP1 in early infancy: case report with literature review. Acta Pediatr 2008;97:1145-9. [17] Nagashima G, Fujimoto T, Suzuki R, et al. Dural invasion of meningioma: a histological and immunohistochemical study. Brain Tumor Pathol 2006;23:13-7. [18] Perry A, Louis DN, Scheithauer BW, et al. Meningiomas. In: Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, editors. Tumours of the nervous system. Geneva, Switzerland: WHO Press; 2007. p. 164-72. [19] Johnson MD, O'Connell M, Facik M, Maurer P, Jahromi B, Pilcher W. Cerebrospinal fluid stimulates leptomeningeal and meningioma cell proliferation and activation of STAT3. J Neurooncol 2012;107:121-31. [20] Walcott BP, Kahle KT, Simard JM. Novel treatment targets for cerebral edema. Neurotherapeutics 2012;9:65-72. [21] Nabi IR. The polarization of the motile cell. J Cell Sci 1999;112: 1803-11. [22] Small JV, Herzog M, Anderson K. Actin filament organization in the fish keratinocyte lamellipodium. J Cell Biol 1995;129:1275-86. [23] Wang YL. Exchange of actin subtypes at the leading edge fibroblasts: possible role of treadmilling. J Cell Biol 1985;101:597-602. [24] Le Goss Clark WE. On the Pacchionian bodies. J Anat 1920;55(Pt 1): 40-8. [25] Fox RJ, Walji AH, Mielke B, et al. Anatomic details of intradural channels in the parasagittal dura: a possible pathway for flow of cerebrospinal fluid. Neurosurgery 1996;39:84-90. [26] Kerber CW. Newton. The macro and microvasculature of the dura. Neuroradiology 1973;6:175-9. [27] Roland J, Bernard C, Bracard S, et al. Microvascularization of the intracranial dura mater. Surg Radiol 1987;9:43-9. [28] Mack J, Squier W, Eastman JT. Anatomy and development of the meninges: implications for subdural collections and CSF circulation. Pediatr Radiol 2009;39:200-10. [29] Johnson MD, Woodard A, Okediji EJ, Toms SA, Allen GS. Lovastatin is a potent inhibitor of meningioma cell proliferation: evidence for inhibition of a mitogen associated protein kinase. J Neurooncol 2002;56:133-42. [30] Johnson MD, Okediji E, Woodard A. Transforming growth factor-β effects on meningioma cell proliferation and signal transduction pathways. J Neurooncol 2004;66:159-66.
www.elsevier.com/locate/humpath
Original contribution
Na-K-2Cl cotransporter and aquaporin 1 in arachnoid granulations, meningiomas, and meningiomas invading dura Mahlon D. Johnson MD, PhD ⁎, Mary O'Connell BS Department of Pathology, Division of Neuropathology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA Received 25 August 2012; revised 26 September 2012; accepted 28 September 2012
Keywords: NKCC1; Aquaporin 1; Arachnoid granulations; Meningioma; Invasion
Summary Meningioma invasion of the dura may contribute to the high rate of recurrence. Recently, ion channels that affect cell shape and movement have been implicated in cancer invasion. Combined Na-K2Cl cotransporter (NKCC1) and aquaporin 1 (AQP1) expression in arachnoid granulations and meningiomas with and without dural invasion has not been characterized. Arachnoid granulations associated with dura were collected from 10 adult formalin-fixed dura/leptomeninges. Thirty-four frozen meningiomas were evaluated by Western blot. An additional 58 formalin-fixed, paraffin-embedded meningiomas including 36 World Health Organization grade I, 15 grade II, and 7 grade III meningiomas were evaluated by immunohistochemistry. By Western blot, NKCC1 was found in 17 (100%) of 17 World Health Organization grade I, 13 (87%) of 15 grade II, and both grade III meningiomas. Distinct AQP1 was not detected in the meningioma lysates tested. By immunohistochemistry, extensive NKCC1 but no AQP1 immunoreactivity was detected in the arachnoid granulation cells. Extensive NKCC1 was detected in meningioma cells in 56 and in capillaries in 43 by immunohistochemistry. In those tumors with dural or bone/soft tissue invasion, NKCC1 immunoreactivity was seen in invading cells in all cases and in their capillaries in the majority. AQP1 was detected in meningioma cells in 29 and in capillaries in all. AQP1 was also detected in cells and capillaries invading the dura or bone in 10 and 18 of 18, respectively. This was extensive in all subtypes of meningiomas studied. These findings suggest that NKCC1 and AQP1 participate in meningioma biology and invasion. NKCC1 might be targeted by FDA-approved NKCC1 inhibitors. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Invasion of the dura by meningioma cells is thought to contribute to the high rate of meningioma recurrence [1–3]. ⁎ Corresponding author. Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Box 626, Rochester, NY 14623. E-mail address: [email protected] (M. D. Johnson). 0046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2012.09.020
The mechanisms underlying this invasion are poorly understood. Activation of collagenases and metaloproteases has been demonstrated in meningiomas and may contribute [4–7]. However, recently, ion and water channels that affect cell shape and movement have been implicated in neoplastic cell migration and metastasis [8–10]. Neutral Na-K-2Cl cotransporter (NKCC1) is a membrane-bound cation chloride cotransporter family membrane encoded by the SLC12 gene family. NKCC1 mediates
NKCC1 and AQP1 in meningiomas potassium and chloride movement across the plasmalemma in cells of several tissues including the brain. It may also be involved in choroid plexus formation of cerebrospinal fluid. Abnormalities in astrocyte and neuronal cation/chloride homeostasis may contribute to many pathophysiologic conditions including cerebral edema and seizure disorders [11,12]. However, NKCC1 expression and function in the leptomeninges and meningiomas have not been systematically studied, although NKCC1s have recently been identified in arachnoid cysts and thought to contribute to fluid accumulation in these lesions [13]. Aquaporins include a family of 19 water channels that allow the transfer of water and small molecules along the cell membrane. Of these, aquaporins 1, 4, and 9 have been identified in the mammalian brain. Aquaporin 1 (AQP1) has been identified on choroid plexus epithelium and tumors of the choroid plexus. Aquaporin 4 is located primarily on astrocyte end-feet lining capillaries [14,15]. Recently, AQP1 has been identified in cells of a cystic meningioma, the dural attachment and sites of invasion of meningiomas [16,17]. In the present study, we evaluated the expression of NKCC1 and AQP1 in capillaries and venous channels in the dura, meningiomas, and meningiomas with dural invasion.
2. Materials and methods 2.1. Tissue Frozen tissue from 34 meningiomas including 17 World Health Organization (WHO) grade I and 15 WHO grade II and 2 grade III meningiomas were collected at the University of Rochester Medical Center or obtained primarily from the Cooperative Human Tissue Network (Philadelphia, PA) with institutional review board approval (Table 1). Arachnoid granulations associated with dura were collected from 10 adult formalin-fixed dura/leptomeninges from consecutive autopsies between 2010 and 2012. Fifty-eight formalin-fixed, paraffin-embedded meningiomas including 36 WHO grade I meningiomas, 15 WHO grade II, and 7 WHO grade III including 9 with dural invasion and 9 cases with bone including 3 cases bone with soft tissue invasion were identified in the University of Rochester Medical Center archives from 2000 to 2011 and classified [18] (Table 2).
2.2. Western blots For Western blots, meningioma lysates were obtained by mechanical homogenization in RIPA Lysis Buffer (Upstate Biotechnology) with 1:100 Protease Inhibitor Cocktail (Sigma, St Louis, MO) and then frozen at –85°C. The tissue slurries were thawed on ice and vortexed vigorously. Solids were removed by centrifugation. Protein concentrations were quantified using a Bradford assay (BioRad Protein Assay reagent), and then 50 μg protein from each was loaded on
1119 Table 1 Summary of meningiomas used for NKCC1 and AQP1 Western blots Histologic type
Mean age (y)
Sex
n
WHO grade I Meningothelial Transitional Fibrous Total WHO grade II Meningothelial Transitional Fibrous Microcystic Total WHO grade III Fibrous Transitional Total
57
14F, 3M 7 7 3 17
54
11F, 4M 6 4 4 1 15
52
1F, 1M 1 1 2
Abbreviations: F, female; M, male.
7.5% acrylamide gel and transferred to 0.45-μm nitrocellulose membrane. The membrane was blocked 1 hour in 5% milk in Tris-Cl buffer with Tween 20 and then reacted with an affinity purified primary antibody overnight at 4°C. This was followed by horseradish peroxidase–conjugated secondary antibody treatment. Detection was achieved with Western Lightening (Perkin Elmer, Waltham, MA, USA) on Xomat film (Kodak, Rochester, NY, USA). Western blots were analyzed with a polyclonal antibody to NKCC1 (Santa Cruz Biotechnology Inc, Santa Cruz, CA). Loading was assessed with an antibody to actin (Cell Signaling, Beverly MA, USA).
Table 2 Summary of tissues used for NKCC1 and AQP1 immunohistochemistry Histologic type
Mean age (y) Sex
Dura with arachnoid 58 granulations WHO grade I 58 Meningothelial Transitional Fibrous Psammomatous Total WHO grade II 60 Meningothelial Transitional Fibrous Total WHO grade III 68 Meningothelial/Undifferentiated Papillary Total Abbreviations: F, female; M, male; U, unknown.
8F, 2M
n 10
31F, 5M 12 18 4 2 36 9F, 5M, 1U 8 5 2 15 3F, 4M 4 3 7
1120
M. D. Johnson, M. O'Connell
Fig. 1 NKCC1 expression in WHO grade I and II meningiomas. Representative Western blot analysis from 8 WHO grade I (M1-M8), 7 grade II, and 2 grade III (M26-M34) meningiomas showing 170-kd NKCC1 (top) and actin (bottom). NKCC1 immunoreactivity was found in 17 (100%) of 17 WHO grade I, 13 (87%) of 15 grade II, and 2 (100%) of 2 grade III meningiomas and was extensive in all common subtypes.
2.3. Immunohistochemistry Each case was analyzed with a polyclonal antibody to human AQP1 (Abcam Inc, Cambridge, MA) goat polyclonal antibody to NKCC1 (Santa Cruz Biotechnology) with MAC4 universal HRP-polymer (Biocare, Concord, CA) or Goat on Rodent HRP-polymer kit (Biocare) with diaminobenzidene chromogen and hematoxylin counterstain (Biocare). For antigen retrieval, tissue sections were incubated in a thermoresistant chamber with X Reveal Decloaker (Biocare Medical) at 120 to 123°C and pressure of 20 to 24 psi, according to the manufacturers' specifications. Immunoreactivity in tumor cells (excluding blood vessels) was assessed by 2 of us as “0” for no distinct immunoreactivity, “1+” if 1% to 30% of cells were immunoreactive, “2+” if 25% to 50%, and “3+” if greater than 50% of cells were positive. Percentage immunoreactivity in blood vessel endothelium was graded as “0” for no distinct immunoreactivity was found, “1+” if 1% to 30% of cells were immunoreactive, and “2+” if more than 30% of blood vessels had immunoreactivity.
3. Results
difference in grade or subtype. In 6 grade I cases analyzed by Western blot, where formalin-fixed tissue was available, all 6 cases also showed NKCC1 immunoreactivity. Immunoreactivity was membranous and also cytoplasmic. Meningioma cells invading the dura and dural channels also exhibited extensive NKCC1 (Table 3; Fig. 4A and C). Extensive NKCC1 was also seen in cells in all 9 cases with bone (Table 3; Fig. 5A and B) or bone with soft tissue extension. It was also seen in capillaries in 7 of 9 meningiomas invading bone without or with soft tissue. AQP1 immunoreactivity was seen in capillaries in all meningiomas (Table 3 and Fig. 3B) and in meningioma cells (Table 3 and Fig. 3D) of 19 meningiomas. In meningiomas invading dura, invasion of endothelium-lined vascular channels was found in 4 of 9 cases. In each case, the capillaries showed extensive AQP1 immunoreactivity. AQP1 was also detected in capillaries and some meningioma cells in a case invading the brain parenchyma. In meningioma cells invading bone or bone with soft tissue, AQP1 was seen in cells in 6 of 9 and was extensive in capillaries in 9 of 9 (Table 3 and Fig. 5C).
4. Discussion
3.1. Western blots As shown in Fig. 1, NKCC1 was found in 17 (100%) of 17 WHO grade I, 13 (87%) of 15 grade II, and 2 (100%) of 2 grade III meningiomas. This was extensive in all subtypes of meningiomas studied. In the 6 cases where formalin-fixed tissue was available, NKCC1 immunoreactivity was extensive. A distinct 30-kd AQP1 band was not detected by Western blot for these tissues.
3.2. Immunohistochemistry As summarized in Table 3, extensive NKCC1 but no AQP1 was detected in all arachnoid granulation cells (Table 3 and Fig. 2). NKCC1 immunoreactivity was detected in meningioma cells by immunohistochemistry in all 40 meningiomas without documented dural or bone invasion and was extensive (3+) in 38 of 40 (Table 3 and Fig. 3A). NKCC1 was found in capillaries in 29 of 40. Because the immunoreactivity was extensive, there was no apparent
The presence of NKCC1 channels has not been described in the arachnoid or meningiomas. Its widespread membranous localization in arachnoid granulations and meningiomas suggests that NKCC1 channels are important to the biology of normal and neoplastic leptomeningeal cells. Several studies suggest that NKCC1 channels participate in neoplastic processes including cell division, apoptosis, and migration [8–10,12]. The presence of NKCC1 in capillaries throughout meningiomas raises the possibility that NKCC1 channels facilitate movement of cerebrospinal fluid in meningiomas and may form an intercellular network for cerebrospinal fluid distribution throughout these tumors. Previously, we have shown that cerebrospinal fluid is a mitogen for meningiomas cells [19]. However, it is not known whether cerebrospinal fluid reaches central portions of tumors or merely stimulates cell proliferation at the margin of meningiomas. The extensive NKCC1 channel distribution reported here, along with variable AQP1, raises the possibility that growth regulatory cerebrospinal fluid components may reach much of the tumor and that distribution could be targeted by Federal
NKCC1 and AQP1 in meningiomas
1121
Drug Administration−approved NKCC1 inhibitors such as bumetamide [20]. Recent studies suggest that ion channels facilitate the migration and metastasis of some carcinomas and gliomas by altering cell volume and shape [8–10]. At the infiltrating edge of tumors, NKCC1 cotransporters facilitate intracellular Table 3
accumulation of potassium and chloride resulting in changes in the osmotic gradient across the cell membrane and attendant intracellular shift in fluids via AQP1 and other channels resulting in changes in tumor cell volume and shape. This results in displacement of the adjacent parenchyma. The subsequent efflux of ions and fluid mediated by voltage-gated
Summary of NKCC1 and AQP1 arachnoid granulations, meningiomas, and meningiomas with dural invasion NKCC1
Arachnoid granulation cells Grade I meningiomas
Grade II meningiomas
AQP1
cells
capillaries
10/10 3+ 1/27 1+ 1/27 2+ 25/27 3+ 10/10 3+
NA 10/27 0 17/27 1+
1/10 0 4/10 1+ 5/10 2+ 2/3 1+ 1/3 2+ 1/3 0 2/3 1+
Grade III meningiomas
3/3 3+
Grade I meningiomas invading dura
3/3 3+
Grade II meningiomas invading dura
4/4 3+
4/4 1+
Grade III meningiomas invading dura
2/2 3+
Grade I meningiomas invading bone
6/6 3+
Grade II meningiomas invading bone
1/1 3+ 2/2 3+
1/2 0 1/2 1+ 2/6 0 2/6 1+ 2/6 2+ 1/1 2+ 2/2 2+
Grade III meningiomas invading bone
Abbreviation: NA, not applicable. a Capillaries in meningiomas.
a
cells
capillaries a
10/10 0 14/27 0 4/27 1+ 9/27 2+ 5/10 0 3/10 1+ 2/10 2+ 2/3 0 1/3 1+ 1/3 0 1/3 1+ 1/3 2+ 2/4 0 1/4 1+ 1/4 2+ 2/2 0
NA
2/6 0 3/6 1+ 1/6 2+ 1/1 2+ 1/2 0 1/2 1+
27/27 2+
10/10 2+
3/3 2+
1/3 1+ 2/3 2+
1/4 1+ 3/4 2+
2/2 2+
6/6 2+
1/1 2+ 2/2 2+
1122
Fig. 2 NKCC1 immunoreactivity in arachnoid granulations. NKCC1 immunoreactivity, largely membranous, was detected in all arachnoid cap cells by immunohistochemistry (hematoxylin counterstain and diaminobenzidine chromogen, original magnification ×400).
M. D. Johnson, M. O'Connell chloride, calcium-activated potassium channels, and aquaporins subsequently “collapses” the volume/shape of the infiltrating cells, whereas actin polymerization alters cell shape allowing the advancing edge of a cell, that is, lamellipodium, to pull the advancing cell forward into the parenchyma [21–23]. The role of NKCC1 and AQP1 channels in meningioma cell invasion of the dura and brain is unknown. However, the apparent insinuation of NKCC1 and AQP1 expressing meningioma cells into unlined and lined vascular spaces raises the possibility that NKCC1/ AQP1-mediated changes in meningioma cell shape may contribute to meningioma invasion of the dura, skull, and soft tissue. Nonetheless, additional in vitro and in vivo studies evaluating meningioma invasion of the dura and brain after pharmacologic or genetic inhibition of NKCC1 and AQP1 activity are required to test this hypothesis. The dura contains a complex network of capillaries, unlined and venous channels that connect with the arachnoid granulations and superior sagittal sinus [24–28]. Their role
Fig. 3 NKCC1 and AQP1 immunoreactivity in meningiomas. NKCC1 immunoreactivity (A) was largely membranous and in meningioma cells, whereas AQP1 immunoreactivity (B) was primarily capillaries in this meningothelial meningioma. In some meningiomas with extensive NKCC1, immunoreactivity (C) also had AQP1 immunoreactivity (D) in meningioma cells as well as capillaries (hematoxylin counterstain and diaminibenzidine chromogen, original magnification ×400 [A and B] and ×200 [C and D]).
NKCC1 and AQP1 in meningiomas
Fig. 4 NKCC1 in meningioma cells invading dura. A, NKCC1 immunoreactivity was extensive in WHO grade I meningioma cells invading dura in endothelium-lined channels. B, In some cases, meningioma cells were also found in unlined dural spaces adjacent to a blood vessel (hematoxylin and eosin). C, NKCC1 was also seen in these meningioma cells (hematoxylin counterstain and diaminobenzidine chromogen, original magnification ×400 [A and C]).
1123
Fig. 5 NKCC1 and AQP1 in meningioma cells invading the skull. A, NKCC1 immunoreactivity in WHO grade I meningioma invading bone Haversian canal. B, NKCC1 immunoreactivity was extensive in WHO grade I meningioma cells invading the skull bone. C, AQP1 was found in variable numbers of meningioma cells and meningioma capillaries invading the bone (hematoxylin counterstain and diaminobenzidine chromogen, original magnification ×400).
1124 as a passageway for meningioma cell invasion or as a reservoir for recurring meningiomas cells is not established. These channels may also be a conduit for cerebrospinal fluid, water, and cytokines. Of note, electron microscopy has shown that these venules have pores [24]. Previously, we have shown that cerebrospinal fluid stimulates proliferation of human leptomeningeal and meningioma cells of all grades [19,29,30]. Growth factor/cytokines and their receptor tyrosine kinases likely participate in these effects [29,30]. However, other factors such as cation and chloride homeostasis may also participate. AQP1 was consistently found in endothelial cells of capillaries, but not larger blood vessels, and variably detected in meningioma cells. AQP1 has been reported in a cystic meningioma in a newborn and in meningioma cells at the point of dural invasion of the dura [16,17]. In the present study, AQP1 was also detected in capillaries in meningioma cells invading the dura. We also found aquaporin expression at sites of dural invasion, but expression was not confined to that site. By Western blot, a definite 30-kd band was not detected in the meningiomas analyzed. This likely reflects the limited amounts of AQP1 detected by immunohistochemistry in meningiomas. NKCC1 and AQP1 were detected in all subtypes and grades of meningiomas, suggesting a role in meningioma biology rather than in progression. Although Merlin was not tested in many cases, this also suggests their presence both in tumor with and in tumor without Merlin. Consequently, pharmacologic agents targeting these channels might have widespread applicability as an adjunct chemotherapy. In summary, these studies have shown that NKCC1 and AQP1 are widely distributed in meningiomas and meningioma cells invading the dura. Considering recent findings implicating ion and water channels in invasion and metastasis, additional studies evaluating their role in meningioma invasion of surrounding tissues are warranted.
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