J Neurosurg 77:445-450, 1992
Insulin and insulin-like growth factors in central nervous system tumors Part V: Production of insulin-like growth factors I and II in vitro ROBERTA P. GLICK, M.D., TERRY G. UNTERMAN, M.D., MARY VAN DER WOUDE, F.I.B.A., AND LISA ZOLLNER BLAYDES~ B.S. Departments of Neurosurgery, Medicine, Anatomy and Cell Biology, and Medical Research, University of Illinois College of Medicine, The Hektoen Institute, Cook County Hospital, and Veterans Administration West Side Medical Center, Chicago, Illinois ~" The authors have previously reported the presence of insulin-like growth factor (IGF) receptors in central nervous system (CNS) tumors and the production of IGF's and their binding proteins by CNS tumors in situ. This study was designed to investigate whether CNS tumor cells are capable of autocrine secretion of IGF-I and IGF-II in vitro. Production of IGF's was studied by specific radioimmunoassay of tumor-cell-conditioned serum-free media from 34 CNS tumors: 12 gliomas, 12 meningiomas, and 10 miscellaneous tumors. Normal human serum and cerebrospinal fluid served as controls. Insulin-like growth factor I was detected in five of 12 meningiomas but in none of the gliomas studied. In contrast, IGF-II was detected in four of 12 gliomas and in six of 11 meningiomas studied. Four miscellaneous tumors produced IGF-I and/or IGF-II. These results suggest that CNS tumors differentially produce IGF-I and IGF-II in vitro. Preferential production of IGFs may be an important marker of the tumor-cell differentiation or malignancy and may be useful as a clinical diagnostic tool. These results add further support to the concept that IGF's may play a role in the regulation of the behavior of CNS tumors.
KEY WORDS central nervous system radioimmunoassay
UMOR cells are characterized by abnormalities in growth and metabolism. The autocrine theory of tumor development suggests that neoplastic cells may produce specific growth factors that bind to appropriate receptors on these cells, resulting in autostimulation of cell growth. ~z58 In a series of investigations we have considered the possibility that insulinlike growth factors (IGF's) may function as autocrine growth factors for central nervous system (CNS) tumors. Insulin-like growth factors are polypeptide mitogens that resemble proinsulin in structure, stimulate protein synthesis, stimulate the production of extracellular matrix in certain cells, and promote cellular proliferation and/or promote the differentiation of a variety of ceils, including oligodendroglia. 17.38,42,60 Two forms of IGF's have been purified from human serum. The first, IGF-I, also known as somatomedinC, is a basic peptide which is growth hormone-dependent and is thought to mediate many of the growth-
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9 tumor
insulin-like growth factor
promoting effects of growth hormone. 59 The second, IGF-II, is a neutral peptide that is produced in many tissues in fetal life and continues to be expressed in abundance in the adult brain. 45'46 Both insulin and IGF receptors have been identified and characterized in the normal adult and fetal brain, 33,34'47-49where they appear to play a role in normal neural growth and development. Insulin-like growth factors have also been shown to function as growth factors in a variety of tumors outside the CNS. 12,24,43,51"55 Based on these observations, we asked whether insulin and the IGF's are CNS tumor growth factors. We have previously demonstrated the differential expression of specific and functional insulin and IGF receptors in CNS tumors, and the effects of these growth factors on tumor-cell proliferation and differentiation, j9'22 In addition, we recently demonstrated the production of specific IGF binding proteins by CNS tumor cells in vitro) 6 To further characterize 445
R. P. Glick, el al. the role of IGF's as CNS tumor growth factors, we sought to determine whether CNS tumors are capable of autocrine secretion of these growth factors. We have recently established specific radioimmunoassays for IGF-I and IGF-II and demonstrated the presence of IGF's in CNS tumor cyst fluid consistent with production of these growth factors in situ. 2~ In the present study, we sought to confirm whether CNS tumor cells produce IGF's and utilized these specific radioimmunoassays to investigate the production of these growth factors in vitro; that is, into tumor-cellconditioned serum-free media. Materials and Methods
Cell Culture Tumor specimens obtained at surgery were embedded in paraffin and fixed for histopathology to confirm the tumor diagnosis, and a portion of the specimen was prepared for cell culture using the methods of Kornblith 32 and Kimmel, et al. 3~ A total of 34 CNS tumors were studied, including 12 gliomas (11 malignant and one benign), 12 meningiomas, and 10 miscellaneous tumors: two pituitary tumors, two medulloblastomas, one primitive neuroectodermal tumor (PNET), one acoustic neuroma, one cerebral ependymoma, one neuroblastoma, one giant-cell tumor, and one metastatic melanoma. Four of these tumors were obtained from pediatric patients, including one medulloblastoma, the one low-grade glioma, the PNET, and the neuroblastoma. For cell culture, freshly isolated tissue was placed in sterile tissue culture medium and minced by scalpel; the cells were then dissociated by passage through needles of decreasing diameter. Single-cell suspensions were plated in 75-sq cm tissue culture flasks at 37~ 5% CO2, and fed with a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium plus 10% (vol/vol) fetal calf serum, 10 U/ml of penicillin, and 100 ug/ml of streptomycin. The medium was changed 5 days after the cells were plated, and then every 3 days until cultures approached confluency. All studies were performed either with cells in primary culture or after a single passage with 0.25% (vol/vol) trypsin, as previously described.~9 In initial studies with malignant gliomas, cells also were seeded onto tissue culture slide chambers for immunohistochemical staining with antiserum against glial fibrillary acidic protein, as previously described; z9 approximately 80% of cells observed were positive for this antigen. Preparation of Conditioned Medium To prepare the conditioned medium, flasks of nearly confluent cells were washed twice with 10 ml Hank's balanced salt solution, then refed with 12 eu cm of serum-free medium which consisted of the same ingredients as the serum media minus the serum. Then 2 ml of medium was withdrawn immediately and stored at -70~ Aliquots of tumor-cell-conditioned serum-free
446
medium were obtained 24, 48, and 72 hours later. The samples were centrifuged for 10 minutes at 1200 G and stored at -70"C for subsequent analysis. Medium collected immediately after refeeding contained no immunoreactive IGF. Human Serum and Cerebrospinal Fluid For comparison, serum was collected and pooled from five healthy adult volunteers, aged 25 to 45 years, and aliquots were stored at -20~ Cerebrospinal fluid (CSF) was collected from two patients with indwelling intraventricular catheters for management of hydrocephalus and from one patient undergoing myelography for benign intervertebral disc disease. Iodination of Insulin-Like Growth Factors I and H Recombinant human IGF-I and IGF-II were iodinated by the lactoperoxidase method 54to a specific activity of 250 to 350 Ci/gm. Labeled IGF's were repurified by immunoaffinity chromatography as previously described 57 and stored at -20"C for use within 4 weeks. Extraction of Insulin-Like Growth Factors Levels of IGF-I and IGF-II in tumor-cell-conditioned medium were determined by radioimmunoassay after acid incubation and solid-phase extraction according to the methods of Kao, et al., 28 to remove IGF binding proteins. Preliminary studies demonstrated that extraction with a C18 solid-phase matrix did not completely remove binding proteins, which can interfere with radioimmunoassay, 7"9''~ and resulted in poor recovery of insulin-like growth factors. Accordingly, we developed a refined extraction procedure utilizing a C2 solidphase matrix. In this procedure, 500 tA of conditioned medium, CSF, or human serum was acidified overnight with 1500 ul of 1 M acetic acid at 4"C (total volume 2000 ul), then loaded onto C2 solid-phase cartridges which were rinsed first with acetic acid and then with 3 ml of 20% acetonitrile and 1% trifluoroacetic acid to remove residual binding proteins. Next, IGF's were eluted with 45% acetonitrile and 1% tfifluoroacetic acid; extracts were then dried by vacuum during centrifugation and reconstituted for either radioimmunoassay or binding protein studies. Western ligand blotting procedures were performed as previously described2~,57and confirmed that this extraction procedure removes IGF binding proteins from a variety of body fluids, including serum, CSF, and CNS tumor cyst fluid and from tumor-cell-conditioned medium. In contrast, recovery of [~2~I]-labeled IGF-I and IGF-II was 92% + 3% (mean _+ standard error of the mean) and 77% _+ 4%, respectively, for this procedure. Radioimmunoassay of Insulin-Like Growth Factors I and H Nonequilibrium radioimmunoassay of 1GF-I and IGF-II was performed according to the method of Furlanetto, et al. ,8 Recombinant human IGF-I and IGF-II were used as standards. For immunoassay, tumor-cellJ. Neurosurg. / Volume 77 / September, 1992
Insulin-like g r o w t h f a c t o r p r o d u c t i o n by C N S t u m o r cells
FIG. 1. Graphs showing the results of radioimmunoassays for insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II). B/B0 = ratio of binding in assay with a sample to maximum binding in assay without a sample. Left: Competitive binding curves in the immunoassay for IGF-I. Data are shown for human recombinant IGF-I and IGF-II standards and for C2 extract of conditioned medium from one meningioma. Right: Competitive binding curves in the immunoassay for IGF-II. Data are shown for IGF-I and IGF-II and for C2 extracts of conditioned medium from one malignant glioblastoma.
conditioned medium extract was incubated for 1 hour at 22"C with either 1:12,000 diluted anti-IGF-I antiserum or monoclonal antibody against IGF-II, before the addition of approximately 12 x 103 cpm of [~2~I]labeled IGF-I or IGF-II, and was incubated for 20 hours at 4*(7. Antigen-antibody complexes were precipitated by overnight incubation with a second antibody and normal rabbit or mouse serum, followed by the addition of polyethylene glycol (62.5 gm/liter) in NaC1 (9 gin/ liter) and 1500-G centrifugation at 4"C. Radioactivity was measured with a gamma counter.* All samples were assayed in duplicate, and at least three dilutions of each sample were tested. The sensitivity of the assay for IGF-I is 100 pg. The intra-assay coefficients of variation in this assay for standards containing 130, 200, and 440 pg are 6.5%, 3.9%, and 2.6%, respectively, and the interassay coefficients of variation for the same standards are 6.9%, 8.2%, and 4.3%, respectively. The sensitivity of the assay for IGF-II is 33 pg. The intra-assay coefficients of variation in the IGF-II assay for standards containing 200, 440, and 580 pg are 3.9%, 2.6%, and 2.1%, respectively, while the interassay coefficients of variation for the same standards are 8.6%, 6.2%, and 4.6%, respectively. Levels of IGF-I and IGF-II in normal human serum detected by these assays (388 and 564 ng/ml, respectively) were consistent with values reported by others. ~ Due to the limited supply and the expense of materials, the radioimmunoassay could not be performed at every time point for all 34 tumors. Therefore, we performed several time-course experiments to determine the optimum incubation time to assay for maximum IGF production. Samples of conditioned media taken * Gamma counter, Model 28063, manufactured by Micromedic, Huntsville, Alabama. J. Neurosurg. / Volume 77 / September, 1992
at 0-, 24-, 48-, and 72-hour intervals were studied in two meningiomas, two glioblastomas, and the PNET. Production of IGF-I and IGF-II was found to be timedependent and reached maximum at 72 hours. Therefore, radioimmunoassay was performed on 72-hour samples of the other 29 tumors. Results
The immunoassay for IGF-I was specific, since crossreactivity of IGF-II in the assay for IGF-I was less than 1%. Following C2 extraction, tumor-cell-conditioned medium competed in parallel with unlabeled recombinant IGF-I for binding to antiserum against IGF-I, further demonstrating that the removal of binding proteins was complete (Fig. 1 left). Similarly, the immunoassay for IGF-II was specific, since cross-reactivity of IGF-I in the assay for IGF-II also was less than 1%. In addition, extracted tumor-cell-conditioned medium competed in parallel with unlabeled recombinant IGFII for binding to specific monoclonal antibody (Fig. 1 right). Thus, it was possible to measure levels of both IGF-I and IGF-II in tumor-cell-conditioned medium. The numbers of tumors with immunoreactive IGF-I and IGF-II present in tumor-cell-conditioned medium are shown in Table 1. Insulin-like growth factor I was detected in the medium of five of the 12 meningiomas but not in those of the 12 gliomas studied. Insulin-like growth factor I was also detected in three miscellaneous pediatric tumors: one medulloblastoma, one neuroblastoma, and one PNET. Measurable levels of IGF-I in tumor-cell-conditioned media ranged from 0.22 to 7.3 ng/ml. In comparison, this assay detected 1.3 ng/ml of IGF-I in CSF. In contrast to IGF-I, IGF-II was detected in four of 12 gliomas (all malignant) and in six of 11 meningiomas studied; four meningiomas produced both factors. Insulin-like growth factor II was also detected in three 447
R. P. Glick, et al. TABLE 1 Tumors with measurable levels of lGF-1 and tGF-II in tumorcell-conditioned serum-free media from 34 CNS tumors*
TumorType
No. of Tumors Total WithIGF-I With1GF-II 12 5 6
meningioma glioma 12 0 ~t miscellaneous medulloblastoma 2 1 0 pituitary tumor 2 0 0 PNET 1 1 1 acousticneuroma 1 0 0 neuroblastoma 1 1 1 ependymoma 1 0 0 giant-celltumor 1 0 1 malignantmelanoma 1 0 0 *Measurable level = 0.2 ng/ml or greater. IGF = insulin-like growth factor;CNS = central nervoussystem; PNET = primitive neuroectodermaltumor.
miscellaneous tumors: one PNET, one neuroblastoma, and one giant-cell tumor. Measurable levels of IGF-II ranged from 0.27 to 4.53 ng/ml. In comparison, this assay detected 3.3 ng/ml of IGF-II in CSF. Discussion
In the present study, we sought to further characterize the role of the IGF's as CNS tumor growth factors and to determine whether CNS tumor cells are capable of autocrine secretion of these growth factors in vitro. Specific radioimmunoassay detected the presence of IGF-I in the conditioned media of many of the meningiomas but in none of the gliomas studied. In contrast, IGF-II was detected in one-third of the gliomas and one-half of the meningiomas studied. Media collected immediately after rinsing cells did not contain immunoreactive IGF's, supporting the concept that IGF-I and IGF-II were produced by the tumor cells. Furthermore, our time-course experiments demonstrated that IGF levels rose accordingly from 0 to 72 hours, also supporting the in vitro production of IGF's. In order to minimize the possibility that the biological properties of CNS tumor cells might be altered during cell culture, and to avoid overgrowth with contaminating fibroblasts, only cells in fresh primary culture or those obtained after a single passage were used for these studies. Indeed, meningiomas and gliomas appear to produce IGF-I and IGF-II differentially in vitro. Increasing evidence suggests that IGF's play a role in the normal growth and differentiation of the CNS. 1,47,4S Specific receptors for IGF-I and IGF-II have been identified in normal human and rat brain tissue. 34'57Studies in cell culture have demonstrated that IGF-I stimulates the growth of fetal brain cells,33increases the expression of cytoskeletal proteins and enhances neurite formation, 39 and promotes the proliferation and differentiation of normal rat oligodendroglia cells.3s These findings support the concept that these growth factors may 448
play a role in the growth and development of both neuronal and glial components of the nervous system. Evidence from a number of laboratories supports the concept that the IGF's may function as autocrine growth factors in a variety of tumors outside the CNS. Several such tumors (fibrosarcoma, Wilms' tumor, lung cancer, breast cancer, and colon cancer) secrete IGFlike peptides, 3'~2'24'2~'43demonstrate an increased abundance of IGF messenger ribonucleic acid (mRNA), 5~'$5 or are stimulated by IGF's in vitroY Recent studies have demonstrated that neuroblastoma cells produce IGF-I and IGF-II peptides, which function in an autocrine manner to promote the growth of these cells. ~5'53 Similarly, production of IGF-I by the rat C6 glioma cell line has recently been reportedfl9 In an early study, Sandberg, et al., 46 demonstrated the presence of mRNA's for IGF's in human gliomas and, more recently, Lichtor, et at., 35reported IGF-n mRNA in three meningiomas. Our present demonstration of IGF production in meningiomas and gliomas confirms the actual biosynthesis and secretion of this growth peptide by CNS tumor cells as seen in other autocrine loops?2 The observation that rneningiomas differentially produce IGF-I is of particular interest and may help to explain several clinical features of these tumors. Early studies identified IGF's as growth-hormone-dependent polypeptides, which stimulate growth activity in cartilage. J7.60More recent investigations have demonstrated that IGF's also stimulate bone formation by osteoblasts? It is interesting to speculate that local production of IGF's may contribute to the thickening of cortical bone and hyperostosis associated with certain meningiomas. Furthermore, since IGF's promote the production and accumulation of extracellular matrix proteinsf '6~ it is also possible that IGF's may play a role in the development of the prominent stromal component present in many meningiomas. We have previously demonstrated the in situ localization of IGF binding proteins in the extracellular matrix in meningiomas by immunohistochemistry,56 supporting this hypothesis. Finally, IGF's may well mediate some of the effects that sex steroids appear to exert on meningiomas. Studies in breast cancer cells have shown that estrogens induce both cellular proliferation and IGF-I secretion, while antiestrogens and glucocorticoids have the opposite effects. 2~ Sex steroids may also influence the production of certain IGF binding proteins by breast can. cer cells.25In addition, the presence of IGF receptors in breast cancer has been correlated positively with both estrogen and progesterone receptor content. 4~ We and others have previously demonstrated the presence of estrogen and progesterone receptors in meningiomas, 4'~4'2~ and both clinical and experimental studies suggest that these sex steroids may influence the growth of meningiomas. 2"27"3~'36'4~176 In addition, we reported the presence of IGF receptors in meningiomas ~9 and the fact that meningiomas secrete IGF binding proteins including IGF binding protein-1, a 30J. Neurosurg. / Volume 77/September. 1992
Insulin-like growth factor production by CNS tumor cells kD protein which is regulated by sex steroid hormones in other tissues 6'8 and has been shown to modulate the effects of IGF's on target tissues. '3''6 Thus, it is interesting to speculate that sex steroid hormones may influence the clinical behavior of meningiomas by regulating the production of IGF's and/or specific IGF binding proteins. Conclusions In summary, the IGF's are thought to be important in normal neuronal and glial growth and differentiation. The results of the present study demonstrate that human CNS tumors produce immunoreactive IGF peptides in vitro. Taken together with our previous reports regarding the differential expression of IGF receptors in CNS tumors, the stimulation of proliferation and differentiation of tumor cells by IGF's, and the production of IGF's and their binding proteins in situ and in vitro, these results provide further support to the concept that IGF's may play an important role in regulating the growth and behavior of CNS tumors. Acknowledgments We thank Venugobal Thongada and Mark Para for their technical assistance, Drs. Harry Yang and George Pappas for the performance of immunohistochemistry, Mrs. Ernestine Daniels for her preparation of the manuscript, and Dr. James Ausman for his critical review of the manuscript. References 1. Baskin DG, Wilcox BJ, Figlewicz DP, et al: Insulin and insulin-like growth factors in the CNS. Trends Neurosci 11:107-111, 1988 2. Bickerstaff ER, Small JM, Guest IA: The relapsing course of certain meningiomas in relation to pregnancy and menstruation. J Neural Neurosurg Psychiatry 21:89-91, 1958 3. Blatt J, White C, Dienes S, et al: Production of an insulinlike growth factor by osteosarcoma. Biochem Biophys Res Common 123:373-376, 1984 4. Cahill DW, Bashirelahi N, Solomon LW, et at: Estrogen and progesterone receptors in meningiomas. J Neurosnrg 60:985-993, 1984 5. Canalis E, McCarthy TU Centrella M: The role of growth factors in skeletal remodeling. Endocrinol Metab Clln North Am 18:903-918, 1989 6. Clemmons DR, Camacho-Hubner C, Coronado E, et al: Insulin-like growth factor binding protein secretion by breast carcinoma cell lines: correlation with estrogen receptor status. Endocrinology 127:2679-2686, 1990 7. Clemmons DR, Van Wyk J J, Ridgway EC, et at: Evaluation of acromegaly by radioimmunoassay of somatomedin-C. N Engl J Med 301:1138-1142, 1979 8. Conover CA, Liu F, Powell D, et al: Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation. J Clin Invest 83:852-859, 1989 9. Daughaday WH, Kapadia M, Mariz I: Serum somatomedin binding proteins: physiologic significance and intereference in radioligand assay. J Lab Clin Med 109: 355-363, 1987 10. Daughaday WH, Rotwein P: Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene J. Neurosurg. / Volume 77 / September, 1992
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Manuscript received October 1, 1991. Accepted in final form March 3, 1992. This work was supported by grants from the Illinois Cancer Council to Dr. Glick, the Department of Veteran Affairs to Dr. Unterman, and National Institutes of Health First Award R29-DK-41430 to Dr. Unterman. Dr. Glick is the recipient of a Clinical Investigator Award from the American Association of Neurological Surgeons. Address reprint requests to: Roberta P. Gliek, M.D., Department of Neurosurgery, Cook County Hospital, 1835 West Harrison, Chicago, Illinois 60612.
J. Neurosurg. / Volume 77/September, 1992
Insulin and insulin-like growth factors in central nervous system tumors Part V: Production of insulin-like growth factors I and II in vitro ROBERTA P. GLICK, M.D., TERRY G. UNTERMAN, M.D., MARY VAN DER WOUDE, F.I.B.A., AND LISA ZOLLNER BLAYDES~ B.S. Departments of Neurosurgery, Medicine, Anatomy and Cell Biology, and Medical Research, University of Illinois College of Medicine, The Hektoen Institute, Cook County Hospital, and Veterans Administration West Side Medical Center, Chicago, Illinois ~" The authors have previously reported the presence of insulin-like growth factor (IGF) receptors in central nervous system (CNS) tumors and the production of IGF's and their binding proteins by CNS tumors in situ. This study was designed to investigate whether CNS tumor cells are capable of autocrine secretion of IGF-I and IGF-II in vitro. Production of IGF's was studied by specific radioimmunoassay of tumor-cell-conditioned serum-free media from 34 CNS tumors: 12 gliomas, 12 meningiomas, and 10 miscellaneous tumors. Normal human serum and cerebrospinal fluid served as controls. Insulin-like growth factor I was detected in five of 12 meningiomas but in none of the gliomas studied. In contrast, IGF-II was detected in four of 12 gliomas and in six of 11 meningiomas studied. Four miscellaneous tumors produced IGF-I and/or IGF-II. These results suggest that CNS tumors differentially produce IGF-I and IGF-II in vitro. Preferential production of IGFs may be an important marker of the tumor-cell differentiation or malignancy and may be useful as a clinical diagnostic tool. These results add further support to the concept that IGF's may play a role in the regulation of the behavior of CNS tumors.
KEY WORDS central nervous system radioimmunoassay
UMOR cells are characterized by abnormalities in growth and metabolism. The autocrine theory of tumor development suggests that neoplastic cells may produce specific growth factors that bind to appropriate receptors on these cells, resulting in autostimulation of cell growth. ~z58 In a series of investigations we have considered the possibility that insulinlike growth factors (IGF's) may function as autocrine growth factors for central nervous system (CNS) tumors. Insulin-like growth factors are polypeptide mitogens that resemble proinsulin in structure, stimulate protein synthesis, stimulate the production of extracellular matrix in certain cells, and promote cellular proliferation and/or promote the differentiation of a variety of ceils, including oligodendroglia. 17.38,42,60 Two forms of IGF's have been purified from human serum. The first, IGF-I, also known as somatomedinC, is a basic peptide which is growth hormone-dependent and is thought to mediate many of the growth-
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insulin-like growth factor
promoting effects of growth hormone. 59 The second, IGF-II, is a neutral peptide that is produced in many tissues in fetal life and continues to be expressed in abundance in the adult brain. 45'46 Both insulin and IGF receptors have been identified and characterized in the normal adult and fetal brain, 33,34'47-49where they appear to play a role in normal neural growth and development. Insulin-like growth factors have also been shown to function as growth factors in a variety of tumors outside the CNS. 12,24,43,51"55 Based on these observations, we asked whether insulin and the IGF's are CNS tumor growth factors. We have previously demonstrated the differential expression of specific and functional insulin and IGF receptors in CNS tumors, and the effects of these growth factors on tumor-cell proliferation and differentiation, j9'22 In addition, we recently demonstrated the production of specific IGF binding proteins by CNS tumor cells in vitro) 6 To further characterize 445
R. P. Glick, el al. the role of IGF's as CNS tumor growth factors, we sought to determine whether CNS tumors are capable of autocrine secretion of these growth factors. We have recently established specific radioimmunoassays for IGF-I and IGF-II and demonstrated the presence of IGF's in CNS tumor cyst fluid consistent with production of these growth factors in situ. 2~ In the present study, we sought to confirm whether CNS tumor cells produce IGF's and utilized these specific radioimmunoassays to investigate the production of these growth factors in vitro; that is, into tumor-cellconditioned serum-free media. Materials and Methods
Cell Culture Tumor specimens obtained at surgery were embedded in paraffin and fixed for histopathology to confirm the tumor diagnosis, and a portion of the specimen was prepared for cell culture using the methods of Kornblith 32 and Kimmel, et al. 3~ A total of 34 CNS tumors were studied, including 12 gliomas (11 malignant and one benign), 12 meningiomas, and 10 miscellaneous tumors: two pituitary tumors, two medulloblastomas, one primitive neuroectodermal tumor (PNET), one acoustic neuroma, one cerebral ependymoma, one neuroblastoma, one giant-cell tumor, and one metastatic melanoma. Four of these tumors were obtained from pediatric patients, including one medulloblastoma, the one low-grade glioma, the PNET, and the neuroblastoma. For cell culture, freshly isolated tissue was placed in sterile tissue culture medium and minced by scalpel; the cells were then dissociated by passage through needles of decreasing diameter. Single-cell suspensions were plated in 75-sq cm tissue culture flasks at 37~ 5% CO2, and fed with a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium plus 10% (vol/vol) fetal calf serum, 10 U/ml of penicillin, and 100 ug/ml of streptomycin. The medium was changed 5 days after the cells were plated, and then every 3 days until cultures approached confluency. All studies were performed either with cells in primary culture or after a single passage with 0.25% (vol/vol) trypsin, as previously described.~9 In initial studies with malignant gliomas, cells also were seeded onto tissue culture slide chambers for immunohistochemical staining with antiserum against glial fibrillary acidic protein, as previously described; z9 approximately 80% of cells observed were positive for this antigen. Preparation of Conditioned Medium To prepare the conditioned medium, flasks of nearly confluent cells were washed twice with 10 ml Hank's balanced salt solution, then refed with 12 eu cm of serum-free medium which consisted of the same ingredients as the serum media minus the serum. Then 2 ml of medium was withdrawn immediately and stored at -70~ Aliquots of tumor-cell-conditioned serum-free
446
medium were obtained 24, 48, and 72 hours later. The samples were centrifuged for 10 minutes at 1200 G and stored at -70"C for subsequent analysis. Medium collected immediately after refeeding contained no immunoreactive IGF. Human Serum and Cerebrospinal Fluid For comparison, serum was collected and pooled from five healthy adult volunteers, aged 25 to 45 years, and aliquots were stored at -20~ Cerebrospinal fluid (CSF) was collected from two patients with indwelling intraventricular catheters for management of hydrocephalus and from one patient undergoing myelography for benign intervertebral disc disease. Iodination of Insulin-Like Growth Factors I and H Recombinant human IGF-I and IGF-II were iodinated by the lactoperoxidase method 54to a specific activity of 250 to 350 Ci/gm. Labeled IGF's were repurified by immunoaffinity chromatography as previously described 57 and stored at -20"C for use within 4 weeks. Extraction of Insulin-Like Growth Factors Levels of IGF-I and IGF-II in tumor-cell-conditioned medium were determined by radioimmunoassay after acid incubation and solid-phase extraction according to the methods of Kao, et al., 28 to remove IGF binding proteins. Preliminary studies demonstrated that extraction with a C18 solid-phase matrix did not completely remove binding proteins, which can interfere with radioimmunoassay, 7"9''~ and resulted in poor recovery of insulin-like growth factors. Accordingly, we developed a refined extraction procedure utilizing a C2 solidphase matrix. In this procedure, 500 tA of conditioned medium, CSF, or human serum was acidified overnight with 1500 ul of 1 M acetic acid at 4"C (total volume 2000 ul), then loaded onto C2 solid-phase cartridges which were rinsed first with acetic acid and then with 3 ml of 20% acetonitrile and 1% trifluoroacetic acid to remove residual binding proteins. Next, IGF's were eluted with 45% acetonitrile and 1% tfifluoroacetic acid; extracts were then dried by vacuum during centrifugation and reconstituted for either radioimmunoassay or binding protein studies. Western ligand blotting procedures were performed as previously described2~,57and confirmed that this extraction procedure removes IGF binding proteins from a variety of body fluids, including serum, CSF, and CNS tumor cyst fluid and from tumor-cell-conditioned medium. In contrast, recovery of [~2~I]-labeled IGF-I and IGF-II was 92% + 3% (mean _+ standard error of the mean) and 77% _+ 4%, respectively, for this procedure. Radioimmunoassay of Insulin-Like Growth Factors I and H Nonequilibrium radioimmunoassay of 1GF-I and IGF-II was performed according to the method of Furlanetto, et al. ,8 Recombinant human IGF-I and IGF-II were used as standards. For immunoassay, tumor-cellJ. Neurosurg. / Volume 77 / September, 1992
Insulin-like g r o w t h f a c t o r p r o d u c t i o n by C N S t u m o r cells
FIG. 1. Graphs showing the results of radioimmunoassays for insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II). B/B0 = ratio of binding in assay with a sample to maximum binding in assay without a sample. Left: Competitive binding curves in the immunoassay for IGF-I. Data are shown for human recombinant IGF-I and IGF-II standards and for C2 extract of conditioned medium from one meningioma. Right: Competitive binding curves in the immunoassay for IGF-II. Data are shown for IGF-I and IGF-II and for C2 extracts of conditioned medium from one malignant glioblastoma.
conditioned medium extract was incubated for 1 hour at 22"C with either 1:12,000 diluted anti-IGF-I antiserum or monoclonal antibody against IGF-II, before the addition of approximately 12 x 103 cpm of [~2~I]labeled IGF-I or IGF-II, and was incubated for 20 hours at 4*(7. Antigen-antibody complexes were precipitated by overnight incubation with a second antibody and normal rabbit or mouse serum, followed by the addition of polyethylene glycol (62.5 gm/liter) in NaC1 (9 gin/ liter) and 1500-G centrifugation at 4"C. Radioactivity was measured with a gamma counter.* All samples were assayed in duplicate, and at least three dilutions of each sample were tested. The sensitivity of the assay for IGF-I is 100 pg. The intra-assay coefficients of variation in this assay for standards containing 130, 200, and 440 pg are 6.5%, 3.9%, and 2.6%, respectively, and the interassay coefficients of variation for the same standards are 6.9%, 8.2%, and 4.3%, respectively. The sensitivity of the assay for IGF-II is 33 pg. The intra-assay coefficients of variation in the IGF-II assay for standards containing 200, 440, and 580 pg are 3.9%, 2.6%, and 2.1%, respectively, while the interassay coefficients of variation for the same standards are 8.6%, 6.2%, and 4.6%, respectively. Levels of IGF-I and IGF-II in normal human serum detected by these assays (388 and 564 ng/ml, respectively) were consistent with values reported by others. ~ Due to the limited supply and the expense of materials, the radioimmunoassay could not be performed at every time point for all 34 tumors. Therefore, we performed several time-course experiments to determine the optimum incubation time to assay for maximum IGF production. Samples of conditioned media taken * Gamma counter, Model 28063, manufactured by Micromedic, Huntsville, Alabama. J. Neurosurg. / Volume 77 / September, 1992
at 0-, 24-, 48-, and 72-hour intervals were studied in two meningiomas, two glioblastomas, and the PNET. Production of IGF-I and IGF-II was found to be timedependent and reached maximum at 72 hours. Therefore, radioimmunoassay was performed on 72-hour samples of the other 29 tumors. Results
The immunoassay for IGF-I was specific, since crossreactivity of IGF-II in the assay for IGF-I was less than 1%. Following C2 extraction, tumor-cell-conditioned medium competed in parallel with unlabeled recombinant IGF-I for binding to antiserum against IGF-I, further demonstrating that the removal of binding proteins was complete (Fig. 1 left). Similarly, the immunoassay for IGF-II was specific, since cross-reactivity of IGF-I in the assay for IGF-II also was less than 1%. In addition, extracted tumor-cell-conditioned medium competed in parallel with unlabeled recombinant IGFII for binding to specific monoclonal antibody (Fig. 1 right). Thus, it was possible to measure levels of both IGF-I and IGF-II in tumor-cell-conditioned medium. The numbers of tumors with immunoreactive IGF-I and IGF-II present in tumor-cell-conditioned medium are shown in Table 1. Insulin-like growth factor I was detected in the medium of five of the 12 meningiomas but not in those of the 12 gliomas studied. Insulin-like growth factor I was also detected in three miscellaneous pediatric tumors: one medulloblastoma, one neuroblastoma, and one PNET. Measurable levels of IGF-I in tumor-cell-conditioned media ranged from 0.22 to 7.3 ng/ml. In comparison, this assay detected 1.3 ng/ml of IGF-I in CSF. In contrast to IGF-I, IGF-II was detected in four of 12 gliomas (all malignant) and in six of 11 meningiomas studied; four meningiomas produced both factors. Insulin-like growth factor II was also detected in three 447
R. P. Glick, et al. TABLE 1 Tumors with measurable levels of lGF-1 and tGF-II in tumorcell-conditioned serum-free media from 34 CNS tumors*
TumorType
No. of Tumors Total WithIGF-I With1GF-II 12 5 6
meningioma glioma 12 0 ~t miscellaneous medulloblastoma 2 1 0 pituitary tumor 2 0 0 PNET 1 1 1 acousticneuroma 1 0 0 neuroblastoma 1 1 1 ependymoma 1 0 0 giant-celltumor 1 0 1 malignantmelanoma 1 0 0 *Measurable level = 0.2 ng/ml or greater. IGF = insulin-like growth factor;CNS = central nervoussystem; PNET = primitive neuroectodermaltumor.
miscellaneous tumors: one PNET, one neuroblastoma, and one giant-cell tumor. Measurable levels of IGF-II ranged from 0.27 to 4.53 ng/ml. In comparison, this assay detected 3.3 ng/ml of IGF-II in CSF. Discussion
In the present study, we sought to further characterize the role of the IGF's as CNS tumor growth factors and to determine whether CNS tumor cells are capable of autocrine secretion of these growth factors in vitro. Specific radioimmunoassay detected the presence of IGF-I in the conditioned media of many of the meningiomas but in none of the gliomas studied. In contrast, IGF-II was detected in one-third of the gliomas and one-half of the meningiomas studied. Media collected immediately after rinsing cells did not contain immunoreactive IGF's, supporting the concept that IGF-I and IGF-II were produced by the tumor cells. Furthermore, our time-course experiments demonstrated that IGF levels rose accordingly from 0 to 72 hours, also supporting the in vitro production of IGF's. In order to minimize the possibility that the biological properties of CNS tumor cells might be altered during cell culture, and to avoid overgrowth with contaminating fibroblasts, only cells in fresh primary culture or those obtained after a single passage were used for these studies. Indeed, meningiomas and gliomas appear to produce IGF-I and IGF-II differentially in vitro. Increasing evidence suggests that IGF's play a role in the normal growth and differentiation of the CNS. 1,47,4S Specific receptors for IGF-I and IGF-II have been identified in normal human and rat brain tissue. 34'57Studies in cell culture have demonstrated that IGF-I stimulates the growth of fetal brain cells,33increases the expression of cytoskeletal proteins and enhances neurite formation, 39 and promotes the proliferation and differentiation of normal rat oligodendroglia cells.3s These findings support the concept that these growth factors may 448
play a role in the growth and development of both neuronal and glial components of the nervous system. Evidence from a number of laboratories supports the concept that the IGF's may function as autocrine growth factors in a variety of tumors outside the CNS. Several such tumors (fibrosarcoma, Wilms' tumor, lung cancer, breast cancer, and colon cancer) secrete IGFlike peptides, 3'~2'24'2~'43demonstrate an increased abundance of IGF messenger ribonucleic acid (mRNA), 5~'$5 or are stimulated by IGF's in vitroY Recent studies have demonstrated that neuroblastoma cells produce IGF-I and IGF-II peptides, which function in an autocrine manner to promote the growth of these cells. ~5'53 Similarly, production of IGF-I by the rat C6 glioma cell line has recently been reportedfl9 In an early study, Sandberg, et al., 46 demonstrated the presence of mRNA's for IGF's in human gliomas and, more recently, Lichtor, et at., 35reported IGF-n mRNA in three meningiomas. Our present demonstration of IGF production in meningiomas and gliomas confirms the actual biosynthesis and secretion of this growth peptide by CNS tumor cells as seen in other autocrine loops?2 The observation that rneningiomas differentially produce IGF-I is of particular interest and may help to explain several clinical features of these tumors. Early studies identified IGF's as growth-hormone-dependent polypeptides, which stimulate growth activity in cartilage. J7.60More recent investigations have demonstrated that IGF's also stimulate bone formation by osteoblasts? It is interesting to speculate that local production of IGF's may contribute to the thickening of cortical bone and hyperostosis associated with certain meningiomas. Furthermore, since IGF's promote the production and accumulation of extracellular matrix proteinsf '6~ it is also possible that IGF's may play a role in the development of the prominent stromal component present in many meningiomas. We have previously demonstrated the in situ localization of IGF binding proteins in the extracellular matrix in meningiomas by immunohistochemistry,56 supporting this hypothesis. Finally, IGF's may well mediate some of the effects that sex steroids appear to exert on meningiomas. Studies in breast cancer cells have shown that estrogens induce both cellular proliferation and IGF-I secretion, while antiestrogens and glucocorticoids have the opposite effects. 2~ Sex steroids may also influence the production of certain IGF binding proteins by breast can. cer cells.25In addition, the presence of IGF receptors in breast cancer has been correlated positively with both estrogen and progesterone receptor content. 4~ We and others have previously demonstrated the presence of estrogen and progesterone receptors in meningiomas, 4'~4'2~ and both clinical and experimental studies suggest that these sex steroids may influence the growth of meningiomas. 2"27"3~'36'4~176 In addition, we reported the presence of IGF receptors in meningiomas ~9 and the fact that meningiomas secrete IGF binding proteins including IGF binding protein-1, a 30J. Neurosurg. / Volume 77/September. 1992
Insulin-like growth factor production by CNS tumor cells kD protein which is regulated by sex steroid hormones in other tissues 6'8 and has been shown to modulate the effects of IGF's on target tissues. '3''6 Thus, it is interesting to speculate that sex steroid hormones may influence the clinical behavior of meningiomas by regulating the production of IGF's and/or specific IGF binding proteins. Conclusions In summary, the IGF's are thought to be important in normal neuronal and glial growth and differentiation. The results of the present study demonstrate that human CNS tumors produce immunoreactive IGF peptides in vitro. Taken together with our previous reports regarding the differential expression of IGF receptors in CNS tumors, the stimulation of proliferation and differentiation of tumor cells by IGF's, and the production of IGF's and their binding proteins in situ and in vitro, these results provide further support to the concept that IGF's may play an important role in regulating the growth and behavior of CNS tumors. Acknowledgments We thank Venugobal Thongada and Mark Para for their technical assistance, Drs. Harry Yang and George Pappas for the performance of immunohistochemistry, Mrs. Ernestine Daniels for her preparation of the manuscript, and Dr. James Ausman for his critical review of the manuscript. References 1. Baskin DG, Wilcox BJ, Figlewicz DP, et al: Insulin and insulin-like growth factors in the CNS. Trends Neurosci 11:107-111, 1988 2. Bickerstaff ER, Small JM, Guest IA: The relapsing course of certain meningiomas in relation to pregnancy and menstruation. J Neural Neurosurg Psychiatry 21:89-91, 1958 3. Blatt J, White C, Dienes S, et al: Production of an insulinlike growth factor by osteosarcoma. Biochem Biophys Res Common 123:373-376, 1984 4. Cahill DW, Bashirelahi N, Solomon LW, et at: Estrogen and progesterone receptors in meningiomas. J Neurosnrg 60:985-993, 1984 5. Canalis E, McCarthy TU Centrella M: The role of growth factors in skeletal remodeling. Endocrinol Metab Clln North Am 18:903-918, 1989 6. Clemmons DR, Camacho-Hubner C, Coronado E, et al: Insulin-like growth factor binding protein secretion by breast carcinoma cell lines: correlation with estrogen receptor status. Endocrinology 127:2679-2686, 1990 7. Clemmons DR, Van Wyk J J, Ridgway EC, et at: Evaluation of acromegaly by radioimmunoassay of somatomedin-C. N Engl J Med 301:1138-1142, 1979 8. Conover CA, Liu F, Powell D, et al: Insulin-like growth factor binding proteins from cultured human fibroblasts. Characterization and hormonal regulation. J Clin Invest 83:852-859, 1989 9. Daughaday WH, Kapadia M, Mariz I: Serum somatomedin binding proteins: physiologic significance and intereference in radioligand assay. J Lab Clin Med 109: 355-363, 1987 10. Daughaday WH, Rotwein P: Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene J. Neurosurg. / Volume 77 / September, 1992
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Manuscript received October 1, 1991. Accepted in final form March 3, 1992. This work was supported by grants from the Illinois Cancer Council to Dr. Glick, the Department of Veteran Affairs to Dr. Unterman, and National Institutes of Health First Award R29-DK-41430 to Dr. Unterman. Dr. Glick is the recipient of a Clinical Investigator Award from the American Association of Neurological Surgeons. Address reprint requests to: Roberta P. Gliek, M.D., Department of Neurosurgery, Cook County Hospital, 1835 West Harrison, Chicago, Illinois 60612.
J. Neurosurg. / Volume 77/September, 1992
