Animal models for brain tumors: historical perspectives and future directions

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J Neurosurg 80:865-876, 1994 Animal models for brain tumors: historical perspectives and future directions DANIEL L. PETERSON, M.D., PETER J. SnERIOAN, PH.D., AND WILLIS E. BROWN, JR., M.D. Division of Neurosurgery and Department of Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, Texas ~," The scientific understanding of the biology of human brain tumors has advanced in large part through the use of animal models. For most of this century, investigators have been evaluating the inciting factors in brain tumor development, and applying this knowledge to direct tumor growth in laboratory animals. Virus-induced, carcinogen-induced, and transplant-based models have been vigorously investigated. As knowledge of the molecular biology of neoplasia has advanced, transgenic technology has been introduced. The authors review the development of animal models for brain tumor, and focus on the role of transgenic models in elucidating the complex process of central nervous system neoplasia. KEy WORDS 9 animal model . brain neoplasm transgenie mouse ESPITE tremendous efforts by neurosurgeons, ba- sic scientists, oncologists, radiation therapists, and others, the prognosis for the patient with a malignant primary brain tumor has changed mini- mally over the last two decades. Improvement in the quality of survival for patients with glioma remains one of the foremost challenges facing this generation of neurosurgeons. As stated by Ausman, et aL, 1 in 1970, "there seems to be little possibility of improving the surgical or radiation therapies of malignant brain tu- mors." It appeared at that time that the development of new and better animal models of glioma would be needed for in vivo evaluation of potential chemother- apeutic and adjunctive therapies for brain tumors. The extensive contributions involving animal models by in- vestigators such as Bigner and Wilson have directed the biological study and adjunctive therapy of brain tumors over the past 25 years. This review is meant to supplement and update information in this already well-documented field. The justification and need for accurate, representa- tive animal models of glioma remain today just as great as in 1970, although the advent of brachytherapy and stereotactically coupled focused beam irradiation offer room for some optimism. While countless chemother- apeutic agents have been tested, few have been found useful in the adjunctive therapy of malignant glioma. As our understanding of the molecular genetics of brain tumors expands, the direction of adjunctive therapy will follow in a biologically oriented manner. The pioneering work of Martuza and colleagues 36 at the Molecular Neurogenetics Unit at the Massachusetts General Hospital, and the work of Oldfield and col- leagues 3~ at the National Institute of Neurological Dis- orders and Stroke in the area of retroviral herpes sim- plex virus-l-thymidine kinase gene transfer-induced ganciclovir sensitivity will lead the therapy of malig- nant glioma into the nucleus, and into the next century. The elegance and complexity of this work deserve an animal model that will adequately represent the growth characteristics of gliomas. In this article, we outline the development of animal models for brain tumor to date, and speculate on future directions for such animal models. Requirements for Animal Model of Glioma A valid animal model for brain tumors must display a variety of specifications: it must be composed of glial-derived neoplastic cells; the growth rate of the tumor and its malignancy characteristics should be pre- dictable and reproducible; the species used should be small and inexpensively maintained so that large num- bers may be evaluated for reliable chemotherapeutic testing; the time to tumor induction should be relatively short and the survival time after induction should be standardized; the tumor should have glioma-like intra- parenchymal growth and blood-brain characteristics showing invasion, neovascularity, and no encapsula- tion; there should be no seeding of tumor in the sub- cutaneous, epidural, or subdural compartments; and the J. Neurosurg. / Volume 80 / May, 1994 865 D. L. Peterson, R J. Sheridan, and W. E. Brown, Jr. tumor should be able to grow in culture and must be safe for laboratory personnel. 2~ Most importantly, the therapeutic responsiveness of the model must imitate that of human brain tumors) > Although many avail- able animal models have some utility, none has fulfilled all of these requirements. Mammalian CNS Tumors Spontaneous tumors of the central nervous system (CNS) occur in nonhuman mammals at a frequency thought to approximate that of humans. The incidence of CNS tumors in dogs is considered to be about 0.1% to 0.5%. 82 Roughly 60% of these tumors are neuro- ectodermal, with about 15% being meningeal. The in- cidence of tumors has been found to be somewhat higher in brachycephalic breeds such as boxers and bulldogs. 75 In contrast, approximately 80% of feline primary CNS tumors are of meningeal origin, s2 Pri- mary CNS tumors appear to be quite rare in nonhuman primates. 122 Interestingly, an autopsy series of 14,000 rhesus monkeys revealed no CNS tumors] ~-3 and an- other study of 1247 aged Old World monkeys (baboons and Macaque monkeys) found no CNS tumors. 62 Spon- taneous CNS tumors are also exceedingly rare in labo- ratory rodents. Guerin autopsied 16,500 rats and found 567 tumors, among which only four were primary CNS tumors, and Mawdesley-Thomas and Newman 8~ re- ported an autopsy series of 41,000 Sprague-Dawley rats with 38 brain tumors, an incidence of 0.09%; ap- proximately one-third were meningiomas, one-third were gliomas, and the remainder were poorly differ- entiated or sarcomatous. In a study by Fitzgerald, et al.)8 involving autopsy of 7803 Sprague-Dawley rats, the incidence of CNS tumors was 0.44%. The most frequently arising spontaneous astrocytic animal tumor, the VM murine astrocytoma, was first reported in 1971. 39 These tumors were discovered as an incidental finding during the examination of over 10,000 mouse brains used in experimental scrapie re- search in an inbred mouse strain. The incidence of as- trocytic tumors in this line is only 1.5% by 600 days of age, making it impractical for therapeutic studies. Cell lines derived from tumors in this strain have been well characterized, and have allowed the development of a useful transplantation model. Carcinogen-Induced Tumors The experimental production of skin cancer in labo- ratory animals with coal tar in 1915 initiated the study of chemical structure and its relationship to carcino- genesis. 114 It also led to the evaluation of tissue-specific susceptibility of different carcinogens. The first studies directed at brain oncogenesis involved a subarachnoid injection of a tar emulsion in the rabbit, but no brain tumors developed. The first successful brain tumor in- duction was reported by Seligman and Shear 114 in 1939. They implanted pellets of methylcholanthrene into the brains of 20 mice; gliomas developed in 11, menin- geal fibrosarcomas in two, and seven had no tumors. The growth characteristics of these tumors were sub- sequently evaluated by subcutaneous passage) 31 Zim- merman ~3" reported on a study by Cohn, who grew frag- ments of a methylcholanthrene-induced ependymoma in chick eggs. After several passages, the tumors be- came an amorphous mass of cells with a morphological appearance that bore no resemblance to the original glial neoplasm. Interestingly, the tumor morphology re- turned to that of an ependymoma after subcutaneous transplantation from chick egg to mouse) 3~ This find- ing brought attention to the influence that the environ- ment of a tumor has on its microscopic structure. In the 1960% a new class of synthetic carcinogen, the N-nitrosoureas, was developed. The incidence, type, and location of N-nitrosourea-induced tumors was found to vary greatly with dose, age, strain, and physiological state of the animal, such as pregnancy or malnutrition. Druckrey, et at., 31 evaluated the ef- fects of ethylnitrosourea (ENU) on laboratory rats, and found that the oncogenic effect of ENU was much greater in younger rats as compared to older rats. This observation led to the injection of ENU into pregnant rats, which demonstrated the transplacental oncogenic- ity of ENU. Schmidek, et a l l ~8 performed 36 weekly injections of N-nitrosomethylurea (NNMU) into 108 male rats. Of the 58 animals that survived the injec- tions, 22 developed primary brain tumors between 17 and 43 weeks after termination of NNMU administra- tion. A spectrum of glial tumors resulted, but the low incidence of tumors (22 in 108 animals) invalidated this model for therapeutic studies. Koestner, et at., 67 administered a single intravenous ENU dose of 50 mg/kg to Sprague-Dawley rats on the 20th day of ges- tation. All 25 offspring developed tumors of the ner- vous system, with a total of 102 neural and seven non- neural tumors. Classification of the tumors by light and electron microscopy revealed 32 oligodendrogliomas, four astrocytomas, 19 mixed gliomas, five anaplastic gliomas, 14 ependymomas, one meningioma, and 27 neurinomas. Brucher and ErmeP 9 were able to dem- onstrate induction of neuroblastoma in Wistar rats by transplacental administration of NNMU. The advan- tage of tumors induced transplacentally is that they may be obtained with a single injection; the result is a litter composed of several tumor-bearing animals of the same age. These and similar studies provided re- liable and reproducible tumors of neural origin for mor- phological and biological investigation, xenografts, and colony-formation assays. 129 Disadvantages of the NNMU and ENU animal models include extensive variability in tumor type, location, induction times, and malignancy characteristics. Virus-Induced Thmors The role of viruses in the induction of human neural tumors has been vigorously investigated. Although in- tact virus particles have been found in human brain tu- mors, z9 an etiological relationship has never been es- tablished. With regard to animal models, a host of CNS tumors have been induced by oncogenic viruses. The observation that viruses that are nontumorigenic in their permissive host can become tumorigenic in a non- 866 J. Neurosurg. / Volume 80 / May, 1994 Animal models for brain tumors permissive host (when inoculated at high titers) has led to many models of CNS neoplasia. Virus-induced tu- mors have a number of advantages: the inoculation pro- duces minimal trauma to brain tissue; an autochthonous tumor develops with a natural blood supply; the tumors can be cultured for in vitro studies; and these tumors may be predictably induced. There are disadvantages, however: the viruses can be difficult to maintain in the laboratory; they may be harmful to man; and the re- sultant tumors vary greatly in histopathology, growth kinetics, and location. Ribonucleic Acid Viruses The most commonly used viruses in brain tumor in- duction are retroviruses, particularly the avian sarcoma viruses. 2~ As early as 1936, V~izquez-L6pez t24 in- duced intracerebral sarcoma in adult chickens with in- tracerebral inoculation of Rous sarcoma virus (RSV)-I, an avian sarcoma virus first discovered in 1911J ~ The type of tumor induced by viruses depends largely on the site of inoculation and the age of the animal at the time of inoculation. A superficial cerebral or vermian location of inoculation favors sarcomas, whereas in- oculation in the subependymal plate region favors gli- oma development. 27 Neonatal animals are much more susceptible to tumor development and progression than adult animals. 26 An early model of experimental brain tumors was developed in the dog using intracerebral injection of the Schmidt-Ruppin strain of the RSV. 7,~~176 This system was so refined by Bigner, et al., 6 that a 0.01-ml con- centrated suspension of virus particles was able to produce 100% tumor induction in newborn beagles. Tumors derived from various glial precursors and intracranial sarcomas resulted. When tested against chemotherapeutic agents, the RSV beagle model was responsive to BCNU (carmustine) and CCNU (lomus- tine), but not cyclophosphamide, which correlates with clinical experience. 29 This work also revealed a sub- stantial amount of information regarding ultrastruc- tural aspects of brain tumors, including identification and characterization of blood-brain barrier defects, en- dothelial disruption, and perivascular tumor spreadJ 25 No virions were found in mammalian hosts, demon- strating that biologically detectable replication of the RSV need not be present in virus-induced tumors, and that viral oncogenesis can occur independent of par- ticle replication. This tumor model is typical of a non- permissive host system, and in every case the RSV ge- nome could be rescued by inoculation of mammalian tumors into the permissive avian host. An alternative method of viral brain tumor induction was presented by Tabuchi, e t a l . , 119 in 1985. They in- fected fibrohlasts derived from chick embryos with the Schmidt-Ruppin strain of RSV, then inoculated the fi- broblasts into the right frontal lobe of Japanese mon- keys (Macaca fascata). Tumors were induced after a latency period of 15 to 67 days in 73% of the experi- mental animals. It was concluded that the inoculated chick embryo fibroblasts regressed early after implan- tation and infection of brain cells. This conclusion was supported by two experimental findings: 1) RSV par- ticles were not seen under electron microscopy, a find- ing consistent with incorporation into a nonpermissive host (in this case, the monkey); and 2) chromosomal analysis of these tumors subsequently grown in culture revealed a diploid number of 42, indicating that the origin of the tumor cells was from the monkey rather than the chick. Unfortunately, 66% of the tumors examined immunohistochemically proved to be sar- comas. Deoxyribonucleic Acid Viruses Human adenovirus 12 has been used to induce tu- mors in mice, rats, and hamsters after intracerebral and intraorbital inoculation. 8s Neuroblastoma and retino- blastoma are the primary tumor types induced, and per- manent cell lines have been established from both of these tumors. The survival of animals bearing tumors induced by this virus is variable, and these models have proven most useful in cytokinetic studies. Simian ad- enovirus (SA7) in the hamster induces choroid plexus papillomas and medulloblastomas; the latter have been maintained in culture. 24,~5 The chicken-embryo-lethal- orphan (CELO) virus is an endogenous adenovirus of chicken eggs, whose infection is inapparent in its per- missive host. Mancini and colleagues 76 inoculated in- tracerebrally a 0.02-ml suspension of purified CELO virus into newborn Syrian hamsters. All 17 animals that survived the inoculation developed CNS sympto- tautology and tumors arising from ependymal sur- faces. These tumors were initially thought to be papillary ependymomas, but choroid plexus papillomas seem a more likely histopathology. 2~ Some animal brain tumor models have arisen from apparently unrelated research. Papovaviruses isolated from brain tissue of humans afflicted with progressive multifocal leukoencephalopathy (PML) have been shown to be tumorigenic when inoculated intracere- brally in newborn Syrian hamsters. 91,9z Tumor types in- duced include cerebellar medulloblastomas, thalamic gliomas, intraventricular papillary ependymomas, a men- ingeal tumor, and a pineocytoma. London, et al. ]3 in- oculated JC polyomavirus derived from human cases of PML into four Colombian owl monkeys; primary brain tumors developed at 16 and 25 months in two of the animals. One tumor was a high-grade glioma, resembling human glioblastoma, and the other con- tained a mixture of neoplastic glial and neuronal cell types. The simian vacuolating virus SV40 was isolated from monkeys and is not oncogenic in monkeys, its permissive host. 118 It has been shown to produce dose- dependent tumorigenesis when inoculated intracere- brally in newborn Syrian hamsters; 32,33 these tumors were interpreted histopathologically as choroid plexus papillomas or ependymomas. Inoculation of SV40 did not induce tumors in young dogs, rats, cats, or rabbits. The transforming genes from SV40, namely, the large T- and small t-antigens, have been sequenced and cloned. The large T-antigen gene has been used extort- J. Neurosurg. / Volume80 / May, 1994 867 D. L. Peterson, E J. Sheridan, and W. E. Brown, Jr. sively to effect malignant transformation of cells in cul- ture 117 as well as to direct oncogenesis in vivo, as will be discussed later. Models Based on Cell and Tissue Transplantat ion Transplantation of tumor cells or tissue into recipient animals has generated numerous brain tumor models. As discussed by Bullard and Bigner, 2~ there are essen- tially two types of transplant models: syngeneic tumors induced by viruses or chemicals and maintained via cell culture or subcutaneous passage for prolonged periods, and heterotransplantation models in which tumor from another species (or a different strain of the same spe- cies) is transplanted into either an immunoincompetent recipient or an immunologically privileged site, such as the cheek pouch, brain, or the anterior chamber of the eye? 8 Transplantation techniques have evolved tremen- dously. Early workers used a needle and stylet to inject minced tumor fragments freehand. This imprecise and nonquantitative method was improved upon by Wilson, et aL, ~27 who achieved a low mortality rate using ste- reotactic implantation of quantitatively standardized cell suspensions from a cell culture. Further improve- ments in implantation technique, including better ste- reotactic localization, concentrated cell suspensions (104 to 107 cells/10/xl), improved injection needles, flushing of the operative field, and augmentation of the cell suspension with 0.5% to 1.0% agar, have resulted in a 90% to 100% yield of intracerebral tumor growth and have reduced the incidence of extracranial and spi- nal metastases to 0% to 5%. 66 Syngeneic Transplantation Models In 1941, Zimmerman and Arnold TM transplanted methylcholanthrene-induced ependymoblastoma in- tracerebrally in mice and obtained 90% to 100% tumor take. From frozen stocks of this original tumor, Aus- man, et al., 1 derived several stable cell lines, including the extensively studied ependymoblastoma A. They transplanted minced tissue intracerebrally from subcu- taneously passaged ependymoblastoma and demon- strated quantitative differences in tumor sensitivity to different drugs, measuring increased survival com- pared with control tumor-bearing animals. This work greatly stimulated the study of chemotherapeutic agents from brain tumor in animal models. While screening of chemotherapeutic agents against ependy- moblastoma was initially promising, 45 its popularity has declined. Two chemotherapeutic agents, CCNU and BCNU, were found to have cure rates in animal models of 80% to 90% and 30% to 40%, 4~ respectively, and procarbazine was similarly effective; however, these drugs rarely produce cures in man. This tumor was shown to be exquisitely sensitive to dianhydro- galactitol in experimental studies, 69 while results with this drug in human studies have been disappointing. The prevailing opinion is that this model overpredicts the activity of certain agents. 7~ Furthermore, human ependymoblastomas account for less than 1.0% of all intracranial tumors, and the histology and kinetics of murine ependymoblastoma are not comparable to hu- man tumors. The C6 line of tumor cells has been used extensively in transplantation-based glioma studies. The origin of this cell line remains somewhat in question. It was in- duced in rats through transplacental exposure to N-ni- trosourea; however, the particular strain of rat is not precisely documented. The cell line is originally men- tioned by Benda, et al.,4 but the strain is not described. Although Dr. Benda is deceased, a coauthor recalls that the rat strain was not inbred (G Sato, personal com- munication, 1993). The conventional wisdom is that the strain of origin of the C6 cell line is randomly bred Wistar rats. 5,112 The C6 cell line demonstrates astrocytic morphology and has been widely studied in neurobiology because of the presence of glial cell markers such as glial fibril- lary acidic protein (GFAP) and S-100 protein. It has been used extensively in vitro, and numerous inves- tigators have published in vivo transplantation models using C6 cells as techniques have become more re- fined. 3v Kaye, et aL, 64 showed that 106 C6 cells sus- pended in 1.0% agarose will grow intracranially as a xenograft model in adult mice of several strains. They used a monoclonal antibody to C6 to show conclusively that microscopic invasion of tumor cells occurred into surrounding brain. Numerous investigators have re- ported models relying on C6 cells grown intracere- brally in adult rats, and it appears that C6 cells grown in adult Wistar rats show more homology to human glioma (with parenchymal invasion, neovascularity, and necrosis) than C6 cells grown in other strains, including Sprague-Dawley and Long-Evans rats, where they grow as sharply demarcated, encapsulated tumors.25,37,107 The 9L gliosarcoma cell line was developed from tumors induced in Wistar and Fischer rats injected with NNMU. Several clonal tumor cell lines originated and were reported to have glial morphology with stable growth patterns and biological characteristics? ,u2 Sar- comatous change occurred with serial passage. 3 While it may not represent glioma as well as other lines, the 9L cell line has been used extensively in screening tests in vitro, including the colony formation assay, and has been used as a model for spinal axis tumor dissemi- nation, ms Additionally, the 9L cell line has been used extensively to evaluate the effect of various agents on the blood-brain barrier. This cell line has lost its ability to invade the parenchyma of the brain, and therefore grows mainly in the Virchow-Robin spaces. This growth pattern may be due to the inability of the cells to effect neovascularity, to produce enzymes that re- model the extracellular matrix, or to produce a solid matrix. Schwartz, et aL, 113 used these characteristics of 9L cells as control features to evaluate the in vitro and in vivo effects of a variety of retrovirally transfected oncogenes (ras, neu, and sis) on the 9L line. The 9L/ras and 9L/neu cells produced more rapidly growing, large, solid, highly vascularized tumors in vivo than did non- transformed 9L cells. The 9L/sis cells produced tumors 868 d. Neurosurg. / Volume 80 / May, 1994 Animal models for brain tumors intermediate between control and 9L/ras and 9L/neu cells. Transfection of these oncogenes did not alter the growth characteristics of 9L cells in vitro, as compared with controls; this implies that the tumor progression was not merely a result of accelerated cellular metabo- lism. The authors suggested that the reason for tumor progression is that the oncogene-transfected cells are capable of enhanced angiogenesis or increased respon- siveness to local growth factors. The question of whether these oncogenes enabled the 9L cells to invade the parenchyma was cleverly addressed. The 9L/on- cogene cells were cotransfected with the prokaryotic .gene for/3-galactosidase, an enzyme in which activity is marked by blue staining in the presence of the ap- propriate substrate X-gal (5-bromo-4-chloro-3-indoyl- /3-galactoside). The 9L cells that expressed the foreign oncogene in addition to the /3-galactosidase activity were then co-injected with C6 cells. The resultant tu- mors showed blue staining in the center, with a non- staining infiltrating surround, and demonstrated that the expression of these oncogenes did not alter the non- infiltrative growth pattern of the 9L cells. The BT4A cell line was initiated in BD IX fetal rat brains by transplacental exposure to ENU; the resultant tumor cell line was then stabilized by serial passage in these syngeneic animals. This cell line provides 100% tumor take in BD IX rats and has the properties of single-cell invasion, the presence of S-100 protein, and histological characteristics in common with dif- ferentiated oligodendroglioma, ss Additionally, because the BT4A cell line shows low immunogenicity and moderate sensitivity to chemotherapy, it is a promising line for further studies. 83 Other similar lines include the RG22~ and the T9 s rat glioma. Bradford and colleagues 13-~5 have worked exten- sively with the spontaneous VM murine astrocytoma. From an initial cell line described by Serano, et al., 115 they isolated and characterized the 497-P(1) cell line. These cells stained for GFAP and other glial markers in vitro, and have been shown to be highly tumorigenic following implantation, with the tumors developing ne- crosis, pseudopallisading, and local invasion. Median survival time of the VM mouse model following trans- plantation was 12.5 days. Bradford, et al., TM assessed the sensitivity of VM 497-P(1) intracerebral tumors to BCNU, CCNU, vincristine, and procarbazine. While treatment with BCNU and CCNU showed substantial increases in life expectancy (142.5% and 132.5%, respectively), no cures were obtained unlike the meth- ylcholanthrene-induced ependymoblastoma model. Vin- cristine produced a moderate response, and procar- bazine was ineffective. The results with procarbazine in this model do not parallel its activity against human gliomas, a discrepancy seen in many tumor models. Wilson 126 suggested in 1978 that the failure of tumors in some animal models, particularly rat models, to re- spond to procarbazine was due to differences in the metabolism of the drug in that animal. The ineffective- ness of procarbazine against the murine 497-P(1) tu- mor model is thought to be due to inherent cellular re- sistance of the 497-P(1) cells. Heterologous Transplantation Models I-teterologous transplantation of human brain tumors was essentially unsuccessful until the advent of the nude mouse, a mutant that is genetically thymus- deficient and therefore immunoincompetent. Early workers showed minimal success with transplantation of brain tumors into the immunologicallyprivileged anterior chamber of the eye in guinea pigs 4~ or into immunologically suppressed animals such as corti- sone-treated rabbits. 48 The nude mouse was first re- ported by Pantelouris 96 in 1968, and since then a plethora of human tumors have been transplanted in athymic mice, with variable success. Tumors grown in this way frequently retain many of the biological char- acteristics of the original biopsy specimens including histopathology, biochemical markers, and hormone pro- duction. 63 In 1977, Rana, et al., t~ injected one human glioblas- toma into three athymic mice. In each mouse, the tumor grew and developed the morphological traits of the original specimen: multinucleated giant cells and pseu- dopallisading around areas of necrosis. Subsequently, several authors have reported results with human brain tumor xenografts in nude mice. Bradley, et al.,17 suc- cessfully grew subcutaneous xenografts from 11 (44%) of 25 adult gliomas, and other authors have reported higher success rates. Shapiro, et al., 116 placed tissue fragments from seven anaplastic gliomas into nude mice. All of the tumors grew initially, but only two resulted in stable tumor cell lines; both of the serially transplantable cell lines were identified histopathologi- cally as gliosarcomas. Jones, et al., ~3 reported a 94% success rate with the transplantation of anaplastic as- trocytomas into nude mice; 16 of 17 xenografts pro- duced a stable tumor cell line. Latency periods from transplantation to initial tumor growth varied from 24 to 364 days. Morphologically, most of these tumors showed cellularity greater than that of the original specimens, and virtually all of them showed a general absence of vascular proliferation. The vascular tissue in these tumors is considered to be of mouse origin, and the difference in species is thought to account for this lack of neovascularity. Regarding the biological characteristics of these transplanted tumors, cellular- ity, growth rate, and necrosis were either "retained, changed or deleted in an unpredictable way. ''63 Some tumors exhibited increased GFAP expression, some maintained GFAP expression similar to the original tu- mor, and some lost GFAP expression with heterotrans- plantation. Evaluation of the chromosomal content of malignant human glioma xenografts in nude mice gen- erally showed karyotypes similar to the original tissue, although some minor changes were seen. 1~ Schold and colleagues 22'1~ have extensively characterized the therapeutic responses of human glioma lines in athy- mic mice, and have found variable correlations with efficacy in clinical trials. The morphological, genetic, and biological differences in heterotransplanted tumors may be due to normal tumor evolution or augmented selection of certain cell subpopulations in an artificial environment. As Bullard and Bigner 2~ state, "(the) na- Z Neurosurg. / Volume 80 / May, 1994 869 D. L. Peterson, P. J. Sheridan, and W. E. Brown, Jr. ture of what determines tumorigenicity in the nude mouse is not clearly dcfincd and may reprcscnt factors not pertinent to human tumors." Other tumor types grown in nude mice include me- . 4 1 , ~ 2 5 4 - , " . ' : ' l " dulloblastoma 9 and clanlopharyngxoma.- Fried- man, et al., 42 reported on a variety of medulloblastoma cell lines grown both intracerebrally and subcutane- ously in nude mice, and showed prolonged median survival times when tumor-bearing mice were treated with melphalan, cyclophosphamide, iphosphamide, and thiotepa, while CCNU and busulfan were ineffec- tive. Studies such as these bear strong implications for clinical trials. While transplant models have become the mainstay of in rive brain tumor research, it is important to realize that they have several inherent limitationsJ ~2 Human brain tumors are extremely heterogeneous, and clonal cell lines do not represent this tendency. Tumor cell lines maintained in culture can, with serial passage, show variability in mitotic rates and transformation potential, and frequently demonstrate sarcomatous degeneration. Direct transplantation from animal to animal somewhat minimizes this variability, although work with the nude mouse has shown that serial sub- cutaneous passage commonly results in altered tu- mor morphologies. Variability persists in even the most carefully controlled clonal transplant studies. Unfor- tunately, despite stereotactic implantation and injection of consistent numbers of cells, survival time may be influenced by errors in technique, such as injection of dead or insufficient cells or seeding of extracranial sites. For these reasons, the search for newer and better models continues. Transgenic Models The ability to introduce specific genes into the germ lines of mammals is one of the major recent techno- logical advances in molecular biology. Jaenisch and Mintz, 6a in 1974, first reported experiments aimed at inserting foreign genes into mammals; they injected SV40 deoxyribonucleic acid (DNA) directly into the blastocyst cavity of developing mouse embryos. The resultant animals showed a mosaic pattern of SV40 DNA integration; however, the germ line did not dem- onstrate SV40 DNA. Other marginally successful el- forts at genome transfer experiments included embryo cell mixing and nucleus transfer. 4~i,57,sl Subsequently, infection of mouse embryos with retroviruses encoding foreign genes became possible ~9 and the standard means of introducing foreign genes into embryos has become direct microinjection. Microinjection has been made possible by the tre- mendous advances in DNA cloning technology, It is the most widely used and most successful means of generating animals with integrated foreign genes, termed "transgenic animals." By far the most com- monly utilized species for transgenic experiments is the mouse, although transgenic pigs, rabbits, sheep, and rats have been produced. 5~ The highly detailed tech- niques of transgene microinjection have been pub- lished previously, 52,5~,93,97 but a brief review is war- ranted. Fertilized e,,os~,~,, from timed malings are harvested from the oviducts of pregnant females, and briefly subjected to hyaluronidase to remove cumulus cells, yielding single-cell embryos at the pronuclear stage of development. Using micromanipulators, the male pronucleus is injected with roughly 500 linear copies of purified DNA coding for the gene of interest. These eggs are then transferred microsurgically into the oviducts of females made pseudopregnant by mating with vasectomized males. The resultant offspring are born after a 19-day gestation. The foreign DNA is usu- ally incorporated into the chromosome at random breaks, with multiple copies arranged in a head-to-tail fashion. If the foreign gone has undergone stable chro- mosomal integration at the one-cell stage, the trans- genic DNA will be present in every cell at the adult mouse. These animals, referred to as "transgenic founders," are identified by screening for the presence of the foreign gone in DNA from a segment of the tail. Transgenic founders may then be bred to give rise to progeny that will inherit the transgene in a stable, men- delian fashion. The success rate in developing transgenic animals is relatively poor. Microinjected transgenes integrate into the genome at random chromosomal breaks and, in the best of circumstances, the rate of incorporation in one-cell embryos approaches 20%. Only one-half of all transgenes successfully incorporated are expressed; those that are unexpressed are likely incorporated into translationally silent areas of genomic DNA. Occa- sionally, the chromosomal position of transgene inser- tion may result in expression of the transgene in un- expected tissues. Because of these factors, even in the best-designed experiment, only about 5% of offspring bear the transgene and express it in an appropriate tissue-specific fashion, s2 Early experiments with transgenic animals found that the crucial problem in their utility was the ex- tremely low or variable expression of the transgene products; this stimulated investigation into the biology of gene expression. It was soon realized that inclusion of prokaryotic vector sequences, necessary for clonal expansion of the foreign gene of interest in bacteria, inhibited transgene expression. 54 The importance of DNA elements that regulate gone expression, namely, the promoter and enhancer sequences, came under a great deal of scrutiny. It was found that the foreign DNA insert must contain a strong promoter sequence, which optimizes the potential for expression of the transgene. Tissue-specific enhancers are DNA se- quences whose presence near a gone of interest greatly enhances the expression of that gene. Enhancers may be upstream, downstream, or located in intervening nontranscribed segments of the gene (introns), and may be relatively short, such as the 72 base pair enhancer in the SV40 genome, s6 or substantially longer. They may be located immediately flanking the gene of in- terest or, as in the case of albumen, an element located 10 kb upstream of the promoter is required for liv- er-specific expression. 99 Appropriate promoter and en- hancer sequences are required in the transgenic 870 J. Neurosurg. / Volume 80 / Ma~; 1994 Animal models for brain tumors construct in order to enable tissue- or cell-specific ex- pression of the transgenc. One of the great remaining mysteries of molecular biology is the role and function of introns, the nontranscribed intervening sequences present in eukaryotic genes. Transgenic studies have shown that the incorporation of genomic sequences, including introns, gives a much higher expression of transgene DNA than does complementary DNA which is composed of coding sequences only and is derived from messenger ribonucleic acid. 74>J5 The types of transgenic models for CNS tumors have evolved over the last 10 or so years, and malay of the published models have arisen somewhat serendipi- tously. The first reported models utilized the expression of the SV40 early region genes, including the large T- and small t-antigens. Surprisingly, when the SV40 large T-antigen gene, regulated by its own promoter and en- hancer sequences, is placed in transgenic mice, tumors of the choroid plexus reproducibly develop.~D. L. Peterson, P. J. Sheridan, and W. E. Brown, Jr. these cells appears limited to GnRH; its release is stimulated by depolarization and norepinephrine and inhibited by tetrodotoxin, demonstrating the presence of fast Na + channels. Morphologically, these cells ex- hibit neuronal characteristics, with spontaneous exten- sion of neurites that end in growth cones or contact distant cells. Unfortunately, the gonads and accessory sex organs in these animals were underdeveloped, and they were infertile, suggesting that transgene expres- sion interfered with sexual maturation. Cell lines of the pituitary gonadotroph lineage have also been derived by targeted oncogenesis in transgenic mice. Windle, et aL, 12s developed a construct coding for 1.8 kb of the 5' flanking region of the gene for the human glycoprotein a-subunit linked to the SV40 T- antigen. More than one-half of the animals developed anterior pituitary tumors, and several stable cell lines were derived from these tumors. Windle, etal., antici- pated that the resulting cell lines would represent go- nadotrophs or thyrotrophs; interestingly, the tumors and cultured cells expressed only the a-subunit and did not express any of the/3-subunits. Several of the cell lines expressed the a-subunit in response to GnRH, but not thyrotropin-releasing hormone. It is thought that, as these animals develop with T-antigen expression in stem cells, the cells destined to become gonadotrophs or thyrotrophs are transformed before complete differ- entiation. The authors postulated that r expres- sion occurs only in completely differentiated anterior pituitary cells. This correlates with the previous obser- vation that a-subunit expression occurs in development before luteinizing hormone-/3 expression. It is interest- ing that a common type of pituitary tumor secretes only the a-subunit and is presumed to develop long after full differentiation of the anterior pituitary. This model therefore provides evidence for the concept of trans- formation-mediated dedifferentiation. The regulatory sequences of the phenylethanolamine N-methyltransferase gene were utilized by Hammang, et aL, 49 to direct T-antigen-related neurosecretory tu- morigenesis. These animals developed bilateral retinal tumors as well as adrenal pheochromocytomas by 12 weeks of age. A line of immortalized retinal neurons was derived from this model that demonstrate retinal neuron-specific markers, as well as neurofilaments. 2 To date, there are no reports of T-antigen-transformed de- paminergic neurons, but one could postulate the value of such a line in the experimental study of Parkinson's disease. The SV40 antigen-related models have provided a great deal of insight into regulatory element specificity and tumorigenesis in general; however, molecular ge- netic analysis of brain tumors provides a much more complicated story. An ever-increasing number of proto-oncogenes have been identified in brain tumors. Amplification of oncogenes, s alterations in ploidy, chromosomal deletions, 61 substitutions, double min- utes, 9 and tumor suppressor gene mutations are among the complex genetic alterations seen. Some of the spe- cific oncogenes amplified in malignant gliomas are neu, N-myc, 43 C-myc, 3s gli, al and C-erb-~3 (epidermal growth factor receptor). 11,34,56 Mutations of the tumor suppressor p53 are well documented, 44 and other tumor suppressor genes are postulated to exist on chromo- somes 10 and 22. 35,61.121 Intuitively, models expressing tumor-associated on- cogenes in a tissue-specific fashion may provide more insight into the complex process of oncogenesis than the T-antigen models. Many transgenic models have used tissue-specific expression of tumor-related onco- genes to assess their role in tumorigenesis. The work of Pattengale, et al.,97 in the field of mammary tumor- igenesis is the most well known; they constructed trans- genes of the neu, ras, and myc oncogenes driven by the murine mammary tumor virus (MuMTV) promoter, and developed highly reproducible murine mammary tumors. Using different lineages derived from each transgenic construct, they demonstrated that the geno- type would predict the phenotype of the resultant mam- mary tumor. 23 Histopathologically, the tumors could be divided into three categories; small cell, large cell, and intermediate cell. The tumors for the MulVlTV/ras lin- eage were all small cell, the MuMTV/myc tumors were mainly composed of large cells, and the cells of the MuMTV/neu line were of intermediate size. Unfortunately, such extensive work using glial- directed expression of oncogenes has not yet been re- ported; however, glial-specific regulatory sequences have been identified and utilized in transgenic studies. Mucke, et aL,87 reported a transgenic mouse model that shed light on the astrocytic response to focal trauma. The murine GFAP promoter was fused to the bacterial reporter gene/3-galactosidase. The/3-galactosidase ac- tivity can be readily detected in tissue sections and has no harmful effects in vivo. This model showed baseline GFAP activity in the hippocampus, glial limitans, and along white-matter tracts including the optic nerves, and GFAP expression was proven to be induced within 1 hour of focal injury (needlestick in right frontal lobe). Our laboratory is currently developing transgenic lines that express the temperature-sensitive mutant p53 tu- mor suppressor gene N-myc and the epidermal growth factor receptor gene under the control of the GFAP promoter. Myelin basic protein (MBP) regulatory se- quences have been studied in transgenic models, and two groups have published models demonstrating cor- rection of the phenotype of the shiverer mouse. 65,1~ The homozygous shiverer (shi/shi) mouse is an auto- somal recessive mutant characterized by the almost to- tal lack of CNS myelin and all MBP's. By 2 weeks of age, the animals show the neurological dysfunction of shivering, develop tonic convulsions, and die at an early age. Readhead, et aL, TM and Kimura, etaL, 65 have published models demonstrating that introduction of the gene for MBP in homozygous shiverer embryos re- sults in substantial resolution of their neurological phe- notype, although MBP production was only 25% to 50% of that in wild-type mice. The only published transgenic model for glial neo- plasia was reported by Hayes, et al. 53 In this excellent study, the transgene construct was composed of the neu oncogene under the control of the MBP transcriptional 872 J. Neurosurg. / Volume 80 / May, 1994 Animal models for brain tumors regulatory elements, with the goal of obtaining trans- formed oligodendrocytes. The neu oncogene is a mem- brane protein that belongs to the tyrosine kinase family of oncogenes (which includes the epidermal growth factor receptor homolog, C-erb-[3). Of note, the neu (.~acogene was originally isolated and cloned from a se- ries of rat glioblastoma cell lines induced by ENU? 12 The MBP/neu animals displayed a low incidence of tumor development, but tumors from three animals were examined histologically and immunocytochemi- cally. The pathological appearance of the tumors was that of a high-grade glioma, with necrosis, giant cells, and fine eosinophilic processes. None of the tmnors exhibited the classic histopathological features of oli- godendrogliomas. One tumor provided a stable cell line in culture; these cells, when cultured in variable media concentrations, may resemble either type 2 astrocytes or oligodendrocytes that result from the differentia- tion of the O-2A bipotential progenitor cell. The O-2A cells differentiate into oligodendrocytes constitutively, and into astrocytes in the presence of high serum concentrations. 72,m~ The MBP/neu cells will express oligodendrocyte-specific antigens and appear morpho- logically similar to oligodendrocytes when cultured in media that favors oligodendrocyte survival. When cul- tured in high serum concentrations, they express abun- dant GFAP as well as MBP and do not react with oli- godendrocyte-specific antibodies. The retained MBP expression in these cells implies that they were trans- formed farther along the oligodendrocyte lineage than at the level of the O-2A progenitor, These cells hold promise for further studies in that they express sig- nificant amounts of MBE We speculate that, by modu- lating the transforming effecl of neu, these cells may be able 1o myelinate axons in culture. The transgenic models described here have not yet provided opportunities for the evaluation of new ad- junctive therapies, such as new chemotherapeutic agents, hyperthermia, or alternative forms of irradia- tion. They do, however, provide a great deal of insight into the processes of differentiation and oncogenesis. It is evident that one problem with the current models is that stem cells are transformed at an early stage of development, prior to differentiation. Transformation of cells in the adult animal would enhance the utility of the model in therapeutic screening. Future efforts will be directed at identifying tissue-specific regulatory sequences inducible by exogenous substances or con- ditions. An example of an inducible promoter is that for the uniquitous protein metallothionein, whose promoter can be induced by metals or glucocorticoids. The identification of a glial-specific inducible promot- er would allow more controlled oncogenesis. 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Cancer Res 1:91 ~)-938, 1941 Manuscript received December 10, 1992. Accepted in final form December 29, 1993. Address reprint requests to: Willis E. Brown, Jr., M.D., Di- vision of Neurosurgery, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7843. 876 J. Neurosurg. / Volume 80 / May, I994


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