Disseminating Tumor Cells and Their Interactions ... - Cancer Research

[CANCER RESEARCH 52, 1018-1025. Februars 15. 1992]

Disseminating Tumor Cells and Their Interactions with Leukocytes Visualized in the Brain1 Lois A. Lampson,2 Patrick Wen, Victoria A. Roman, James H. Morris,3 and Jacob A. Sarid Departments of Medicine (Neurologi') [L. A. L., P. W., V. A. R.¡,Pathology ¡J.H. M.J, and Surgery (J. A. S.J, Brigham and Women's Hospital, and the Departments of Neurology [L. A. L., P. W., V. A. R.J, Pathology fJ. H. M.J, and Surgery fJ. A. SJ, Han-ard Medical School, Boston, Massachusetts 02115

ABSTRACT Brain tumors are increasingly prevalent. Recent advances focus atten tion on individual, disseminated tumor cells that cannot be imaged or eliminated. Cells of the immune system may be ideally suited to attack individual tumor cells, but more basic understanding is needed. We describe a rat model, using the lac'/, reporter gene, that allows identifi cation of individual tumor cells, and tumor-leukocyte interactions in vivo. The model demonstrates how widely tumor can disseminate, without secondary tumorigenesis or recruitment of nonneoplastic cells. It dem onstrates that leukocytes have access to disseminating tumor. Among its many applications, this work lays a foundation for developing cellmediated immunotherapy against individual brain tumor cells.

INTRODUCTION Brain tumors are the second most common group of cancers in children and are increasingly prevalent among the elderly (1). As new therapies are better able to eliminate a brain tumor mass, outlying or infiltrating tumor cells become more impor tant as sources of tumor recurrence (2). These disseminated cells may be particularly appropriate targets for cell-mediated immunotherapy. Cells of the immune system are able to move through normal tissue and recognize and selectively destroy abnormal targets. Although brain tumors may secrete immunosuppressive factors (3, 4), the local concentrations would be reduced at sites of disseminated tumor. Yet, in most cases individual outlying or infiltrating cells cannot be identified in situ by conventional histológica! or immunocytochemical techniques. Here, we have adapted the use of the Escherichia coli lacZ reporter gene to visualize individual tumor cells (5-10), following the cells as they dissem inate through the brain. Double labeling is used to reveal the interactions between the disseminating tumor and immunocompetent cells. We describe the extent and pattern of tumor spread and patterns of tumor-leukocyte interactions. MATERIALS AND METHODS Modified Tumor Cells. The parent cell line was the 9L gliosarcoma line (11). The cells were infected with the BAG replication-deficient retroviral vector carrying the E. coli lacZ gene encoding /3-gal4and the Tn5 neomycin resistance gene (which confers resistance to the drug G418), following the procedures of Walsh and Cepko (5). The Ebag2 retrovirus-producing cell line was the generous gift of Dr. Cepko. Three ml of Ebag2 supernatant (from cells grown in Dulbecco's modified Eagle's medium/10% fetal calf serum) were used to infect IO6 9L cells Received 7/8/91 ; accepted 12/3/91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by grants to L. A. L. from the National Institute of Neurological and Communicative Diseases and Stroke (NS25638. NS24878) and from the Alex Coffin Fund for Brain Tumor Research. A preliminary report appeared as Ref. 31. 2 To whom requests for reprints should be addressed, at Division of Neurology, 116 LMRC, Brigham and Women's Hospital. Boston. MA 02115. 3 Present address: Department of Neuropathology. Radcliffe Infirmary, Wood stock Road, Oxford OX2 6HE. England. 4 The abbreviations used are: fi-gal. fi-galactosidase; MHC. major histocompatibility complex; LCA, leukocyte common antigen: CSF, cerebrospinal fluid.

(plated 1 day previously onto a 100 x 15 mm Petri dish) in the presence of polybrene (final concentration, 8 ng/ml). After 4 h, 9 ml of fresh medium were added. After an additional 48 h, the 9L cells were split 1:4 into fresh Dulbecco's modified Eagle's medium/10% fetal calf serum containing G418 (1 mg/ml). The cells were kept growing under these selective conditions for 14 days. The G418-resistant colonies were pooled and grown out in a fresh Petri dish, from which six colonies were selected for testing. The chosen colonies were grown in 24-well plates, fixed within the wells, and evaluated for constitutive /3-gal expression by a histochemical reaction (below), performed within the wells. Five of the colonies showed strong staining for /J-gal, and the sixth showed weaker staining. One colony, now called 9L/lacZ, which constitutively produces high levels of the enzyme, was selected for in vivo study. Stability in Vitro. The /3-gal production by the 9L/lacZ line has remained stable through freezing and thawing and passage in culture. Most recently, over 2 years after the line was established, an aliquot that had been thawed and grown up for injection into rats was found to contain 98% /3-gar cells. (With prolonged growth in culture, greater numbers of negative variants do appear, as would be expected, requiring recloning of the line. However, this has not been a problem in our hands during the period required to grow cells for injection from frozen aliquots.) Tumor Growth in Vivo. Tumor cells were injected stereotactically into the right caudate nucleus of syngeneic CD Fischer rats (12). Each rat received 2 x IO4cells in a volume of 10 ¡t\,delivered with a 10-^1 Hamilton syringe. For the time course study, 20 rats received injections of 9L/lacZ cells. One or two rats were sacrificed on days 1-4, 6-10, 13-17, and 22. (Ten of the rats received 9L cells expressing an alter native marker in addition to the 9L/lacZ cells. No differences in the properties reported were noted, and only 0-gal expression was evaluated for these studies; these rats were not included in the evaluation of the frequency with which /3-gal-negative variants arise from the 9L/lacZ line.) To evaluate tumor growth, 6-ji sections were cut through the region of brain containing the injection site (after processing as described below). Sections taken at intervals (every other section at the earliest times and greater intervals at later days) were stained for /3-gal activity, as described below. Intervening sections were stored at —¿70°C for subsequent labeling with monoclonal antibodies. Typically, 20 slides, encompassing the area of maximum tumor growth, were evaluated for each rat. Light hematoxylin counterstain was used on most slides, and standard hematoxylin and eosin stains were made on additional slides from each brain. Tissue Processing. At the time of sacrifice, rats were perfused with 2% paraformaldehyde fixative (5). The brains were removed and cryoprotected with 30% sucrose. Cryostat sections, 6 M thick, were mounted on poly-D-lysine-coated slides. A histochemical reaction for /J-gal, using 5-bromo-4-chloro-3-indolyl-0-D-galactopyranoside (Xgal) as the substrate (5), was performed on the sections. For double labeling, after processing for /3-gal histochemistry, slides were immediately proc essed for immunocytochemistry for LCA, using the OX1 mouse mono clonal antibody, or for class II MHC antigens, using the OX6 mouse monoclonal antibody (12). Both monoclonals were used in the form of culture supernatant (Accurate Scientific). Irrelevant antibodies or plasmacytoma-secreted mouse immunoglobulin were used as negative con trols (12). Slides were examined using a Nikon Optiphot microscope equipped with a x 1 objective and flip-out condenser for scanning (final magnification. xlO) and with differential interference contrast (DIC or "Nomarski") optics for photomicrography at higher power (final mag nification, X200-1000).

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after tumor growth in the brain for 3 weeks (13-15). This is also consistent with reports from other laboratories, where the lacZ gene has been introduced by either retroviral infection or by transfection (5-10). Thus, the developing experience indi cates that /3-gal can serve as a sufficiently stable tumor marker for many kinds of studies.

RESULTS Preliminary Characterization of /8-gal+ Tumor Cells in Vivo Appearance and Specificity of the /8-gal Marker. In order to visualize individual tumor cells, the gliosarcoma-derived line 9L was infected with a retroviral vector containing the E. coliderived lacZ gene for /3-gal (see "Materials and Methods"). To

Patterns of Tumor Spread

evaluate the appearance and specificity of the marker, 9L/lacZ cells, or the 9L parent line, were injected stereotactically into the right caudate nucleus of CD Fischer rats. The rats were sacrificed on day 21, and 6-n cryostat sections cut through the brain encompassing the injection site were processed for /i-gal histochemistry. Rats from the tumor dissemination study (be low), from which the illustrations are taken, as well as other rats studied subsequently, gave the same results. The histochemical reaction revealed /3-gal as a cell-filling cytoplasmic marker in the tumor cells. Single tumor cells could be identified at the lowest power (final magnification, xlO). An overview of tumor growing in the brain is illustrated in Fig. \A. Pictures of tumor taken at higher magnification are illustrated in Fig. 1, B-D. Under the assay conditions used, the intense cytoplasmic reaction product was specific to the 9L/lacZ tumor cells. In the control rats that received injections of unmodified 9L, neither unmodified 9L cells nor surrounding brain cells showed the intense blue-green cell-filling reaction product (not shown). The contrast between the bright blue /O-gal*tumor cells

To examine tumor spread in this model, rats were sacrificed from 1 to 22 days after implantation of 9L/lacZ cells into the right caudate nucleus. Sections cut at regular intervals were analyzed by histochemistry for /3-gal as well as with conven tional stains (see "Materials and Methods"). As described above, /3-gal+ tumor cells were detected in every brain, and independent areas of /3-gal-negative tumor, which would have been revealed by the conventional stains, were not seen. The tumor cells were seen to spread through the brain in several different ways. Illustrations of disseminating tumor in animals sacrificed 2-17 days after tumor injection are shown in Figs. 1-3. In each case, the tumor is identified by the bright blue product of the histochemical stain for /3-gal. Overviews of tumor growing out from a main mass near the injection site are seen in Figs. \A and "LA.Examples of characteristic patterns of

and the surrounding brain is illustrated in all panels of Fig. 1. This specificity of staining is in agreement with reports from other laboratories, including analysis of /3-gal+ C6 glioma cells and other /3-gal+cells growing in the brain (5-10). Stability of the /î-gal Marker in Vivo. The information in this section, based on analysis of the rats that received injections of 9L/lacZ cells for this report (see "Materials and Methods"), is also representative of our subsequent experience with this cell line (13-15). For each rat that received intracerebral injection of 9L/lacZ cells, brain sections were evaluated by standard histológica! stains as well as by histochemical staining for /3-gal (see "Materials and Methods"). Most of the sections were both stained for /3-gal and lightly counterstained with hematoxylin. In addition, conventional hematoxylin and eosin sections were prepared for each brain. Tumor expressing the /3-gal marker was detected in every rat that received an injection of 9L/lacZ cells; the last rat was sacrificed after 22 days. All slides were scanned carefully for additional tumor that might be revealed by the conventional stains. Either hematoxylin and eosin or hematoxylin alone is well able to reveal even fairly small tumor masses (as confirmed by our own studies of the parent 9L line (12, 16), although they would not normally reveal single tumor cells. Independent areas of /3-gal-negative tumor were not seen. Within the largest tumor masses, heterogeneity in the level of blue staining was sometimes seen. Double labeling for /3-gal and leukocyte antigens often revealed that the /3-gal-negative cells in these masses were inflammatory cells rather than tumor cells. An example is seen in Fig. 2/1, where a mass of brown inflammatory cells is seen amid the large mass of blue tumor. Damaged or necrotic tissue may also be present in these regions. For example, in Fig. 1/1, the blue staining is less intense where the edge of the needle wound merges with the main tumor mass. Thus, the 9L/lacZ cells retained their ability to express the 0-gal marker for at least 22 days in vivo, which is consistent with other reports (5-10). Our subsequent studies have con firmed the low incidence of outgrowth by 0-gal-negative variants

dissemination beyond the main tumor mass are seen in Figs. IB and 3. High-power views of disseminating tumor in char acteristic locations are found in Fig. 1, B-D. Detailed Description of Tumor Spread. In these studies, tumor was injected stereotactically near the lateral ventricle (Fig. \A). Tumor cells grew out from the injection site into both grey and white matter, just as is seen with the parent line (12, 16). The tumor was often seen to spread in the white matter tract underlying the ventricle; a particularly clear example is seen in Fig. 3A. A second common pattern of tumor spread was along the blood vessel walls. A high-power view of tumor surrounding a vessel lumen is seen in Fig. \B. A lower-power overview of tumor growing along several elongated vessels is seen in Fig. IB. In some cases, the vessel lumen was distended to an extent not seen in normal brain. The microscopic examination made it apparent that tumor was growing in the perivascular (Virchow-Robin) space and not within the lumina of the vessels. This pattern of spread has been well documented for the parent cell line (17-18) and for human tumors in the brain (19). Tumor cells also spread within the CSF. Tumor cells were seen within the ventricles (Fig. ID) and at ventricle walls distant from the injection site (Figs. 1C and 3Ä),even on the opposite side of the brain. Examination of sequential sections often revealed that the wall of the ipsilateral ventricle had been breached by the injection. Thus, the data here do not provide evidence for tumor growing out from the brain into the ventri cle. However, the fact that tumor was seen growing through the ventricle wall on the side of the brain opposite that of the injection site does imply that the cells were able to seed the CSF and invade the brain at a distant site. CSF dissemination of the parent line has been described previously (20, 21) and is also seen in human tumors (22, 23). All of the tumor described above was identified by its expression of the /i-gal marker. The presence of the marker confirmed that tumor at even the most distant sites was derived from the original inoculum. In summary, the patterns of spread seen with the /3-gal+cells are the same as those seen with the parent 9L line, which has been characterized extensively (17, 18, 20, 21), as was con firmed by evaluation of rats injected with the parent line in our

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A

Fig. I. Appearance and dissemination of identifiable brain tumor cells. I. bright blue tumor cells growing out from the injection site 6 days after 9L/lacZ cells were injected stereotactically into the right caudate nucleus. There is a typical pattern of tumor spread, including spread through white matter parallel to the ventricle and along blood vessels. B, cross-sectional view of bright blue tumor cells growing around a blood vessel. In C and O, tumor cells have seeded the CSF. In C, bright blue tumor cells are seen at the edge of the ventricle wall. 2 days after tumor implantation. Brown MHC class II* cells within the choroid plexus are revealed by immunocytochemistry. as described in Fig. 2. In D, a clump of bright blue tumor cells is seen within the ventricle, near the wall. Photographed at x 200 (A) or x 400 (B-C). D.I.C. ("Nomarski") optics: light hematoxylin counterstain.

laboratory (12, 16). Yet the 0-gaI marker permitted more rapid and confident identification of small numbers of tumor cells, and identification of even single tumor cells, which is consistent with what has been found in other contexts (5-10). Tumor-Leukocyte Interactions Double labeling was used to define interactions between disseminating tumor and inflammatory cells. Intervening sec tions from the brains evaluated above were stained for 0-gal

plus either of two monoclonal antibodies. The antibody to LCA identifies all subpopulations of leukocytes, including lympho cytes and mononuclear phagocytes. The antibody to a monomorphic determinant on rat MHC class II proteins (OX6) identifies mononuclear phagocytes. LCA+ cells were present from day 1, throughout the period of tumor growth. LCA* cells could be seen both within and at the periphery of the main tumor mass growing near the injec tion site. Although LCA+ cells were always found to be associ-

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Fig. 2. Overviews of disseminating tumor and tumor-leukocyte interactions. A, bright blue .•¿' u.il' tumor cells spreading out from injection site 10 days after tumor injection. (Injection site, which does not appear in the figure, is just below the large mass of tumor.) Brown leukocytes, stained with antibody to LCA, can be seen within and at the periphery of the large tumor mass and at sites of spreading tumor, as described in the text. In B. bright blue .IM!' tumor cells are seen growing along blood vessels, distant from the injection site. 14 days after tumor injection. Brown mononuclear phagocytes, stained with antibody to class II MHC antigen, can be seen in the meninges (top) and in contact with vessel-associated tumor. D.I.C. ("Nomarski") optics: light hematoxylin counterstain.

ated with tumor growing near the injection site, the number of LCA+ cells varied among the different tumors and in different areas of a single tumor. An example is seen in Fig. 2A. Individ ual brown LCA+ cells are seen all around the periphery of the main tumor mass, although at different densities in different areas. LCA+ cells are also seen amid the mass of growing tumor, again at different densities in different areas. LCA+ cells were also seen to be associated with extensions of tumor into adjacent brain. For example, LCA+ cells can be seen to be associated with the extension of tumor seen to the reader's left of the large mass in Fig. 2A.

LCA* cells were also seen at a still greater distance from the injection site, even on the opposite side of the brain. Their numbers were usually reduced at a distance from the main tumor mass. An example of the leukocytes revealed at a distance from the injection site, using an antibody that gave the same result as anti-LCA, is shown in Fig. 2Ä,as discussed below. From day 3, MHC class IT cells had the same distribution as the LCA+ cells. This is consistent with the abundance of mononuclear phagocytes found to be associated with the parent cell line (12, 16) and with human brain tumors (see "Discus sion") (24). An example of MHC class II+ cells associated with

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B Fig. 3. Inflammatory cells need not be directly adjacent to tumor. I. tumor growing in white matter adjacent to the lateral ventricle 4 days after tumor injection. (The injection site, not seen in the figure, is below and to the reader's left.) Immunocytochemistry reveals brown LCA* leukocytes adjacent to the tumor and also extending beyond it. B, tumor growing near the ventricle at day 17. (The injection site, not seen in the figure, is below and to the reader's right.) Bright blue tumor and brown MHC class II* mononuclear phagocytes can be seen in the same area but do not overlap. MHC class II* cells can also be seen in the choroid plexus.

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tumor growing at a distance from the injection site is seen in Fig. 2B. Dissemination of the bright blue tumor cells, especially along blood vessels, is seen. Although not as abundant as in regions closer to the injection site, individual brown MHC class ir cells are seen to be associated with the blue tumor cells. Larger numbers of MHC class II* cells are seen to be associated with tumor at the edge of the brain (Fig. IB, top), consistent with the larger number of inflammatory cells typically found in the meninges. Outside of the injection site, the LCA* and MHC class ir cells were seen only in areas of tumor growth. They were not seen in tumor-free areas of the brain. Nor were they seen beyond the injection site in control animals receiving buffer but not tumor (12, 16). Although LCA+ and MHC class II* cells were limited to areas of tumor growth, they were not always directly adjacent to tumor cells. Tumor cells alone, concentrations of inflam matory cells alone, and inflammatory cells closely associated with tumor cells could all be seen within a given area. Examples are seen in Fig. 3. In Fig. 3A, bright blue tumor is seen spreading along the white matter underlying the lateral ventricle on the injection side. This is a typical pattern of spread under the injection conditions used, as discussed above. Brown LCA* cells can be seen juxtaposed to the tumor mass and also extend ing beyond the tumor. Adjacent sections stained for MHC class II showed the same pattern (not shown). In Fig. 3fi, bright blue tumor cells are again seen growing near the wall of the lateral ventricle, in this case, at a later time (day 17) than in Fig. 3.4. Brown MHC class II* cells are seen between the larger tumor masses and extending to the edge of the ventricle. Within the ventricle, MHC class II+ cells are also seen in the choroid plexus, where MHC class II* mononuclear phagocytes (epiplexus cells) are known to be present in normal brain (25). The distribution of the MHC class II* cells suggests that they may have migrated from the choroid plexus to the tumor area (26). Taken together, the observed distributions demonstrate that LCA* and MHC class II* cells are able to come into contact with disseminating tumor. The distributions are heterogeneous even within areas of a single tumor, and the LCA* or MHC class II* cells are not always directly adjacent to tumor cells. The implications of these distributions are discussed below. DISCUSSION Brain tumors are increasingly prevalent in the population (1). Advances in the treatment of a well-defined tumor mass have led to renewed interest in immunotherapy as an adjunct therapy for the control of residual tumor (15). Yet individual outlying or infiltrating cells cannot usually be identified in situ by con ventional techniques. The E. coli lacZ reporter gene has now been used to reveal cells in many contexts, including glioma cell lines in the brain, and dissemination of micrometastases in lung and other organs (5-10). Here, we extend the reported work, describing how the use of the reporter gene has enabled us to follow patterns of tumor spread through the brain and to reveal interactions between tumor and inflammatory cells. The syngeneic system described is suitable for immunological studies. In our model system, the tumor cells spread through the brain by the same routes as seen with human tumors (Fig. 1) (19, 22, 23). The cells grow by direct extension into grey and white matter and spread along white matter tracts. The tumor also spreads along the blood vessels, in the perivascular space.

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Using yet another route, tumor cells seed the CSF, spread through the ventricular system, and invade the brain at the ventricle wall distant from the injection site. In separate studies, we have found that the tumor is also able to spread through the leptomeninges.5 These patterns of spread are also seen with the parent 9L line (12, 16-18, 20, 21). The presence of the ß-gal marker in all areas of tumor spread confirmed that tumor at even the most distant sites was derived from the original inoculum. When human tumors are seen to be widely disseminated within the brain, it is often suggested that a secondary wave of neoplasia may have been initiated or that dividing cells may have been "recruited" from the surrounding brain. Our studies do not rule out these potential secondary sources of abnormal growth. However, they do illustrate how widely tumor may disseminate through the brain, in a case where all areas of tumor growth can be shown to derive from a single source. A secondary wave of tumorigenesis or recruitment of nonneoplastic cells need not be implied. Human brain tumors show variable levels of leukocyte infil tration at the time of biopsy or autopsy (24). Little is known about tumor-leukocyte interactions, and the potential for their manipulation, during the course of tumor growth or in areas distant from the main tumor mass. In our model, double labeling revealed that tumor-leukocyte interactions can occur at a distance from the injection site, although the numbers of inflammatory cells adjacent to disseminated tumor are usually much lower than at the main tumor mass (compare Fig. 2A to Fig. IB). The antibody staining patterns seen here are consistent with the abundance of mononuclear phagocytes found to be associated with human brain tumors (24) as well as with the parent 9L line (12, 16). A more detailed analysis of leukocyte subpopulations will be reported separately. The important point here is that leukocytes do have access to disseminating tumor cells within the brain. A provocative aspect of the tumor-leukocyte distributions was that, although inflammatory cells were limited to areas of tumor growth, they were not always directly adjacent to tumor cells. Tumor cells alone, concentrations of inflammatory cells alone, and inflammatory cells closely associated with tumor cells could all be seen within a given area (Fig. 3). A related finding was that leukocytes showed a heterogeneous distribu tion within areas of a single tumor mass (Fig. 2A). These distributions suggest that inflammatory cells may ac cumulate in response to secondary or disseminated signals, rather than recognizing the tumor cells per se (27). Possibilities for further study include leukocyte recognition of tissue damage caused by the spreading tumor or of cytokines secreted by the tumor cells or adjacent cells. The presence of a source of inflammatory cells, such as damaged blood vessels or the cho roid plexus (Fig. 3ß),may contribute to greater leukocyte concentrations in some areas. An additional factor, suggested by the distribution of LCA* or MHC class II* cells in Fig. 3 (A and B), is that concentrations of leukocytes not adjacent to tumor may be in the process of moving toward the tumor area. Manipulations that change the patterns of leukocyte traffic in the brain may help to define the underlying mechanisms governing tumor-leukocyte interactions ( 16,28, 29). The effects on tumor growth of increasing the frequency of direct tumorleukocyte interactions must also be defined, as discussed below. The patterns of tumor growth and the presence of inflam matory cells described here are consistent with previous descrip tions of both human tumors and of unmodified 9L cells within

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the brain (12, 16-24). As in other experimental systems, intro duction of the lacZ reporter gene and expression of the /i-gal protein do not appear to have altered the patterns of cell growth (5-10). Yet the model system has allowed identification of individual tumor cells, and definition of tumor-leukocyte inter actions at sites of disseminated tumor, that could not be defined easily by conventional techniques. Our studies have not revealed qualitative differences in the immunological behavior of the /3-gar cells and the parent 9L cell line. While /3-galactosidase is a well-characterized immunogen in other contexts, the parent line itself is also immunogenic (20, 30). Despite this immunogenicity, the /3-gal+ cells, like the parent line, are able to grow and ultimately cause the death of the host after implantation into the brain or skin. The inflammatory response reported does not depend upon the presence of the /3-gal protein. Abundant inflammatory cells are found to be associated with the parent 9L tumor as it grows in the brain or skin and are also associated with human brain tumors (12, 16, 24). The expression of E. co//-derived ß-galby the tumor cells does offer an important advantage for immunological studies. Few well-characterized brain tumor antigens have been defined, either for 9L or other experimental systems. In separate studies, we have shown that, in addition to acting as a tumor marker, 0-gal is able to serve as a well-defined tumor antigen in this system (14). In these studies, no immunization or other immune manip ulation was attempted. Under those conditions, the presence of inflammatory cells did not prevent tumor growth or spread. In recent work, we have shown that cytokine treatment can cause selective changes in the immune environment at each of the anatomic sites of tumor growth, including the grey and white matter, the perivascular space, and the ventricle walls (15, 28, 29). Using the lacZ reporter system as described here, the effects of immune manipulations on the growth and spread of dissem inating brain tumor cells can now be directly evaluated. The model will be valuable for the analysis of many other aspects of tumor immunology and tumor biology as well. ACKNOWLEDGMENTS We thank Dr. Connie Cepko for her gift of the Ebag2 cell line and for sharing her laboratory's procedures and Dana Dunne for her excel lent technical assistance.

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