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Endocrine manipulation of meningiomas with medroxyprogesterone acetate
Surg Neurol 1989;31:96-100
96
Endocrine
Manipulation
of Meningiomas
Medroxyprogesterone
Acetate
Effect of MPA on Growth
of Primary
Meningioma
with Cells in Monolayer
Tissue Culture Ernst R. Waelti,
Ph.D.,
Institute
and Department
of Pathology
Thomas-Mare
Markwalder,
of Neurosurgery,
University
Waelti ER, Markwalder T-M. Endocrine manipulation of meningiomas with medroxyprogesterone acetate. Effect of MPA on growth of primary meningioma cells in monolayer tissue culture. Surg Neurol 1989;31:96-100.
treated with memeningiomas from patients droxyprogesterone acetate as wel1 as from untreated patients were studied in monolayer tissue culture with trials of in vitro hormonal modulation with medroxyprogesterone acetate. The following conclusions were drawn from investigations which comprise 37 cel1 culture assays: (a) tissue cultures of meningiomas inherit the disadvantages of loss of the progesterone receptor and frequent transformation to cells resembling fibroblasts after three to four passages. For these reasons, drug testing as wel1 as the establishment of cel1 cultures that exhibit the characteristics of meningioma are impeded; (6) the progesterone receptor-content of the solid tumors does not reflect the response to medroxyprogesterone acetatetherapy in vitro; (c) medroxyprogesterone acetate-pretreated meningiomas showed sufficient in vitro growth in 38%, and untreated meningiomas grew wel1 in 56% of the cases; (d) medroxyprogesterone acetate-induced inhibition or delay of growth was observed in 35%. These findings have resulted in criticism with respect to the value of meningioma tissue cultures for trials of hormonal manipulation and it is thought that another method, which consists of immunostaining of cycling cells, and has been tested in another study, may be superior to cel1 culture assays with respect to evaluation of the effect of hormonotherapy in meningiomas. Medroxyprogesterone acetate holds an interesting position because it reduces cel1 growth in some meningiomas in vitro.
IntracraniaI
KEY WOFUX: Tissue culture; roids; Medroxyprogesterone ningioma
Progesterone receptor; Sex steacetate; Neurochemistry; Me-
A~~WJJreprint requests to: Ernst R. Waelti, Ph.D., Institute of Pathology, University of Bern, Freiburgstr. 30, CH-3010 Bern, Switzerland. Received February 3, 1988; accepted August 25, 1988. 0 1989
by Elsevier Science Publishing Co., inc
M.D. of Bern, Bern,
Switzerland
In a recently published study, we were able to demonstrate that medroxyprogesterone acetate (MPA, DepoProvera 500, Upjohn SA, Zurich, Switzerland), a semisynthetic gestagen, acted as a competitive binder to progesterone receptors (PR) in meningiomas and significantly reduced PR activity in meningioma cytosols [12). In another study we analyzed the effect of MPA on growth fractions of meningioma cells by use of the monoclonal antibody Ki-67, and were able to show a reduction of growth fractions after hormonotherapy ClOl. The aim of the present contribution is to report out results of MPA treatment of primary meningioma cells in monolayer tissue culture and to evaluate critically the value of in vitro studies versus immunohistochemical analyses of the effect of hormonotherapy.
Materials
and Methods
Cell Culture Meningioma tissue samples were cut into 1-mm fragments and transferred into 25-cm* tissue culture flasks. Cells were fed with Richter’s IMEM supplemented with insulin (200 ng/mL), gentamycin (0.1 mg/mL), and 10% (v/v) fetal calf serum (FCS). The cultures were maintained in a humidified atmosphere of 95 % air-5 % CO2 at 37°C. After a 48-hour incubation, supernatants with the residual unattached tissue fragments were decanted and the growth medium replaced. This procedure was repeated every 2 days until cells reached confluence. At confluence, cells were removed from the flasks with trypsin/EDTA solution. The resulting cel1 suspension was centrifuged for 5 minutes at approximately 150 g. The resuspended cells were split 2 : 1 and passed into new culture flasks. Usually three passages were required to obtain sufficient amounts of cells for the studies. 0090-3019/89/53.50
Effect of MPA on Meningioma
Surg Neurol 1989;3 1:96- 100
In Vitro
Determination of Wbole Cel Uptake of Radiolabeled Progesterone in Meningioma Cel/ Cultures Twenty-four hours before harvesting the primary meningioma cells, the growth medium was replaced with medium containing 2% charcoal-treated (ct) FCS as described by Lippman et al {9]. Cells were removed from the flasks with trypsin/EDTA and resuspended in medium containing 2% ct-FCS as described by Zava et al Cl 71. Aliquots (1 mL) of cel1 suspension (200,000 cells) were transferred into 12 x 75 mm glass tubes. Cells were sedimented by centrifugation (250 g, 10 minutes), and the medium decanted. One milliliter of lO-nm C3Hfpromegestone (R5020, 77 Ci/mmol), plus or minus lOO-fold molar excess of unlabeled R5020 in medium containing 2Yo ct-FCS was added (in duplicate) to the cel1 pellets. The cells were incubated with promegestone for 45 minutes at 37°C in the incubator. The tubes were centrifuged (250 g, 10 minutes) and the radioactive medium removed. Cells were gently resuspended in 1 mL medium (2vo ct-FCS) and maintained at room temperature for 10 minutes. Cells were centrifuged as before, the medium was decanted, and the tubes were immersed in an ice-water bath. The cells were then washed twice with 1 mL cold phosphate buffer (5 mM Na-phosphate, 0.25 M sucrose, 10% glycerol, pH 7.4). One milliliter ethanol was added to each tube to extract the promegestone. After 1 hour incubation and a repeated vortex mixing, the tubes were centrifuged (700 g, 10 minutes, 0°C). The clear ethanol was transferred 3 mL Beckman HPb into scintillation vials containing scintillation cocktail. Radioactivity was measured in Beckman LS 1800 scintillation counter. DNA was assayed by the diphenylamine method of Burton {3], with calf thymus DNA as a standard.
Analysis of Cel Growth: Trials of Modulatìon with MPA Cells were removed from the flasks, suspended in Richter’s IMEM, and plated in 24 multiwell plates (Costar, Europe Ltd., The Netherlands; approximately 10,000 cells/well). The plates were incubated overnight to allow adequate cel1 adherente. The growth medium was then replaced with 2% ct-FCS medium containing 10e6 M pure MPA. MPA was prepared in stock solution of ethanol. The corresponding amount of ethanol was added to control cultures. Medium plus or minus MPA was replaced every 2 days. At various time intervals (3, 5, 7, and 10 days) the medium was removed, and the cells were detached with trypsin/EDTA and counted in a hemocytometer. In a series of experiments the cel1 number was determined by a DNA-assay as described by Taylor et al {I6].
97
Results Thirty-seven samples of meningioma tissue, 21 from MPA-pretreated and 16 from untreated patients, were studied in monolayer tissue culture. From the MPApretreated tumor specimen, 8 of 2 1 showed sufficient in vitro growth, rendering them suitable for manipulation trials with MPA. Nine of 16 meningiomas obtained from untreated patients grew wel1 in culture. About half of the 20 samples that did not grow in vitro after the first or second passages showed adherente on the plates; in the other half, even adherente was not noticed. There was no clear-cut correlation between the histologie type, mitotic index, or presence of nuclear polymorphism and the growth behavior in tissue culture. The PR content of the primary tumors from untreated patients was within the normal range (>707~ of PR-positive samples) and comparable with the PR values obtained in previous investigations (PR,,,, = 54.9 fmol/mg protein [range 0-5861, and 2813 fmol/g tumor [range 0-17,168], respectively [11,12,14]. In MPApretreated primary tumor samples the PR values were significantly lower (PR,,,, = 15.6 fmol/mg protein [range 0-691 and 338.3 fmol/g tumor [range 0-11901, respectively [12]. None of the analyzed primary tumor specimen and cel1 cultures contained significant amounts of cytoplasmic or nuclear estrogen receptor. As observed previously [ 171, primary meningioma cells completely lose the capacity to express PR during in vitro culturing. MPA inhibited in vitro cel1 growth in six instances, four of these tumors were treated with MPA prior to surgical excision. Figure 1 depicts the in vitro growth behavior in the tumor samples from a man in whom a huge olfactory groove meningioma of the meningothelial histologie type was operated on in two stages. The curves (Z) were obtained with the tumor specimen from the first operation, which was carried out after pretreatment with MPA over a 17-day period (total dose: 17,000 mg). In this sample, the PR values were 105 fmol/mg protein (3696 fmol/g tumor) and it contained 0.09% Ki-67 positive cells (range 0.04-0.14). The monoclonal antibody Ki-67, prepared by Gerdes et al Cb], reacts with a nuclear antigen present in proliferating cells but absent in quiescent cells. Since our results of the Ki-67-study were already published in a previous paper, we recommend referral to [ 101 for immunohistochemical and methodical details. The curves (1) illustrate the delayed cel1 growth by day 7 (even more pronounced when MPA was additionally administered in culture) in comparison to the growth curves (ZZ) obtained with the tumor samples from the second operation which was carried out 10 days after discontinuation of MPA-therapy. In the second tumor sample PR-values
Surg Neurol 1989;31:96-100
98
Waelti and Markwalder
70
w A +MPA 0
60 1
A CONTROL
nu
50.
ltm
40.
10 230. 0
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A ;
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Figute 1. In vitro growth curve ofprìmary meningioma cells from MPApretreated (1) and untreated (11) tumor samples (olfactoty groove meningioma operated in two stages; see text). Cells were incubated in duplicate in six wells cell culture clusters (wel1 growth area 9.5 cm2) without and with MPA at a concentration of 1 0m6M. At the time points indicated, cells were harvested, and the cel1growth was assessed by measurement of the total cel1 DNA of eacb wel1 as described by Taylor et al [16]. Points are the mean of two ualues.
were, as expected [lO, 127, much higher (202 fmol/mg protein and 6140 fmol/g tumor, respectively), and it contained 0.28% Ki-67 positive cells (range 0.220.33). The curves (ZZ) show a similar cytostatic effect of MPA on the in vitro cel1 growth by day 7. An example of an untreated meningioma is shown in Figure 2. Presence of 10e6 M MPA in the medium resulted in significant growth inhibition after 5 days. In this case the size of the tumor was not sufficient to allow PR determination of both the solid tumor and the cel1 culture. A PR content of 1 fmol/pg DNA was measured by whole cel1 uptake of radiolabeled progesterone in cel1 culture. In contrast to the case described in Figure 1, the observed diversity of responsiveness to MPA of primary meningioma cells in monolayer tissue culture could not be brought in relation to the measured PR content of the solid tumors in al1 cases. Immunohistochemical demonstration of the percentage of cells out of Go-phase by Ki-67 was only done in the case shown in Figure 1 [lol.
the studies with human breast cancer cel1 lines. Unfortunately, studies of primary meningioma cells in monolayer culture are hampered by two drawbacks: First, only a limited propagation of meningioma cells is possible (usually no more than five cel1 passages). The latter might be due to the fact that cultured primary meningioma cells transform to cel1 types with fibroblastic characteristics and biological activities similar to those of fibroblasts [4,13,17]. Second, meningioma cells in culture exhibit only insignificant amounts of PR or completely lose the capacity to express them, although the original solid tumor may show a high content. Regarding this finding our present data confirm those already reported by Zava et al [13,171. It appears that transformation to a fibroblastic cel1 type and loss of PR are causally related; meningiomas of fibroblastic histological types were reported to contain much less PR than do meningiothelial histologie types {11,14]. Since ligand specificity testing has demonstrated that MPA binds to PR in the cytosolic extracts of meningiomas [ 11,141 and a recent study has shown a significant decrease in the PR-content after MPA-therapy [12], the loss of PR or
Figure 2. In vitro growth curves of primary meningioma cells from an untreated tumor sample (petrous-tentorial meningioma of meningothelial histologie type; see text). Cells were incubated in duplicate in 2S-cm2 cel1 culturejlasks without and with MPA at a concentration of 10m6M. At the time points indicated, cells were hawested, and the cel1growth was assessed by measurement of the total cel1DNA of eacbjask as described by Taylor et al [lb]. One jask was assayed for PR leve1 (1 fmollpg DNA) using tbe assay described in Materials and Metbods. Points are tbe mean of two values.
Discussion A great deal of information about the hormonal responsiveness of human breast cancer has been learned from
3
5
10 cîa:s
Effect of MPA on Meningioma
In Vitro
Surg
Neurol
1989;31:96-100
the retained low PR content may provide an explanation why MPA exerted only marginal or no inhibition at al1 on the in vitro cel1 growth in some of our meningioma cel1 cultures. Apart from this hypothesis, which is in favor of an active role of the PR with respect to the effects of MPA in tissue culture, there was no clear-cut correlation between the measured PR content in the solid tumors and the effect of MPA in vitro. This result suggests that MPA may have cytotoxic effects independent of PR. Regarding the role of steroids in meningioma cel1 cultures, recent studies of other investigators show a divergent picture: Jay et al [8] reported that estradiol and progesterone at concentrations of either 10-‘M or 10m9M stimulated three of four tumors CS]. Olson et al [15] tested the same compounds in combination and observed growth stimulation in two of three meningioma cel1 cultures. Tamoxifen increased tumor cel1 growth in two of three cases, while RU486, a progesterone antagonist, inhibited proliferation of cells in al1 three tumors (inhibition ranging from 8% to 36%) CIS]. The presence of PR in meningioma cells without ER remains a biochemical enigma [l]. It does not fit into the general view that in appropriated target cells PR is synthesized in response to estrogens acting through ER. However, there exists a smal1 number of anomalous tumors that have no ER, yet contain PR. Such a situation is represented by some T47D human breast cancer cells, which contain high PR that are not estrogenrelated [7]. Summariting our data, we can draw the following conclusions from our trials of hormonal manipulation of meningiomas in monolayer tissue culture: (a) tissue cultures of meningiomas inherit the disadvantage of loss of PR and frequent transformation to cells resembling fibroblasts; (b) for these reasons drug testing as wel1 as the establishment of cel1 cultures that show the characteristics of in vivo meningioma are impeded; (c) the response to MPA-treatment in vitro did not reflect the measured PR content of the solid tumors; (d) MPA-pretreated meningiomas showed sufficient in vitro growth in 38% and untreated meningiomas in 56% of the cases; and (e) MPA-induced inhibition or delay of growth was observed in 35% of the cases. Although from our standpoint meningioma tissue cultures may have a certain prognostic value for trials of hormonal manipulation, as demonstrated in Figures 1 and 2, the in vitro results should be judged critically. For these reasons we have used the monoclonal antibody Ki-67 for immunohistochemical demonstration of the effect of MPA-therapy on growth fractions of meningioma cells ElO]. Ki-67 reacts with a human nuclear antigen expressed on al1 proliferating cells. Expression
99
of this antigen occurs preferentially during late Gl, S, M, and G2 phases of the cel1 cycle, while cells in Ga phase consistently lack the antigen [2,5,6]. Thus, the reaction with Ki-67 serves as an indicator of the growth fraction, i.e., the number of cells undergoing active division, of the human neoplasm [bl. In our previous study a decrease of Ki-67 positive cells by a factor 6, 5 and 3, respectively, could be demonstrated after MPA therapy in three patients with meningiomas operated in two stages. In meningioma specimens from patients receiving no MPA therapy, Ki-67-positive cells were present in 1.02% + 0.48%, whereas in samples from MPAtreated tumors the percentage of Ki-67 positive cells was 0.14 + 0.40 [lol. In contrast to cel1 cultures limited by the changes in cel1 behavior caused by the culture environment, Ki-67 allows one to determine the growth fraction in meningiomas in situ. From this point of view the simple immunohistological labeling with monoclonal antibody Ki-67 of cryostat sections might be superior to the timeconsuming cel1 cultures and be a more objective aid for evaluating therapies.
References MA, Blaauw G, Lamberts WJ, Mulder E. Presence 1. Blankenstein of progesterone receptors and absente of estrogen receptors in human intracranial meningioma cytosols. Eur J Cancer Clin Oncol 1983;19:365-70. 2. Burger PC, Shibata T, Kleihues P. The use of the monoclonal antibody Ki-67 in the identification of proliferating cells: application to surgical neuropathology. Am J Surg Pathol 1986;10:61 l7. 3. Burton DA. Studies of conditions and mechanisms of the diphenylamine reaction for the colorimetric determination of deoxyribonucleic acid. Biochem 1956;62:3 15-23. 4. Costero 1, Pomerat CM, Jackson IJ, Barroso-Moguel R, Chevez AZ. Tumors of the human nerveus system in tissue culture. 1. The cultivation and cytology of meningioma cells. J Nat1 Cancer Inst 1953;15:1319-39. F, Lennert K. Growth fractions in malig5. Gerdes J, Dallenbach nam non-Hodgkin’s lymphomas (NHL) as determined in situ with the monoclonal antibody Ki-67. Hematol Oncol 1984;2:365-71. of a mouse 6. Gerdes J, Schwab IJ, Lemke H, Stein H. Production monoclonal antibody reactive with a human nuclear antigen associated with cel1 proliferation. Int J Cancer 1983;3 1:13-20. 7. Horwitz KB, Mockus MB, Lessey BA. Variant T47D human breast cancer cells with high progesterone receptor Ievels despite estrogen and antiestrogen resistance. Cel1 1982;633-42. 8. Jay JR, MacLaughlin DT, Riley KR, Martuza RL.. Modulation of meningioma cel1 growth by sex steroid hormones in vitro. J Neurosurg 1985;62:757-62. 9. Lippman M, Bolan G, Huff K. The effect of estrogens and antiestrogens on hormone responsive human breast cancer in longterm tissue culture. Cancer Res 1976;4595-601. 10. Markwalder TM, Gerber HA, Waelti E, Schaffner T, Markwalder RV. Hormonotherapy of meningiomas with medroxyprogesterone acetate: immunohistochemical demonstration of the effect of MPA on growth fractions of meningioma cells using the monoclonal antibody Ki-67. Surg Neurol 1988;30:97-101.
100
Surg Neurol
Waelti
and Markwalder
1989;31:96-100
ll.
Markwalder TM, Markwalder RV, Zava DT. Estrogen and progestin receptors in meningiomas: clinicopathological correlations. Clin Neuropharmacoi 1984;7:368-74.
12. Markwalder TM, Waelti E, Koenig MP. Endocrine manipulation of meningiomas with medroxyprogesterone acetate. Effect of MPA on receptor status of meningioma cytosols. Surg Neurol 1987;28:3-9. 13. Markwalder TM, Zava DT. Sex steroid hormones and meningioma cel1 growth (letter). J Neurosurg 1986;64:341-2. 14. Markwalder
TM, Zava DT, Goldhirsch
A, Markwalder
RV. Es-
trogen and progesterone receptors in meningiomas in relation clinicaf and pathologie features. Surg Neurol 1983;20:42-7.
to
15. Olson JJ, Beek DW, Schlechte J, Loh P-M. Hormonal manipulation of meningiomas in vitro. J Neurosurg 1986; 65:99-107. 16. Taylor CM, Blanchard B, Zava DT. A mine whole-cel1 uptake of radiolabelled one and their subcellular localization in monolayer culture. J Steroid Biochem
simple method to deterestrogen and progesterbreast cancer cel1 lines in 1984;20: 1083-8.
17. Zava DT, Markwalder TM, Markwalder RV. Biological expression of steroid hormone receptors in primary meningioma cells in monolayer tissue culture. Clin Neuropharmacol 1984;7:382-8.
96
Endocrine
Manipulation
of Meningiomas
Medroxyprogesterone
Acetate
Effect of MPA on Growth
of Primary
Meningioma
with Cells in Monolayer
Tissue Culture Ernst R. Waelti,
Ph.D.,
Institute
and Department
of Pathology
Thomas-Mare
Markwalder,
of Neurosurgery,
University
Waelti ER, Markwalder T-M. Endocrine manipulation of meningiomas with medroxyprogesterone acetate. Effect of MPA on growth of primary meningioma cells in monolayer tissue culture. Surg Neurol 1989;31:96-100.
treated with memeningiomas from patients droxyprogesterone acetate as wel1 as from untreated patients were studied in monolayer tissue culture with trials of in vitro hormonal modulation with medroxyprogesterone acetate. The following conclusions were drawn from investigations which comprise 37 cel1 culture assays: (a) tissue cultures of meningiomas inherit the disadvantages of loss of the progesterone receptor and frequent transformation to cells resembling fibroblasts after three to four passages. For these reasons, drug testing as wel1 as the establishment of cel1 cultures that exhibit the characteristics of meningioma are impeded; (6) the progesterone receptor-content of the solid tumors does not reflect the response to medroxyprogesterone acetatetherapy in vitro; (c) medroxyprogesterone acetate-pretreated meningiomas showed sufficient in vitro growth in 38%, and untreated meningiomas grew wel1 in 56% of the cases; (d) medroxyprogesterone acetate-induced inhibition or delay of growth was observed in 35%. These findings have resulted in criticism with respect to the value of meningioma tissue cultures for trials of hormonal manipulation and it is thought that another method, which consists of immunostaining of cycling cells, and has been tested in another study, may be superior to cel1 culture assays with respect to evaluation of the effect of hormonotherapy in meningiomas. Medroxyprogesterone acetate holds an interesting position because it reduces cel1 growth in some meningiomas in vitro.
IntracraniaI
KEY WOFUX: Tissue culture; roids; Medroxyprogesterone ningioma
Progesterone receptor; Sex steacetate; Neurochemistry; Me-
A~~WJJreprint requests to: Ernst R. Waelti, Ph.D., Institute of Pathology, University of Bern, Freiburgstr. 30, CH-3010 Bern, Switzerland. Received February 3, 1988; accepted August 25, 1988. 0 1989
by Elsevier Science Publishing Co., inc
M.D. of Bern, Bern,
Switzerland
In a recently published study, we were able to demonstrate that medroxyprogesterone acetate (MPA, DepoProvera 500, Upjohn SA, Zurich, Switzerland), a semisynthetic gestagen, acted as a competitive binder to progesterone receptors (PR) in meningiomas and significantly reduced PR activity in meningioma cytosols [12). In another study we analyzed the effect of MPA on growth fractions of meningioma cells by use of the monoclonal antibody Ki-67, and were able to show a reduction of growth fractions after hormonotherapy ClOl. The aim of the present contribution is to report out results of MPA treatment of primary meningioma cells in monolayer tissue culture and to evaluate critically the value of in vitro studies versus immunohistochemical analyses of the effect of hormonotherapy.
Materials
and Methods
Cell Culture Meningioma tissue samples were cut into 1-mm fragments and transferred into 25-cm* tissue culture flasks. Cells were fed with Richter’s IMEM supplemented with insulin (200 ng/mL), gentamycin (0.1 mg/mL), and 10% (v/v) fetal calf serum (FCS). The cultures were maintained in a humidified atmosphere of 95 % air-5 % CO2 at 37°C. After a 48-hour incubation, supernatants with the residual unattached tissue fragments were decanted and the growth medium replaced. This procedure was repeated every 2 days until cells reached confluence. At confluence, cells were removed from the flasks with trypsin/EDTA solution. The resulting cel1 suspension was centrifuged for 5 minutes at approximately 150 g. The resuspended cells were split 2 : 1 and passed into new culture flasks. Usually three passages were required to obtain sufficient amounts of cells for the studies. 0090-3019/89/53.50
Effect of MPA on Meningioma
Surg Neurol 1989;3 1:96- 100
In Vitro
Determination of Wbole Cel Uptake of Radiolabeled Progesterone in Meningioma Cel/ Cultures Twenty-four hours before harvesting the primary meningioma cells, the growth medium was replaced with medium containing 2% charcoal-treated (ct) FCS as described by Lippman et al {9]. Cells were removed from the flasks with trypsin/EDTA and resuspended in medium containing 2% ct-FCS as described by Zava et al Cl 71. Aliquots (1 mL) of cel1 suspension (200,000 cells) were transferred into 12 x 75 mm glass tubes. Cells were sedimented by centrifugation (250 g, 10 minutes), and the medium decanted. One milliliter of lO-nm C3Hfpromegestone (R5020, 77 Ci/mmol), plus or minus lOO-fold molar excess of unlabeled R5020 in medium containing 2Yo ct-FCS was added (in duplicate) to the cel1 pellets. The cells were incubated with promegestone for 45 minutes at 37°C in the incubator. The tubes were centrifuged (250 g, 10 minutes) and the radioactive medium removed. Cells were gently resuspended in 1 mL medium (2vo ct-FCS) and maintained at room temperature for 10 minutes. Cells were centrifuged as before, the medium was decanted, and the tubes were immersed in an ice-water bath. The cells were then washed twice with 1 mL cold phosphate buffer (5 mM Na-phosphate, 0.25 M sucrose, 10% glycerol, pH 7.4). One milliliter ethanol was added to each tube to extract the promegestone. After 1 hour incubation and a repeated vortex mixing, the tubes were centrifuged (700 g, 10 minutes, 0°C). The clear ethanol was transferred 3 mL Beckman HPb into scintillation vials containing scintillation cocktail. Radioactivity was measured in Beckman LS 1800 scintillation counter. DNA was assayed by the diphenylamine method of Burton {3], with calf thymus DNA as a standard.
Analysis of Cel Growth: Trials of Modulatìon with MPA Cells were removed from the flasks, suspended in Richter’s IMEM, and plated in 24 multiwell plates (Costar, Europe Ltd., The Netherlands; approximately 10,000 cells/well). The plates were incubated overnight to allow adequate cel1 adherente. The growth medium was then replaced with 2% ct-FCS medium containing 10e6 M pure MPA. MPA was prepared in stock solution of ethanol. The corresponding amount of ethanol was added to control cultures. Medium plus or minus MPA was replaced every 2 days. At various time intervals (3, 5, 7, and 10 days) the medium was removed, and the cells were detached with trypsin/EDTA and counted in a hemocytometer. In a series of experiments the cel1 number was determined by a DNA-assay as described by Taylor et al {I6].
97
Results Thirty-seven samples of meningioma tissue, 21 from MPA-pretreated and 16 from untreated patients, were studied in monolayer tissue culture. From the MPApretreated tumor specimen, 8 of 2 1 showed sufficient in vitro growth, rendering them suitable for manipulation trials with MPA. Nine of 16 meningiomas obtained from untreated patients grew wel1 in culture. About half of the 20 samples that did not grow in vitro after the first or second passages showed adherente on the plates; in the other half, even adherente was not noticed. There was no clear-cut correlation between the histologie type, mitotic index, or presence of nuclear polymorphism and the growth behavior in tissue culture. The PR content of the primary tumors from untreated patients was within the normal range (>707~ of PR-positive samples) and comparable with the PR values obtained in previous investigations (PR,,,, = 54.9 fmol/mg protein [range 0-5861, and 2813 fmol/g tumor [range 0-17,168], respectively [11,12,14]. In MPApretreated primary tumor samples the PR values were significantly lower (PR,,,, = 15.6 fmol/mg protein [range 0-691 and 338.3 fmol/g tumor [range 0-11901, respectively [12]. None of the analyzed primary tumor specimen and cel1 cultures contained significant amounts of cytoplasmic or nuclear estrogen receptor. As observed previously [ 171, primary meningioma cells completely lose the capacity to express PR during in vitro culturing. MPA inhibited in vitro cel1 growth in six instances, four of these tumors were treated with MPA prior to surgical excision. Figure 1 depicts the in vitro growth behavior in the tumor samples from a man in whom a huge olfactory groove meningioma of the meningothelial histologie type was operated on in two stages. The curves (Z) were obtained with the tumor specimen from the first operation, which was carried out after pretreatment with MPA over a 17-day period (total dose: 17,000 mg). In this sample, the PR values were 105 fmol/mg protein (3696 fmol/g tumor) and it contained 0.09% Ki-67 positive cells (range 0.04-0.14). The monoclonal antibody Ki-67, prepared by Gerdes et al Cb], reacts with a nuclear antigen present in proliferating cells but absent in quiescent cells. Since our results of the Ki-67-study were already published in a previous paper, we recommend referral to [ 101 for immunohistochemical and methodical details. The curves (1) illustrate the delayed cel1 growth by day 7 (even more pronounced when MPA was additionally administered in culture) in comparison to the growth curves (ZZ) obtained with the tumor samples from the second operation which was carried out 10 days after discontinuation of MPA-therapy. In the second tumor sample PR-values
Surg Neurol 1989;31:96-100
98
Waelti and Markwalder
70
w A +MPA 0
60 1
A CONTROL
nu
50.
ltm
40.
10 230. 0
cl
A ;
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%O
A
_/-.
IA IO-
\
A
Th Y
3
5
10 cia:s
Figute 1. In vitro growth curve ofprìmary meningioma cells from MPApretreated (1) and untreated (11) tumor samples (olfactoty groove meningioma operated in two stages; see text). Cells were incubated in duplicate in six wells cell culture clusters (wel1 growth area 9.5 cm2) without and with MPA at a concentration of 1 0m6M. At the time points indicated, cells were harvested, and the cel1growth was assessed by measurement of the total cel1 DNA of eacb wel1 as described by Taylor et al [16]. Points are the mean of two ualues.
were, as expected [lO, 127, much higher (202 fmol/mg protein and 6140 fmol/g tumor, respectively), and it contained 0.28% Ki-67 positive cells (range 0.220.33). The curves (ZZ) show a similar cytostatic effect of MPA on the in vitro cel1 growth by day 7. An example of an untreated meningioma is shown in Figure 2. Presence of 10e6 M MPA in the medium resulted in significant growth inhibition after 5 days. In this case the size of the tumor was not sufficient to allow PR determination of both the solid tumor and the cel1 culture. A PR content of 1 fmol/pg DNA was measured by whole cel1 uptake of radiolabeled progesterone in cel1 culture. In contrast to the case described in Figure 1, the observed diversity of responsiveness to MPA of primary meningioma cells in monolayer tissue culture could not be brought in relation to the measured PR content of the solid tumors in al1 cases. Immunohistochemical demonstration of the percentage of cells out of Go-phase by Ki-67 was only done in the case shown in Figure 1 [lol.
the studies with human breast cancer cel1 lines. Unfortunately, studies of primary meningioma cells in monolayer culture are hampered by two drawbacks: First, only a limited propagation of meningioma cells is possible (usually no more than five cel1 passages). The latter might be due to the fact that cultured primary meningioma cells transform to cel1 types with fibroblastic characteristics and biological activities similar to those of fibroblasts [4,13,17]. Second, meningioma cells in culture exhibit only insignificant amounts of PR or completely lose the capacity to express them, although the original solid tumor may show a high content. Regarding this finding our present data confirm those already reported by Zava et al [13,171. It appears that transformation to a fibroblastic cel1 type and loss of PR are causally related; meningiomas of fibroblastic histological types were reported to contain much less PR than do meningiothelial histologie types {11,14]. Since ligand specificity testing has demonstrated that MPA binds to PR in the cytosolic extracts of meningiomas [ 11,141 and a recent study has shown a significant decrease in the PR-content after MPA-therapy [12], the loss of PR or
Figure 2. In vitro growth curves of primary meningioma cells from an untreated tumor sample (petrous-tentorial meningioma of meningothelial histologie type; see text). Cells were incubated in duplicate in 2S-cm2 cel1 culturejlasks without and with MPA at a concentration of 10m6M. At the time points indicated, cells were hawested, and the cel1growth was assessed by measurement of the total cel1DNA of eacbjask as described by Taylor et al [lb]. One jask was assayed for PR leve1 (1 fmollpg DNA) using tbe assay described in Materials and Metbods. Points are tbe mean of two values.
Discussion A great deal of information about the hormonal responsiveness of human breast cancer has been learned from
3
5
10 cîa:s
Effect of MPA on Meningioma
In Vitro
Surg
Neurol
1989;31:96-100
the retained low PR content may provide an explanation why MPA exerted only marginal or no inhibition at al1 on the in vitro cel1 growth in some of our meningioma cel1 cultures. Apart from this hypothesis, which is in favor of an active role of the PR with respect to the effects of MPA in tissue culture, there was no clear-cut correlation between the measured PR content in the solid tumors and the effect of MPA in vitro. This result suggests that MPA may have cytotoxic effects independent of PR. Regarding the role of steroids in meningioma cel1 cultures, recent studies of other investigators show a divergent picture: Jay et al [8] reported that estradiol and progesterone at concentrations of either 10-‘M or 10m9M stimulated three of four tumors CS]. Olson et al [15] tested the same compounds in combination and observed growth stimulation in two of three meningioma cel1 cultures. Tamoxifen increased tumor cel1 growth in two of three cases, while RU486, a progesterone antagonist, inhibited proliferation of cells in al1 three tumors (inhibition ranging from 8% to 36%) CIS]. The presence of PR in meningioma cells without ER remains a biochemical enigma [l]. It does not fit into the general view that in appropriated target cells PR is synthesized in response to estrogens acting through ER. However, there exists a smal1 number of anomalous tumors that have no ER, yet contain PR. Such a situation is represented by some T47D human breast cancer cells, which contain high PR that are not estrogenrelated [7]. Summariting our data, we can draw the following conclusions from our trials of hormonal manipulation of meningiomas in monolayer tissue culture: (a) tissue cultures of meningiomas inherit the disadvantage of loss of PR and frequent transformation to cells resembling fibroblasts; (b) for these reasons drug testing as wel1 as the establishment of cel1 cultures that show the characteristics of in vivo meningioma are impeded; (c) the response to MPA-treatment in vitro did not reflect the measured PR content of the solid tumors; (d) MPA-pretreated meningiomas showed sufficient in vitro growth in 38% and untreated meningiomas in 56% of the cases; and (e) MPA-induced inhibition or delay of growth was observed in 35% of the cases. Although from our standpoint meningioma tissue cultures may have a certain prognostic value for trials of hormonal manipulation, as demonstrated in Figures 1 and 2, the in vitro results should be judged critically. For these reasons we have used the monoclonal antibody Ki-67 for immunohistochemical demonstration of the effect of MPA-therapy on growth fractions of meningioma cells ElO]. Ki-67 reacts with a human nuclear antigen expressed on al1 proliferating cells. Expression
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of this antigen occurs preferentially during late Gl, S, M, and G2 phases of the cel1 cycle, while cells in Ga phase consistently lack the antigen [2,5,6]. Thus, the reaction with Ki-67 serves as an indicator of the growth fraction, i.e., the number of cells undergoing active division, of the human neoplasm [bl. In our previous study a decrease of Ki-67 positive cells by a factor 6, 5 and 3, respectively, could be demonstrated after MPA therapy in three patients with meningiomas operated in two stages. In meningioma specimens from patients receiving no MPA therapy, Ki-67-positive cells were present in 1.02% + 0.48%, whereas in samples from MPAtreated tumors the percentage of Ki-67 positive cells was 0.14 + 0.40 [lol. In contrast to cel1 cultures limited by the changes in cel1 behavior caused by the culture environment, Ki-67 allows one to determine the growth fraction in meningiomas in situ. From this point of view the simple immunohistological labeling with monoclonal antibody Ki-67 of cryostat sections might be superior to the timeconsuming cel1 cultures and be a more objective aid for evaluating therapies.
References MA, Blaauw G, Lamberts WJ, Mulder E. Presence 1. Blankenstein of progesterone receptors and absente of estrogen receptors in human intracranial meningioma cytosols. Eur J Cancer Clin Oncol 1983;19:365-70. 2. Burger PC, Shibata T, Kleihues P. The use of the monoclonal antibody Ki-67 in the identification of proliferating cells: application to surgical neuropathology. Am J Surg Pathol 1986;10:61 l7. 3. Burton DA. Studies of conditions and mechanisms of the diphenylamine reaction for the colorimetric determination of deoxyribonucleic acid. Biochem 1956;62:3 15-23. 4. Costero 1, Pomerat CM, Jackson IJ, Barroso-Moguel R, Chevez AZ. Tumors of the human nerveus system in tissue culture. 1. The cultivation and cytology of meningioma cells. J Nat1 Cancer Inst 1953;15:1319-39. F, Lennert K. Growth fractions in malig5. Gerdes J, Dallenbach nam non-Hodgkin’s lymphomas (NHL) as determined in situ with the monoclonal antibody Ki-67. Hematol Oncol 1984;2:365-71. of a mouse 6. Gerdes J, Schwab IJ, Lemke H, Stein H. Production monoclonal antibody reactive with a human nuclear antigen associated with cel1 proliferation. Int J Cancer 1983;3 1:13-20. 7. Horwitz KB, Mockus MB, Lessey BA. Variant T47D human breast cancer cells with high progesterone receptor Ievels despite estrogen and antiestrogen resistance. Cel1 1982;633-42. 8. Jay JR, MacLaughlin DT, Riley KR, Martuza RL.. Modulation of meningioma cel1 growth by sex steroid hormones in vitro. J Neurosurg 1985;62:757-62. 9. Lippman M, Bolan G, Huff K. The effect of estrogens and antiestrogens on hormone responsive human breast cancer in longterm tissue culture. Cancer Res 1976;4595-601. 10. Markwalder TM, Gerber HA, Waelti E, Schaffner T, Markwalder RV. Hormonotherapy of meningiomas with medroxyprogesterone acetate: immunohistochemical demonstration of the effect of MPA on growth fractions of meningioma cells using the monoclonal antibody Ki-67. Surg Neurol 1988;30:97-101.
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to
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