[CANCER RESEARCH 45, 2827-2833, June 1985) Growth of Human Lung Tumor in the Brain of the Nude Rat as a Model to Evaluate Antitumor Agent Delivery across the Blood-Brain Barrier1 Edward A. Neuwelt,2 Eugene P. Frenkel, Anthony N. D'Agostino, Desmond N. Carney, John D. Minna, Peggy A. Barnett, and Christopher I. McCormick Department ol Surgery. Division of Neurosurgery, Oregon Health Sciences University, Portland, Oregon 97201 [E. A. N., P. A. B., C. I. M.]; Department of Internal Medicine, Division of Hematology -Oncology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235 [E. P. F.¡;Department of Pathology, Good Samaritan Hospital and Medical Center, Portland, Oregon 97210 [A. N. D.]; and NCI-Navy Medical Oncology Branch, Division of Cancer Treatment/NIH, Naval Hospital, Bethesda, Maryland 20814 [D. N. C., J. D. M.] ABSTRACT human tumors as xenografts. For instance, intracerebral growth of human glioma in immunosuppressed rats results in a highly We have developed a brain tumor model in the nude rat utilizing permeable tumor (4) unlike the situation clinically (6, 7) or in a NCI-N417D human small cell carcinoma of the lung grown both virally induced animal glioma model (8, 9). intracerebrally and s.c. The median latency period from the time The introduction of the athymic nude mouse (10) offered a of intracerebral tumor inoculation to the onset of neurological new opportunity to study the growth and behavior of human symptoms is 13 days with an intracerebral tumor take rate of tumor cells in vivo under controlled conditions (11). A variety of 91% (29 of 32). The median survival is 13 days, and all animals human tumors have been established and serially passed in the were dead by Day 26. The tumor is discrete, well circumscribed, athymic mouse (12). The histology and responsiveness for most with occasional leptomeningeal spread and with minimal evi tumors in the nude mouse are similar to those in humans, and dence of surrounding cerebral edema. Intracerebrally, this tumor validation of this model for the study of a variety of biological is usually impermeable to Evan's blue:albumin (M, 68,500) but features of human tumors has been achieved (13). Tumors so not fluorescein (M, 376). Although variable, the intracerebral grown show a differential sensitivity to cytoreductive chemo- tumor is less permeable to methotrexate than is the same tumor therapeutic agents that reflects the clinical effectiveness of these grown s.c. in the same animal (P < 0.005). The intraarterial and agents in the original patient. For instance, Giovanella et al. (14) i.v. routes of methotrexate administration in the presence and evaluated the efficacy of 9 chemotherapeutic agents on 3 types absence of blood-brain barrier opening were evaluated. Drug of human tumor xenografts implanted in the nude mouse (breast delivery to the intracerebral tumor and ipsilateral brain was cancer, colon cancer, and melanoma); tumor regressions were significantly (P < 0.025) greater when the methotrexate was consistent with the clinical experience in patients except for 2 given intraarterially and was significantly (P < 0.0025) increased false-positive and one false-negative values. Histological evalu after osmotic blood-brain barrier opening. After barrier opening, ation of human tumor xenografts has been reported to maintain methotrexate concentration was enhanced 3- to 4-fold in tumor morphological fidelity with the parent tumor (13). In addition, and 10- to 20-fold in brain around tumor. Thus, the nude rat several reports (15-17) have shown that xenografts of human provides a model to investigate the biology and therapeutic tumors can be consistently transplanted intracerebrally into the responsiveness of human small cell carcinoma of the lung grown nude mouse, and Chambers ef al. (18) have reported the intra- intracerebrally where it develops a blood-tumor barrier similar to cranial growth of cells from small cell carcinoma of the lung in that seen in humans. This model further provides the unique nude mice. One possible disadvantage of the nude mouse is that opportunity to investigate the role of osmotic blood-brain barrier human tumors which have a high propensity to metastasize do opening in the treatment of a tumor which is sensitive to chem- not metastasize in nude animals. otherapeutic agents and for which tumor-specific monoclonal Unfortunately, the small size of the nude mouse makes it a antibodies are available. very difficult model for the study of complex multimodality ther apeutic approaches to ¡ntracerebral tumors. The ideal model would permit human xenografts to be examined at both intra INTRODUCTION cerebral and systemic sites, and the intracerebral site should develop the biological characteristics of spontaneous primary or Although Greene (1, 2) reported heterotransplantation of hu metastatic tumors, i.e., to demonstrate at least a partial BBB3 man brain tumors in 1951, attempts to implant and grow spon (8, 9, 19, 20). In addition, animal size should permit a variety of taneous human tumors intracerebrally in laboratory animals have therapeutic manipulations, including i.a. (9, 21-23) infusions, been inconsistent (3) and generally unsuccessful. Neither condi opening of the BBB (6-8, 24, 25), and applicability for studies of tioning with immunosuppressive agents (4) nor utilization of the "targeted" therapy, such as with monoclonal antibodies (26-29). subarachnoid space of the animal as a growth chamber (5) The nude rat provided the appropriate structural requirements, provides a satisfactory model for the study of gliomas or other and human small cell (oat cell) xenografts provided the accept 1This work was supported by the Oregon Medical Research Foundation, able tumor for the development of a therapeutic model. The National Cancer Institute Grant CA31770, Good Samaritan Hospital Foundation, nude rat has immunological features like those of the nude NIH Grant CA 23115, Meadows Foundation, and the Southwestern Medical Foun- dation-Kinsler Williamson Brown Fund. mouse (30, 31). Thus, the congenitally athymic nude rat accepts 2 To whom requests for reprints should be addressed, at Division of Neurosur allografts and xenografts, and their splenic lymphocytes respond gery (L472), Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201. 3 The abbreviations used are: BBB, blood-brain barrier; MTX, methotrexate; Received 8/6/84; revised 11/14/84. 2/28/85; accepted 3/11/85. CNS, central nervous system; i.a., intraarterial(ly); i.e. intracarotid. CANCER RESEARCH VOL. 45 JUNE 1985 2827 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1985 American Association for Cancer Research. NUDE RAT BRAIN TUMOR poorly to T-cell mitogens. Unlike the nude mouse, the nude rat BBB Opening in the Nude Rat. BBB opening in the nude rat was is less susceptible to infections and is thereby easier to maintain. performed and graded as previously described (8). Five min before BBB The nude rat does not provide as good a growth medium for opening, Evans blue (2%, 2 ml/kg, i.v.) and/or fluorescein (10%, 0.12 to tumor xenografts (31) as the mouse, perhaps as the result of a 0.25 ml, i.v.) was administered. MTX Delivery to Human Tumor in Nude Rats. Animals symptomatic higher level of natural killer cells (32, 33). However, a variety of from intracerebral tumor were randomly divided into 4 groups: Group I, different human cancers (e.g., carcinoma of the breast, gastroin i.v. MTX after i.e. 0.9% NaCI solution (saline) (BBB intact); Group II, i.e. testinal tract, melanoma, and rhabdomyosarcoma) have been MTX after i.e. saline (BBB intact): Group III, i.v. MTX after i.e. mannitol grown in the nude rat (34, 35). (BBB opened); or Group IV, i.e. MTX after i.e. mannitol (BBB opened). In The present studies focused on the small cell carcinoma (oat all groups, MTX (4 mg/kg) was administered immediately after i.e. saline cell) of the humarj lung, since this carcinoma is a common tumor or mannitol over 1 min. Thirty min after MTX administration, a serum which frequently metastasizes to the CNS and is responsive to sample was obtained, and the animals were sacrificed with an overdose a variety of chemotherapeutic agents. In addition, a number of of sodium pentobarbital. Tumor, brain around tumor (1 to 2 mm of brain tumor-specific monoclonal antibodies have been developed to immediately surrounding tumor), brain distant to tumor (coronal section this neoplasm (36,37). The human small cell tumors were grown of the occipital lobe) in the perfused hemisphere as well as in the contralateral brain (coronal section of occipital lobe), s.c. tumor, and in the nude rat, and histology, survival, and the delivery of a other organs and tissues were obtained for MTX determinations. MTX chemotherapeutic agent (MTX) were examined. These studies content in serum and tissue was determined by radioimmunoassay as have shown the feasibility of the nude rat as a xenograft model previously described (22). to examine this common human tumor and to evaluate strategies The effect of the intracerebral implantation technique on BBB integrity of therapy by comparing the delivery of drugs to the tumor in was examined in rats inoculated with lethally irradiated NCI-N417D cells the brain with the tumor at s.c. sites. In particular, this model (8000 to 9000 rads, using a 137Cssource). On Day 14 after intracerebral allows an evaluation of the role of BBB modification in anti-tumor inoculation, Evans blue, fluorescein, and MTX were administered i.v. as agent delivery. described above. Statistical Analysis. MTX levels are expressed in ng/g of tissue or ng/ml of serum as the mean ±SE. Comparisons of the log concentration MATERIALS AND METHODS of tissues in the irradiated inoculum control group (Table 1) and within the 4 treatment groups (Table 2) were made by the one-way analysis of variance followed by the Neuman-Keuls multiple comparisons when the Nude Rat Colony.