Endothelial growth factor present in tissue culture of CNS tumors PATRICK J. KELLY, M.D., ROBERT L. SUDDITH, PH.D., HAROLD T. HUTCHISON, PH.D., KAREN WERRBACH~ AND BERNARDHABER, PH.D. Division of Neurosurgery, Department of Surgery, The University of Texas Medical Branch and The Marine Biomedical Institute, Galveston, Texas v' Human endothelial cells obtained from postpartum umbilical veins and placed in primary tissue cultures were treated with media from cultures of human and experimental central nervous system tumors. Endothelial proliferation was determined by the uptake of 3H thymidine with autoradiography and represented as the thymidine labeling index (TI), which is the proportion of 8H thymidine-labeled endothelial cells to total number of cells counted. There was a marked increase in the TI when tumor-conditioned medium was added to endothelial cultures (range 28.7% to 98.3%) when compared to controls (2.1%) and endothelium with conditioned media from fibroblasts (4.5%). This study demonstrates the presence of a chemical substance produced by tumor cells which results in endothelial proliferation. The system described provides a useful assay technique for the further characterization of this endothelial growth factor. KEYWORDS tissue culture
9 CNStumors 9 endothelium 9 autoradiography
HEendothelial proliferation and neovascularization associated with CNS neoplasms is well recognized.8,",17-~9 A specific chemical factor produced by tumor cells which induces neovascularization and more specifically endothelial mitotic activity has been suggested by experiments in which hamster cheek pouch vessel proliferation was induced by a tumor contained in a millipore filter?, ~5 Folkman8 evaluated angiogenic activity of various experimental and human tumors upon vessels in rat tissue and found that extracts of those tumors resulted in neovascularization.',~~This was also shown by injecting tumor extracts intraocularly and
T
342
9 angiogenesis
9
evaluating neovascularization in the iris? s Endothelial uptake of 8H thymidine and increase in mitotic activity in vivo induced by tumor angiogenesis factor (TAF) has been studied in other experiments? Although these assay systems for TAF axe workable, they are also cumbersome and subject to other variables peculiar to in vivo bioassay techniques. The purpose of our study was to continue this work in a tissue culture assay program in which tumor specimens and endothelium would be grown in separate cultures; the effect of media in which tumors had been growing (conditioned media) or fractions thereof on the endothelial cultures could be evaluated by the incorporation of 8H J. Neurosurg. / Volume 44 / March, 1976
E n d o t h e l i a l g r o w t h f a c t o r in t u m o r tissue c u l t u r e s thymidine? ,lx,~~ We are reporting our results of medium for the initial 24 hours following with the first in vitro assay system for the plating. These were then supplemented with study of tumor trophic factors on endothelial either 1 ml of conditioned medium or 1 ml of fresh Flo containing 20% fetal calf serum. proliferation. The fibroblasts were larger than the astroMethods and Materials cytoma cells, and were therefore plated at 80% of the cell density used for the astroCell Culture cytoma (1.0 • llY astrocytoma cells/10 ml of Endothelial Cells. Endothelial cells were media). Each unit volume of medium was obtained from the umbilical vein of human conditioned by approximately the same umbilical cords by collagenase digestion number of cells. employing the method described by Jaffe, et al.le These were then cultured on glass cover A u toradiography slips in six well plates* in Flo plus 20% fetal Twenty-four hours after the addition of the calf serum and 1.5% CO~. Handled as conditioned media, 0.5 uCi of SH thymidine primary cultures the endothelial cells were the was added to each well. Seventy-two hours vehicle for our assay system; for each endolater, the cover slips were rinsed with Hanks thelial sample we prepared one control and balanced salt solution, and then fixed in 3:1 five experimental cultures. fixative (ethanol:acetic acid) for 10 minutes. Human Tumors. Surgical specimens were Subsequently the slides were coated with diced into l-mm cubes and placed aseptically Kodak NTB-2 nuclear tracking emulsion and in Flo medium and 20% fetal calf serum and exposed for 5 days. After development the transferred to Falcon plastic tissue culture cover slips were stained with either toluidine flasks as lying drop cultures. Twenty-four blue or Giemsa. hours later fresh F~o medium was added to the flask and the tumor cells were allowed to Assay Method grow for 1 week. The tumor type was determined by sections of the original surgical Slides of 8H thymidine labeled endothelia specimen stained with hematoxylin and eosin. were observed under 10• magnification, and Experimental Clonal Tumors. Three dif- the number of cells labeled with SH thyferent clonal lines of C8 rat astrocytoma cells2 midine as a proportion of the total number of were grown in F~o medium containing 10% cells for that field was recorded from 10 ranfetal calf serum.t Fibroblasts were obtained domly selected fields on each slide. The ratio from a biopsy specimen of a human scar and of the cells labeled compared to the total maintained in primary culture with Flo and number of cells counted in each field is 10% fetal calf serum. The fibroblasts used in termed the thymidine labeling index (TI). To these studies had a doubling time of 29 hours, eliminate bias, the worker reading the slide as compared to 20 hours for the C6 astro- was unaware of the group that each particular cytoma. slide represented. Labeled cells are those Conditioned Media. One ml of medium which have more than 20 silver grains from a growing cell line (conditioned localized over the nucleus. The labeling index medium) filtered through a 0.22 p millipore was computed as an average of the 10 low filter was added to 2 ml of F~o and 20% fetal power, random fields for each slide, and these calf serum already in the culture well. One ml data were tabulated for each of the groups of fresh medium was added to one of the six given in Table 1. wells as a control. Thus, all control and exResults perimental cultures contained a total of 2 ml As shown in Table 1 and Fig. 1, there is a dramatic increase in endothelial incor*Well plates manufactured by Linbro Plastics, poration of SH thymidine when conditioned New Haven, Connecticut. ?Rat astrocytoma cells were obtained from S. medium from human or experimental tumors M. Pfeiffer (University of Connecticut), J. De from the central nervous system (CNS) is Veliss (University of California at Los Angeles), added to primary endothelial cultures. It has and from the American Type Culture Collection been our consistent observation that the addi(CCLI07). tion of conditioned medium to endothelial J. Neurosurg. / Volume 44 / March, 1976
343
P. J. Kelly, et al.
FIG. 1. Graphic display of thymidine-labeling index of endothelial culture with control and conditioned media from human and experimental CNS tumors (see also Table 1).
FIG. 2. Autoradiograph showing endothelial cells after the addition of conditioned media from an angioblastic meningioma. Note high degree of thymidine uptake and mitotic figures. 344
culture also produces morphological alterations as seen by light microscopy. The striking increase of thymidine labeling is shown in Fig. 2 where almost every cell is labeled and mitotic figures may be seen. A wide range in TI was noted in the endothelium cultures treated with human tumorconditioned medium, ranging from a Grade III astrocytoma TI of 30.1% to a 98.3% TI resulting from the addition of conditioned medium from an angioblastic meningioma. This tumor had a prominent stain arteriographically and was highly vascular at surgery. It is interesting that the degree of endothelial stimulation does not reflect a tumor's arteriographic staining prominence as demonstrated by the 32.0% and 34.5% TI's that resulted from conditioned media from two arteriographically nonstaining acoustic neurinomas in culture. The experimental tumors in clonal cultures also have a wide range of endothelial trophic influence. Three varieties of C6 rat astrocytoma resulted in TI's of 28.7%, 83.3%, and 96.6% respectively. All of these cultures had been growing for some time, and it is interesting that the ability to produce an endothelial trophic factor was not lost after these cells adapted to a tissue culture environment. There was a slight increase from a control labeling index of 2.1% to a TI of 4.5% when fibroblast conditioned medium was added. Amniotic fluid which contained a number of growth factors did not induce an increase in endothelial uptake of SH thymidine, neither did a medium conditioned by amniotic cells in culture. The mitotic index of the endothelial cells in control cultures was 0.1%, and increased to 7.0% in the presence of tumor conditioned media. This increase in the mitotic activity indicates actual cell division, rather than mere DNA synthesis or repair. We also performed an assay in which conditioned medium from a human astrocytoma was treated by heating to 56 ~ C for 10 minutes; we then subjected this to the same experimental protocol. The resultant TI was 4.4%, which suggests that the endothelial trophic factor is inactivated by heat. Filtration of conditioned media through Amicon filters with a 10,000 dalton molecular weight cut-off does not remove the proliferation effects of such media. These findings rendered the possibility extremely unlikely that the J. Neurosurg. / Volume 44 / March, 1976
Endothelial growth factor in tumor tissue cultures FABLE 1 Endothelial uptake o[ 3H thymidine oj control cultures and cultures with conditioned media from human and experimental CNS tumors* NO.
Cells No. Labeling Exam- Cells Index ined Labeled (%) controls endothelia alone 4279 endothelia with: amniotic fluid 1008 fibroblast conditioned 1254 media human CNS tumors endothelia with conditioned media from: meningioma (syncytial) 832 meningioma (angio4905 blastic) astrocytoma 741 ependymoma 847 neuroblastoma 249 acoustic neurinoma (1) 1446 acoustic neurinoma (2) 972 experimental CNS tumors endothelia with conditioned media from: C-6 (Pfeiffer) 4354 C-6 (JDV) 724 C-6 (CCL107) 5411
89
2.1
11 56
1.1 4.5
327 4820
38.2 98.3 30.1 4o.0 78.7 32.0 34.5
241 328 196 462 282
4195 603 1554
96.6 83.3
28.7
* Data represent the cumulated averages of multiple cultures and assays.
tumor is deprived of its blood supply it may remain in a dormant state. 13 The concept of antiangiogenesis requires definitive characterization of TAF. In our experiment the fact that human tumors and experimental rodent CNS tumors increased the uptake of 3H thymidine by cultured human endothelium suggested that there is no species specificity in the trophic substance. Because these studies were limited to CNS tumors, we tested the effects of media conditioned by primary fetal brain, spinal cord, and retinal cultures, which were uniformly ineffective in the stimulation of endothelial proliferation. Furthermore, tumorconditioned medium had no effect in cultured fibroblasts and this fact suggests that the trophic factor must be fairly specific for endothelial cells. We have described a relatively straightforward in vitro assay system for the further characterization of TAF which we have demonstrated to be present in human or experimental tumor cultures. The relative stability of this as yet unidentified factor in conditioned media should make its future purification and identification possible. Such experiments are presently underway in our laboratory. References
causative factor is a low molecular weight substance such as lactate. Unlike endothelium, primary fibroblasts in culture do not show an increase in thymidine uptake response to tumor-conditioned media. Discussion
Use of the tissue culture system allows the study of cell-to-cell interaction and in our experiment demonstrates the presence of a chemical substance produced by C N S tumor cells which results in the proliferation of endothelial cells. The existence of this tumor angiogenesis factor (TAF) has been postulated i,~1,22 and shown experimentally. 35'x~ We are suggesting that our growth factors may be similar or identical to TAF. The identification of T A F may have farreaching therapeutic implications as Folkman suggested, 7 for it has been shown that when a J. Neurosurg. / Volume 44 / March, 1976
1. Algire GH, Chalkley HW: Vascular reactions of normal and malignant tissues in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic transplants. J Natl Cancer Inst 6:73-85, 1945 2. Benda P, Lightbody J, Sato G, et al: Differentiated rat glial cell strain in tissue culture. Science 161:370-371, 1968 3. Cavallo T, Sade R, Folkman J, et al: Tumor angiogenesis. Rapid induction of endothelial mitoses demonstrated by autoradiography. J Cell Biol 54:408-420, 1972 4. Cavallo T, Sade R, Folkman J, et al: Ultrastructural autoradiographic studies of the early vasoproliferative response in tumor angiogenesis. Am J Pathol 70:345-352, 1973 5. Ehrmann RL, Knoth M: Choriocarcinoma: Transfilter stimulation of vasoproliferation in the hamster cheek pouch - - studied by light and electron microscopy. J Natl Cancer Inst 41:1329-1342, 1968 6. Feigin I, Allen LB, Lipkin L, et al: The endothelial hyperplasia of the cerebral blood vessels with brain tumors, and its sarcomatous transformation. Cancer 11:264-277, 1958 345
P. J. Kelly, et al. 7. Folkman J: Anti-angiogenesis: New concept for therapy of solid tumors. Ann Surg 175:409-416, 1972 8. Folkman J: Tumor angiogenesis: Therapeutic implications. N Engl J Med 285:1182-1186, 1971 9. Folkman J, Hochberg M: Self-regulation of growth in three dimensions. J Exp Med 138:745-753, 1973 10. Folkman J, Merler E, Abernathy C, et al: Isolation of a tumor factor responsible for angiogenesis. J Exp Med 133:275-288, 1971 11. Gimbrone MA Jr, Cotran RS, Folkman J: Human vascular endothelial cells in culture. Growth and DNA synthesis. J Cell Binl 60:673-684, 1974 12. Gimbrone MA Jr, Leapman SB, Cotran RS, et al: Tumor angiogenesis: Iris neovascularization at a distance from experimental intraocular tumors. J Natl Cancer Inst 50:219-225, 1973 13. Gimbrone MA Jr, Leapman SB, Cotran RS, et al: Tumor dormancy in vivo by prevention of neovascularization. J Exp Med 136: 261-276, 1972 14. Gough J: The structure of the blood vessels in cerebral tumours. J Pathol Bacterini 51:23-28, 1940 15. Greenblatt M, Shubi KP: Tumor angiogenesis: Transfilter diffusion studies in the hamster by the transparent chamber technique. J Natl Cancer Inst 41:111-124, 1968 16. Jaffe EA, Nachman RL, Becker CG, et al: Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745-2756, 1973
346
17. Krylova NV: Endothelium characteristics of blood vessels in neoplasms. Blbi Anat 12:497-503, 1973 18. NystrSm S: Pathological changes in blood vessels of human glioblastoma multiforme. Comparative studies using plastic casting, angiography, light microscopy and electron microscopy, and with reference to some other brain tumours. Aeta Pathnl Micrnbinl Stand 49 (Suppl 137):1-83, 1960 19. Pefia CE, Felter R: Ultrastructure of a composite glioma-sarcoma of the brain. Acta Neuropathol (Bed) 23:90-94, 1973 20. Sade RM, Folkman J, Cotran RS: DNA synthesis in endothelium of aortic segments in vitro. Exp Cell Res 74:297-306, 1972 21. Tannock IF: Population kinetics of carcinoma cells, capillary endothelial cells, and fibroblasts in a transplanted mouse mammary tumor. Cancer Res 30:2470-2476, 1970 22. Tannock IF: The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Br J Cancer 22:258-273, 1968 23. Tuan D, Smith S, Folkman J, et al: Isolation of the nonhistone proteins of rat Walker carcinoma. Their association with tumor angiogenesis. Biochemistry 12:3159-3165, 1973 This paper was supported by NIH Grant NS 11255 and Neurosurgery MSRDP Research Funds. Address reprint requests to: Patrick J. Kelly, M.D., Division of Neurosurgery, Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77550.
J. Neurosurg. / Volume44 / March, 1976
9 CNStumors 9 endothelium 9 autoradiography
HEendothelial proliferation and neovascularization associated with CNS neoplasms is well recognized.8,",17-~9 A specific chemical factor produced by tumor cells which induces neovascularization and more specifically endothelial mitotic activity has been suggested by experiments in which hamster cheek pouch vessel proliferation was induced by a tumor contained in a millipore filter?, ~5 Folkman8 evaluated angiogenic activity of various experimental and human tumors upon vessels in rat tissue and found that extracts of those tumors resulted in neovascularization.',~~This was also shown by injecting tumor extracts intraocularly and
T
342
9 angiogenesis
9
evaluating neovascularization in the iris? s Endothelial uptake of 8H thymidine and increase in mitotic activity in vivo induced by tumor angiogenesis factor (TAF) has been studied in other experiments? Although these assay systems for TAF axe workable, they are also cumbersome and subject to other variables peculiar to in vivo bioassay techniques. The purpose of our study was to continue this work in a tissue culture assay program in which tumor specimens and endothelium would be grown in separate cultures; the effect of media in which tumors had been growing (conditioned media) or fractions thereof on the endothelial cultures could be evaluated by the incorporation of 8H J. Neurosurg. / Volume 44 / March, 1976
E n d o t h e l i a l g r o w t h f a c t o r in t u m o r tissue c u l t u r e s thymidine? ,lx,~~ We are reporting our results of medium for the initial 24 hours following with the first in vitro assay system for the plating. These were then supplemented with study of tumor trophic factors on endothelial either 1 ml of conditioned medium or 1 ml of fresh Flo containing 20% fetal calf serum. proliferation. The fibroblasts were larger than the astroMethods and Materials cytoma cells, and were therefore plated at 80% of the cell density used for the astroCell Culture cytoma (1.0 • llY astrocytoma cells/10 ml of Endothelial Cells. Endothelial cells were media). Each unit volume of medium was obtained from the umbilical vein of human conditioned by approximately the same umbilical cords by collagenase digestion number of cells. employing the method described by Jaffe, et al.le These were then cultured on glass cover A u toradiography slips in six well plates* in Flo plus 20% fetal Twenty-four hours after the addition of the calf serum and 1.5% CO~. Handled as conditioned media, 0.5 uCi of SH thymidine primary cultures the endothelial cells were the was added to each well. Seventy-two hours vehicle for our assay system; for each endolater, the cover slips were rinsed with Hanks thelial sample we prepared one control and balanced salt solution, and then fixed in 3:1 five experimental cultures. fixative (ethanol:acetic acid) for 10 minutes. Human Tumors. Surgical specimens were Subsequently the slides were coated with diced into l-mm cubes and placed aseptically Kodak NTB-2 nuclear tracking emulsion and in Flo medium and 20% fetal calf serum and exposed for 5 days. After development the transferred to Falcon plastic tissue culture cover slips were stained with either toluidine flasks as lying drop cultures. Twenty-four blue or Giemsa. hours later fresh F~o medium was added to the flask and the tumor cells were allowed to Assay Method grow for 1 week. The tumor type was determined by sections of the original surgical Slides of 8H thymidine labeled endothelia specimen stained with hematoxylin and eosin. were observed under 10• magnification, and Experimental Clonal Tumors. Three dif- the number of cells labeled with SH thyferent clonal lines of C8 rat astrocytoma cells2 midine as a proportion of the total number of were grown in F~o medium containing 10% cells for that field was recorded from 10 ranfetal calf serum.t Fibroblasts were obtained domly selected fields on each slide. The ratio from a biopsy specimen of a human scar and of the cells labeled compared to the total maintained in primary culture with Flo and number of cells counted in each field is 10% fetal calf serum. The fibroblasts used in termed the thymidine labeling index (TI). To these studies had a doubling time of 29 hours, eliminate bias, the worker reading the slide as compared to 20 hours for the C6 astro- was unaware of the group that each particular cytoma. slide represented. Labeled cells are those Conditioned Media. One ml of medium which have more than 20 silver grains from a growing cell line (conditioned localized over the nucleus. The labeling index medium) filtered through a 0.22 p millipore was computed as an average of the 10 low filter was added to 2 ml of F~o and 20% fetal power, random fields for each slide, and these calf serum already in the culture well. One ml data were tabulated for each of the groups of fresh medium was added to one of the six given in Table 1. wells as a control. Thus, all control and exResults perimental cultures contained a total of 2 ml As shown in Table 1 and Fig. 1, there is a dramatic increase in endothelial incor*Well plates manufactured by Linbro Plastics, poration of SH thymidine when conditioned New Haven, Connecticut. ?Rat astrocytoma cells were obtained from S. medium from human or experimental tumors M. Pfeiffer (University of Connecticut), J. De from the central nervous system (CNS) is Veliss (University of California at Los Angeles), added to primary endothelial cultures. It has and from the American Type Culture Collection been our consistent observation that the addi(CCLI07). tion of conditioned medium to endothelial J. Neurosurg. / Volume 44 / March, 1976
343
P. J. Kelly, et al.
FIG. 1. Graphic display of thymidine-labeling index of endothelial culture with control and conditioned media from human and experimental CNS tumors (see also Table 1).
FIG. 2. Autoradiograph showing endothelial cells after the addition of conditioned media from an angioblastic meningioma. Note high degree of thymidine uptake and mitotic figures. 344
culture also produces morphological alterations as seen by light microscopy. The striking increase of thymidine labeling is shown in Fig. 2 where almost every cell is labeled and mitotic figures may be seen. A wide range in TI was noted in the endothelium cultures treated with human tumorconditioned medium, ranging from a Grade III astrocytoma TI of 30.1% to a 98.3% TI resulting from the addition of conditioned medium from an angioblastic meningioma. This tumor had a prominent stain arteriographically and was highly vascular at surgery. It is interesting that the degree of endothelial stimulation does not reflect a tumor's arteriographic staining prominence as demonstrated by the 32.0% and 34.5% TI's that resulted from conditioned media from two arteriographically nonstaining acoustic neurinomas in culture. The experimental tumors in clonal cultures also have a wide range of endothelial trophic influence. Three varieties of C6 rat astrocytoma resulted in TI's of 28.7%, 83.3%, and 96.6% respectively. All of these cultures had been growing for some time, and it is interesting that the ability to produce an endothelial trophic factor was not lost after these cells adapted to a tissue culture environment. There was a slight increase from a control labeling index of 2.1% to a TI of 4.5% when fibroblast conditioned medium was added. Amniotic fluid which contained a number of growth factors did not induce an increase in endothelial uptake of SH thymidine, neither did a medium conditioned by amniotic cells in culture. The mitotic index of the endothelial cells in control cultures was 0.1%, and increased to 7.0% in the presence of tumor conditioned media. This increase in the mitotic activity indicates actual cell division, rather than mere DNA synthesis or repair. We also performed an assay in which conditioned medium from a human astrocytoma was treated by heating to 56 ~ C for 10 minutes; we then subjected this to the same experimental protocol. The resultant TI was 4.4%, which suggests that the endothelial trophic factor is inactivated by heat. Filtration of conditioned media through Amicon filters with a 10,000 dalton molecular weight cut-off does not remove the proliferation effects of such media. These findings rendered the possibility extremely unlikely that the J. Neurosurg. / Volume 44 / March, 1976
Endothelial growth factor in tumor tissue cultures FABLE 1 Endothelial uptake o[ 3H thymidine oj control cultures and cultures with conditioned media from human and experimental CNS tumors* NO.
Cells No. Labeling Exam- Cells Index ined Labeled (%) controls endothelia alone 4279 endothelia with: amniotic fluid 1008 fibroblast conditioned 1254 media human CNS tumors endothelia with conditioned media from: meningioma (syncytial) 832 meningioma (angio4905 blastic) astrocytoma 741 ependymoma 847 neuroblastoma 249 acoustic neurinoma (1) 1446 acoustic neurinoma (2) 972 experimental CNS tumors endothelia with conditioned media from: C-6 (Pfeiffer) 4354 C-6 (JDV) 724 C-6 (CCL107) 5411
89
2.1
11 56
1.1 4.5
327 4820
38.2 98.3 30.1 4o.0 78.7 32.0 34.5
241 328 196 462 282
4195 603 1554
96.6 83.3
28.7
* Data represent the cumulated averages of multiple cultures and assays.
tumor is deprived of its blood supply it may remain in a dormant state. 13 The concept of antiangiogenesis requires definitive characterization of TAF. In our experiment the fact that human tumors and experimental rodent CNS tumors increased the uptake of 3H thymidine by cultured human endothelium suggested that there is no species specificity in the trophic substance. Because these studies were limited to CNS tumors, we tested the effects of media conditioned by primary fetal brain, spinal cord, and retinal cultures, which were uniformly ineffective in the stimulation of endothelial proliferation. Furthermore, tumorconditioned medium had no effect in cultured fibroblasts and this fact suggests that the trophic factor must be fairly specific for endothelial cells. We have described a relatively straightforward in vitro assay system for the further characterization of TAF which we have demonstrated to be present in human or experimental tumor cultures. The relative stability of this as yet unidentified factor in conditioned media should make its future purification and identification possible. Such experiments are presently underway in our laboratory. References
causative factor is a low molecular weight substance such as lactate. Unlike endothelium, primary fibroblasts in culture do not show an increase in thymidine uptake response to tumor-conditioned media. Discussion
Use of the tissue culture system allows the study of cell-to-cell interaction and in our experiment demonstrates the presence of a chemical substance produced by C N S tumor cells which results in the proliferation of endothelial cells. The existence of this tumor angiogenesis factor (TAF) has been postulated i,~1,22 and shown experimentally. 35'x~ We are suggesting that our growth factors may be similar or identical to TAF. The identification of T A F may have farreaching therapeutic implications as Folkman suggested, 7 for it has been shown that when a J. Neurosurg. / Volume 44 / March, 1976
1. Algire GH, Chalkley HW: Vascular reactions of normal and malignant tissues in vivo. I. Vascular reactions of mice to wounds and to normal and neoplastic transplants. J Natl Cancer Inst 6:73-85, 1945 2. Benda P, Lightbody J, Sato G, et al: Differentiated rat glial cell strain in tissue culture. Science 161:370-371, 1968 3. Cavallo T, Sade R, Folkman J, et al: Tumor angiogenesis. Rapid induction of endothelial mitoses demonstrated by autoradiography. J Cell Biol 54:408-420, 1972 4. Cavallo T, Sade R, Folkman J, et al: Ultrastructural autoradiographic studies of the early vasoproliferative response in tumor angiogenesis. Am J Pathol 70:345-352, 1973 5. Ehrmann RL, Knoth M: Choriocarcinoma: Transfilter stimulation of vasoproliferation in the hamster cheek pouch - - studied by light and electron microscopy. J Natl Cancer Inst 41:1329-1342, 1968 6. Feigin I, Allen LB, Lipkin L, et al: The endothelial hyperplasia of the cerebral blood vessels with brain tumors, and its sarcomatous transformation. Cancer 11:264-277, 1958 345
P. J. Kelly, et al. 7. Folkman J: Anti-angiogenesis: New concept for therapy of solid tumors. Ann Surg 175:409-416, 1972 8. Folkman J: Tumor angiogenesis: Therapeutic implications. N Engl J Med 285:1182-1186, 1971 9. Folkman J, Hochberg M: Self-regulation of growth in three dimensions. J Exp Med 138:745-753, 1973 10. Folkman J, Merler E, Abernathy C, et al: Isolation of a tumor factor responsible for angiogenesis. J Exp Med 133:275-288, 1971 11. Gimbrone MA Jr, Cotran RS, Folkman J: Human vascular endothelial cells in culture. Growth and DNA synthesis. J Cell Binl 60:673-684, 1974 12. Gimbrone MA Jr, Leapman SB, Cotran RS, et al: Tumor angiogenesis: Iris neovascularization at a distance from experimental intraocular tumors. J Natl Cancer Inst 50:219-225, 1973 13. Gimbrone MA Jr, Leapman SB, Cotran RS, et al: Tumor dormancy in vivo by prevention of neovascularization. J Exp Med 136: 261-276, 1972 14. Gough J: The structure of the blood vessels in cerebral tumours. J Pathol Bacterini 51:23-28, 1940 15. Greenblatt M, Shubi KP: Tumor angiogenesis: Transfilter diffusion studies in the hamster by the transparent chamber technique. J Natl Cancer Inst 41:111-124, 1968 16. Jaffe EA, Nachman RL, Becker CG, et al: Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745-2756, 1973
346
17. Krylova NV: Endothelium characteristics of blood vessels in neoplasms. Blbi Anat 12:497-503, 1973 18. NystrSm S: Pathological changes in blood vessels of human glioblastoma multiforme. Comparative studies using plastic casting, angiography, light microscopy and electron microscopy, and with reference to some other brain tumours. Aeta Pathnl Micrnbinl Stand 49 (Suppl 137):1-83, 1960 19. Pefia CE, Felter R: Ultrastructure of a composite glioma-sarcoma of the brain. Acta Neuropathol (Bed) 23:90-94, 1973 20. Sade RM, Folkman J, Cotran RS: DNA synthesis in endothelium of aortic segments in vitro. Exp Cell Res 74:297-306, 1972 21. Tannock IF: Population kinetics of carcinoma cells, capillary endothelial cells, and fibroblasts in a transplanted mouse mammary tumor. Cancer Res 30:2470-2476, 1970 22. Tannock IF: The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumour. Br J Cancer 22:258-273, 1968 23. Tuan D, Smith S, Folkman J, et al: Isolation of the nonhistone proteins of rat Walker carcinoma. Their association with tumor angiogenesis. Biochemistry 12:3159-3165, 1973 This paper was supported by NIH Grant NS 11255 and Neurosurgery MSRDP Research Funds. Address reprint requests to: Patrick J. Kelly, M.D., Division of Neurosurgery, Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77550.
J. Neurosurg. / Volume44 / March, 1976
