Herpes Simplex Virus Thymidine Kinase Gene Therapy in Experimental Rat BT4C Glioma Model

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Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: Effect of the percentage of thymidine kinase-positive glioma cells on treatment effect, survival time, and tissue reactions

Anu-Maaria Sandmair,1,2 Marita Turunen,1,3 Kristiina Tyynela¨,1,3 Sami Loimas,1 Pauli Vainio,4 Ritva Vanninen,4 Matti Vapalahti,2,5 Rolf Bjerkvig,6 Juhani Ja¨nne,1,5 and Seppo Yla¨-Herttuala1,5,7 1A.I. Virtanen Institute, University of Kuopio, Kuopio, Finland; Departments of 2Neurosurgery, 3Oncology, and 4Clinical Radiology, and 5Gene Therapy Unit, University Hospital of Kuopio, Kuopio, Finland; 6Department of Anatomy and Cell Biology, University of Bergen, Bergen, Norway; and 7Department of Medicine, University Hospital of Kuopio, Kuopio, Finland.

Herpes simplex virus thymidine kinase (HSV-tk) gene transfer and ganciclovir (GCV) administration have been suggested for the treatment of malignant gliomas. To understand tissue responses and possible ways to improve the treatment effect, we studied tumor growth, tissue reactions, and survival time after HSV-tk/GCV treatment in a syngeneic BT4C rat glioma model by mixing various ratios of stably transfected HSV-tk-expressing BT4C-tk glioma cells with wild-type BT4C glioma cells (percentage of BT4C-tk cells: 0%, 1%, 10%, 30%, 50%, and 100%), followed by injection into BDIX rat brains (n ϭ 79). With the exception of some animals with end-stage tumors, very little astroglia or microglia reactivity was detected in the wild-type tumors as analyzed by immunocytochemistry using glial fibrillary acid protein (GFAP)-, vimentin-, human histocompatibility leukocyte antigen-DR-, OX-42-, and CD68-specific monoclonal antibodies. After 14 days of GCV treatment, tumors induced with Ն10% BT4C-tk cells showed a significant reduction in tumor size (P Ͻ .05) and prolonged survival time (P Ͻ .01). Astrogliosis, as indicated by a strong GFAP and vimentin immunoreactivity, was seen in the tumor scar area. GFAP and vimentin reactivity was already present after the GCV treatment in tumors induced with 1% BT4C-tk cells. Much less human histocompatibility leukocyte antigen-DR-positive microglia was seen in the treated animals, indicating low microglia reactivity and immunoactivation against the tumor. However, GCV-treated tumors were positive for apoptosis, indicating that apoptosis is an important mechanism for cell death in the BT4C-tk glioma model. Our results suggest that Ն10% transfection efficiency is required for a successful reduction in BT4C glioma tumor size with HSV-tk/GCV treatment in vivo. Tissue reactions after 14 days of GCV treatment are characterized by astrogliosis and apoptosis, whereas microglia response and immunoactivation of the brain cells appear to play a minor role. Stimulation of the microglia response by gene transfer or other means might improve the efficacy of the HSV-tk/GCV treatment in vivo. Cancer Gene Therapy (2000) 7, 413–421

Key words: Apoptosis; gene transfer; bystander effect; glial ﬁbrillary acid protein; astroglia; microglia; nuclear magnetic resonance imaging; thymidine kinase; glioma.

espite aggressive treatment, the prognosis of pa- During the last 15 years, the prognosis of glioblastoma Dtients with malignant glioma is extremely poor. The has not changed despite improvements in surgery and median survival time is 14 months, and a reasonable radiation therapy.3 Thus, new therapeutic approaches quality of life is limited to even shorter periods of time. are urgently needed. Glioblastoma is the most aggressive glioma, with an Gene therapy may offer a new strategy for the treat- average survival time of 10 months.1 In recurrent glio- ment of malignant gliomas. The most widely used gene blastoma, mortality is nearly 100% within a few months.2 therapy approach for the treatment of glioma involves the transfer of herpes simplex virus thymidine kinase (HSV-tk), followed by ganciclovir (GCV) administra- 4–7 Received August 5,1998; accepted July 24, 1999. tion. HSV-tk can be delivered into the brain with Address correspondence and reprint requests to Dr. Seppo Yla¨-Hert- many vectors, such as plasmid-liposomes, retroviruses, 8,9 tuala, A.I. Virtanen Institute, University of Kuopio, P.O. Box 1627, retroviral packaging cells, and adenoviruses. Brain FIN-70211 Kuopio, Finland. tumors are well suited for retrovirus-mediated gene

Cancer Gene Therapy, Vol 7, No 3, 2000: pp 413–421 413 414 SANDMAIR, TURUNEN, TYYNELA¨ , ET AL: EFFECT OF HSV-TK-POSITIVE CELLS ON MALIGNANT GLIOMAS therapy, because neurons show no proliferative activity and retroviral vectors can only transfer genes to prolif- erating cells.5,10 Adenoviruses have also successfully been used for gene transfer to malignant gliomas.11,12 Transfer of the HSV-tk gene renders cells sensitive to GCV treatment. HSV-tk catalyzes the phosphorylation of GCV to a cytotoxic nucleotide analog that blocks DNA replication and kills transfected cells.4,5 The therapeutic effect of HSV-tk is further enhanced by the so-called bystander effect, in which the toxic effect spreads from the transfected cells to the neighboring untransfected cells.11,13 The mechanism of the bystander effect is not fully understood, but may involve the release Figure 1. Structure of the pLTKSN plasmid. The plasmid is com- posed of Moloney murine sarcoma virus LTR (5ЈLTR), an extended of cytotoxic nucleotide analogs to neighboring cells packaging signal (␸ϩ), HSV-tk cDNA, simian virus 40 early region through gap junctions or activation of inflammatory and promoter (SV40 ep), neomycin phosphotransferase (NEO) cDNA, immunological responses mediated by cytokines and and the Moloney murine leukemia virus LTR (3ЈLTR). The rest of the 14 surrounding brain cells. However, very little data are plasmid is derived from pBR322 plasmid with an ampicillin resis- available with regard to tissue reactions (astroglia, mi- tance (amp) gene. croglia, inflammatory cells) in the HSV-tk/GCV-treated tumors. Knowledge about the type and extent of cellular reactivity should help us to improve the efficacy of the HSV-tk/GCV treatment effect. A typical feature of human malignant glioma is that it is virtually invisible to the human immune system. Several rat glioma models have been used to model malignant glioma tumors in experimental studies; however, many widely used syngeneic models, such as 9L gliosarcoma, can be immunogenic.15 Another widely used model, C6 glioma, is not syngeneic and induces immune responses that can affect therapeutic efficacy and survival.15 In the present study, we describe the BT4C rat glioma model for the evaluation of the effectiveness of HSV-tk/ GCV treatment and tissue reactions in the BDIX rat brain. The model resembles human glioma in many aspects, including its ability to grow without inducing any significant tissue reactions against the tumor. Our results show that Ն10% HSV-tk-positive glioma cells are required for a successful treatment effect. Tissue reactions after the HSV-tk/GCV treatment were characterized by astrogliosis and apoptosis, with only moderate involvement of microglia and immunoactivation of brain cells.

MATERIALS AND METHODS Cell lines and viral vectors A BT4C rat glioma cell line was derived from fetal BDIX rat brain cells, which were transferred to monolayer cell culture shortly after in vivo exposure to N-ethylnitrosourea. BT4C cells have been characterized elsewhere.16 The cells were grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM) with 10% fetal calf sera, 1% penicillin/streptomycin, and nonessential amino Figure 2. Effectiveness of HSV-tk gene transfer and GCV treatment acids (Life Technologies, Paisley, UK). A total of 1.2 kb of in stably transfected rat BT4C-tk glioma cells. A: The optimal HSV-tk cDNA17 (GenBank accession no. V00470) was sub- concentration of GCV was determined in BT4C-tk cells by plating cloned into EcoRI-XhoI sites of the pLXSN retroviral plas- 1 ϫ 104 cells on 35-mm plates and incubating the cells with mid18 (kindly provided by Dr. A. Dusty Miller, Fred Hutchin- indicated concentrations of GCV for 4 days. The 50% lethal dose for son Cancer Research Center, Seattle, Wash) (Fig 1). The GCV in transfected cells is ϳ0.1 ␮g/mL. B: The bystander effect was expression of the HSV-tk cDNA is driven by a 5Ј Moloney studied in vitro by mixing different percentages of BT4C-tk cells and murine sarcoma virus long terminal repeat (LTR). A PA317 wt BT4C cells, followed by GCV incubation (2 ␮g/mL) for 6 days. packaging cell line18 was used for the packaging of replication- Each experiment was performed in triplicate and repeated twice deﬁcient amphotropic viruses. A stable producer clone (clone (mean Ϯ SD).

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3.0 D5) was obtained after geneticin selection. Viral titers of 1, 1 ϫ 104 cells were seeded on 35-mm wells (Nunc, Roskilde, 1 ϫ 105 to 1 ϫ 106 colony-forming units/mL were produced by Denmark). Growth medium containing the indicated concen- the selected clone 3.0 D5. A similar virus lacking HSV-tk trations of GCV was added 24 hours later and replaced on day cDNA but containing the neomycin resistance gene was pro- 3. Cells were trypsinized on day 5, and the number of cells in duced in PA317 cells and used as a control “empty” virus (titer each well was counted (Bu¨rker, Marienfeld, Germany). The of 0.5–1 ϫ 106 colony-forming units/mL). Viral titers were bystander effect was evaluated in vitro by mixing increasing determined in 209F fibroblasts as described previously.19 Be- percentages (1%, 5%, 10%, 30%, 50%, and 100%) of BT4C- fore use, the packaging cell lines and virus lots were shown to tk-positive glioma cells with wt BT4C glioma cells. On day 1, be free of any helper virus activity, endotoxin, mycoplasma, or cell mixtures were seeded at a total of 4 ϫ 105 cells per 35-mm other types of microbiological contaminants.20 well. Growth medium containing 2 ␮g/mL GCV was added 24 hours later and replaced every 2 days. After 6 days of GCV In vitro studies treatment, cells were suspended and counted as described above. Results are presented as percentages of the number of Rat BT4C glioma cells were transduced with replication- treated cells from the number of wt cells cultured without deficient HSV-tk retroviruses or control empty retroviruses. GCV under identical conditions. Stably transduced BT4C-tk and BT4C control cell lines were 18 isolated using standard techniques. Sensitivity to GCV treat- In vivo studies ment of the BT4C-tk cell line and wild-type BT4C control cell line was determined in vitro using five different drug concen- Intracranial implantations of BT4C glioma cells were per- trations: 0 ␮g/mL, 0.01 ␮g/mL, 0.1 ␮g/mL, 1 ␮g/mL, 10 ␮g/mL, formed in inbred male BDIX rats (290–410 g). Because the and 100 ␮g/mL (Cymevene, Syntex Nordica Ab, So¨derta¨lje, BT4C glioma cell line was originally derived from BDIX rats, Sweden). Stably transduced BT4C cell lines and wild-type (wt) the model does not induce any adverse host-tumor tissue or BT4C cells were grown in DMEM as described above. On day immunological reactions.21 The rats were anesthetized intra-

Figure 3. Characterization of the glioma model using MRI (A,D,G), hematoxylin-eosin staining (B,E,H), and GFAP immunocytochemistry (C,F,I). A–C: A group I animal at 10 days after the injection of wt BT4C glioma cells (i.e., at the time when the GCV treatment was started). Despite the presence of the tumor, GFAP staining shows no astrocytosis around the tumor. D–F: A group I animal at 21 days after the wt BT4C glioma cell injection shows a large tumor with slight hydrocephalus. Only weak GFAP reactivity is present (arrow). G–I: A group V animal (30% BT4C-tk-positive cells) at 13 days after the glioma cell injection and 3 days after the beginning of GCV treatment. MRI detects a small tumor, and GFAP staining shows astroglia reaction around the tumor (arrows). MRI (T1-weighted coronal slices after contrast enhancement) was performed Յ24 hours before the animals were sacriﬁced. Control immunostainings with class- and species-matched Igs and incubations in which primary Abs were omitted were negative (data not shown). Original magniﬁcation is ϫ5 for (A,B,D,E,G,H) and ϫ100 for (C,F,I).

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peritoneally (i.p.) with 0.4 mL/100 g of a solution containing Tumor sizes were determined with a computerized image 4.25% chloral hydrate and 0.97% pentobarbital and placed in analysis system (MCID Software, Imaging Research, St. Ca- a stereotactic apparatus (Kopf, Berlin, Germany). A burr hole tharines, Ontario, Canada) from histological sections by mea- was done 1 mm posterior to the bregma and 2 mm to the right suring the tumor area (pixels) at the level of the injection site. of the midline. The dura was incised with a needle. For cell The tumor area was standardized against the total brain area injections, BT4C-tk and wt glioma cells were trypsinized, measured from the same section. counted, and collected by centrifugation. The cells were resus- pended in DMEM to a final concentration of 10,000 cells in 5 Magnetic resonance imaging (MRI) ␮L. Cells were injected (104 cells in 5 ␮L of DMEM) with a MRI was used for the visualization of the tumors. For imaging, 10-␮L Hamilton syringe (Hamilton, Bonaduz Ab, Switzerland) that was placed in a microinjection unit of the stereotactic the rats were anesthetized as described above. The rats re- frame. Injection was made at the depth of 3 mm within the ceived contrast enhancer i.p. (Magnevist, License Schering AG 22 Pharma, Berlin, Germany; 0.3 mL in 0.7 mL of physiological right corpus callosum. To avoid backflow of the tumor cells, salt solution), and MRI was performed with a superconducting the injection was done during a 5-minute period; the needle whole body system (Siemens, Vision, Erfangen, Germany) was left in place for another 5 minutes before removal. Skin operating at 1.5 T using a small field of view surface coil. T1- incision was closed with stitches (4-0 Dexon, Davis and Geck, and T2-weighted spin-echo sequences were used. The imaging Hampshire, UK). parameters for the T1-weighted images were as follows: 500/20 The rats were divided into six groups, which were used for (repetition time, TR/echo time, TE; ms) with four acquisitions morphometric and immunocytochemical analysis as follows: and imaging time of 8:36 minutes. Proton and T2-weighted Group I (n ϭ 10) received wt BT4C glioma cells; group II (n ϭ images were acquired with a TR/TE of 2000/20 and 76 ms 3) received BT4C control cells transfected with empty virus; (imaging time of 8:36 minutes) with one acquisition. Slice group III (n ϭ 4) received 1% BT4C-tk cells and 99% wt BT4C thickness was 2 mm with both sequences, and a 60- to 100-mm cells; group IV (n ϭ 5) received 10% BT4C-tk cells and 90% field of view was used. A 256 ϫ 256 imaging matrix yielded wt BT4C cells; group V (n ϭ 5) received 30% BT4C-tk cells in-plane resolution of 0.23–0.39 mm. and 70% wt BT4C cells; and group VI (n ϭ 13) received only BT4C-tk cells. For the survival study, the following number of Statistical analysis animals was used: Group I (n ϭ 6), group II (n ϭ 3), group III (n ϭ 6), group IV (n ϭ 7), group V (n ϭ 6), and group VI (n ϭ The Kruskal-Wallis test for pairwise comparisons between 11). In the survival study, group V consisted of animals injected tumor sizes in the treatment groups was made using BMDP 1.0 with 50% BT4C-tk cells and 50% wt BT4C cells. All animals in software for Windows (Imaging Research, St. Catharine’s, groups II-VI received i.p. GCV treatment (25 mg/kg) twice a Ontario, Canada). Statistical analyses for survival data were day for 2 weeks. The treatment was started 10 days after the performed using the SPSS for Windows (SPSS Inc., Chicago, glioma cell injection. Group I (control group) showed the IL) log-rank test for Kaplan-Meier plots. natural course of the disease. Three additional control animals with wt tumors were sacrificed 10 days after the tumor cell injections to study early tissue reactions against the tumor. RESULTS Animals for morphological analyses were sacrificed after the GCV treatment period. Animals in the survival study were HSV-tk and GCV treatment in BT4C glioma cells followed for 6 months. in vitro The efficacy of the HSV-tk gene transfer and GCV Immunocytochemistry and image analysis treatment was verified in BT4C cell cultures. A total of ␮ The rats were perfused with 100 mL of phosphate-buffered 1 g/mL GCV was able to kill 91% of the BT4C-tk saline and 4% paraformaldehyde for 10 minutes. The brains glioma cells in 4 days, whereas no effect was detected in were dissected and divided at the injection site into two wt BT4C cells at a low concentration of GCV (Fig 2A). coronal pieces. The tissue samples were further fixed in 4% In separate experiments, no effect of GCV treatment on paraformaldehyde for 18 hours and rinsed overnight in 15% BT4C control cells transfected with empty virus was sucrose.23 Paraffin sections were either stained with hematox- detected (data not shown). The bystander effect was ylin-eosin (BDH, Poole, Dorset, UK) or immunostained with evaluated in vitro by mixing different percentages of monoclonal antibodies (mAbs) against glial fibrillary acid BT4C-tk-positive glioma cells with wt BT4C glioma protein (GFAP) (dilution 1/100, Boehringer-Mannheim, cells. Cultures were then subjected to GCV treatment Mannheim, Germany), CD68 (dilution 1/100, Dako, Glostrup, for 6 days. Our results showed that 10% and 30% of Denmark), EBM-11 (dilution 1/25, Dako), OX-42 (dilution 1/500, Serotec, Kidlington, Oxon, UK), or human histocom- BT4C-tk glioma cells were able to kill 78% and 86% of patibility leukocyte antigen (HLA)-DR CR43 (dilution 1/100, all cells (Fig 2B). Dako). With some antibodies (Abs), microwave oven incubation in citric acid buffer was used to improve the immunostain- Characterization of the BT4C glioma model in vivo ings. An avidin-biotin-horseradish peroxidase system (Vector Histology, immunocytochemistry, and MRI were used Elite, Vector Laboratories, Burlingame, Calif) was used for for the characterization of the BT4C rat glioma model in signal detection. Apoptosis was detected using an ApopTag vivo (Fig 3). All rats in the control group (group I) Plus kit (Oncor, Gaithersburg, Md). Rat spleens and lymph ϫ 4 nodes and normal human tonsils and brain tissue obtained injected with 1 10 wt BT4C glioma cells developed from biopsy material were used as positive controls for immu- macroscopic tumors. It has been determined previously nostainings. The negative controls included incubations with that the mean survival time of the animals with wt glass- and species-matched unrelated immunoglobulins (Igs) tumors is 39 days (range 18–60 days, A.-M.S., K.T., and and incubations in which primary Abs were omitted.23,24 S.Y.-H. unpublished observations). The site, size, and

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Table 1. Tumor Sizes and Tissue Responses in Different Treatment Groups

Group II Group I (BT4C control Group III Group IV Group V Group VI (BT4C wt empty virus (1% BT4C- (10% BT4C- (30% BT4C- (100% BT4C- cells) cells) tk cells) tk cells) tk cells) tk cells)

Number of animals Total 10 3 4 5 5 13 With intracerebral 10 3 4 0 1 0 tumors at the site of injection With tumors at 10 3 4 2 1 0 the brain cortex Tumor size 5190 Ϯ 3748 501 Ϯ 43 792 Ϯ 679 22 Ϯ 30* 16 Ϯ 35* 0 Ϯ 0* Astroglia response ϩ ϩϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩϩ Microglia response ϩϪϪϪϪϪ The size (pixels) of the tumors was measured with an image analysis system as described in Materials and Methods. To analyze tissue reactions during the natural course of the disease, group I animals were allowed to develop end-stage tumors and were not included in the statistical analysis. Tumor size in group II versus group III: not signiﬁcant; group II versus group IV: P Ͻ .05; group II versus group V: P Ͻ .01 (Kruskal-Wallis test). Astroglia and microglia responses were evaluated after theءء:P Ͻ .05; group II versus group VIء immunostaining of 10 randomly selected sections from each animal for GFAP and vimentin (astroglia) and for CD68 and OX-42 (microglia). Ϫ, absent or weakly positive in less than half of the sections; ϩ, weakly positive in more than half of the sections; ϩϩ, moderately positive in all sections; ϩϩϩ, strongly positive in all sections.

shape of the tumors in the contrast-enhanced MRI observed at the injection site in all group VI animals images correlated well with the histological findings (Fig (Fig 4L). 3). Immunocytochemistry showed that at the early stage, GFAP and vimentin immunocytochemistry (Fig 3C; animals with wt tumors had no immunostaining in the Fig 4, B and C) in group I showed a limited astroglia tumor area for GFAP or vimentin (see below), indicat- reaction in the wt tumors. With only 1% BT4C-tk- ing a low level of astroglia reactivity against the tumor positive cells (group III), the frequency of positive (Table 1; Fig 3C). At the late stage, some positive GFAP and vimentin immunostainings had clearly in- astroglia reactivity was observed (Table 1; Fig 3F). Very creased around the tumor (Fig 4, E and F). This finding little microglia reactivity, as evaluated by CD68, HLA- indicates that even if the tumor cannot be eradicated, DR, and OX-42 immunocytochemistry, was present in the treated tumor stimulates astroglia reactions. Only a the tumor area (Table 1, see below). Thus, the rat BT4C few HLA-DR-positive cells (Fig 5, A and B) or OX-42- glioma model resembles human glioblastoma in that it positive cells (data not shown) were detected in any of does not induce strong tissue reactions against the the tissue sections, indicating that microglia activation tumor. However, GFAP (Fig 3I) and vimentin (data not and/or an influx of blood monocyte-macrophages does shown) immunostainings were clearly detectable around not play a major role in defense and tissue reactions at the tumor after gene therapy only 3 days after the the time of sacrifice (Table 1). However, apoptotic cells beginning of the GCV treatment. were more frequently detected in the treated tumors than in the wt glioma (Fig 5, C and D). The findings Characterization of the treatment effect in the BT4C suggest that apoptosis is an important mechanism for glioma model in vivo cell death after HSV-tk/GCV treatment. Macroscopic tumors were present in all group III (1% The treatment effect was already visible in group IV BT4C-tk-positive cells) rats (Table 1; Fig 4, D–F). In (10% BT4C-tk-positive cells), in which only two animals group IV (10% BT4C-tk-positive cells), two of five rats had residual tumors at the brain cortex. These cortical had small residual tumors at the brain cortex that were tumors were counted as recurrent tumors and were probably caused by spilling of the tumor cells through included in the measurements (Table 1). The tumor the injection canal. Intracerebral tumors in the deeper sizes were small compared with the tumors in group I-III areas of brain were not detected in group IV animals, animals (Table 1; Fig 4, G–I). The treatment effect was but a low cell density scar was present at the site of also evident in group V (30% BT4C-tk-positive cells). injection (Table 1; Fig 4, G–I). In group V (30% Thus, it appears that Ն10% BT4C-tk-positive cells can BT4C-tk-positive cells), one of five rats had a small lead to a successful reduction in tumor size. However, in tumor at the brain cortex. The same animal also had one treated animal in Group V, remaining glioma cells some remaining tumor cells within the scar area. No were still detected at the brain cortex; only 100% other tumors were detected in group V animals. The BT4C-tk cells could be fully eradicated with the GCV group VI (100% BT4C-tk-positive cells) animals had no treatment (Table 1). tumors (Table 1; Fig 4, J–L). A scar formation was All animals that were left for a long-term follow-up in

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Figure 4. Characterization of HSV-tk and GCV treatment effect in tumors induced with various ratios of BT4C-tk and wt BT4C glioma cells in vivo. Representative micrographs from groups induced with 0, 1, 10, and 100% of BT4C-tk glioma cells with wt BT4C glioma cells, followed by treatment with GCV, are shown. A–C: Group I (control group with wt BT4C cells); D–F: Group III (1% BT4C-tk-positive cells); G–I: Group IV (10% BT4C-tk-positive cells); J–L: Group VI (100% BT4C-tk-positive cells). Hematoxylin-eosin staining (A,D,G,J), and GFAP (B,E,H,K) and vimentin (C,F,I,L) immunostainings. Control immunostainings with class- and species-matched Igs and incubations in which primary Abs were omitted were negative (data not shown). Original magnification is ϫ5 for (A,D,G,J), ϫ100 for (B,C,E,F,H,K,L), and ϫ200 for (I). group VI (HSV-tk 100%) survived for Ͼ6 months. BDIX rats. The model was chosen because the wt BT4C Survival was also significantly (P Ͻ .01) prolonged with cells mimic human malignant glioma in that they induce animals with 10% and 50% HSV-tk-positive cells (Fig very few tissue reactions when injected into the brain of 6). No difference was found in survival between wt BT4C a BDIX rat. Also, MRI can be used to follow-up the glioma cells and empty virus-transfected control BT4C growth of the tumors without sacrificing the animals, and cells (Fig 6). BT4C glioma cells can easily be transfected with Molo- ney murine-derived retroviruses or adenoviruses.25 MRI was also used to ensure the existence of tumors before DISCUSSION GCV treatment. Thus, the model is very useful for studies related to human glioblastoma. Current treatment strategies have failed to improve the Our results show that, both in vitro and in vivo, Ն10% prognosis of patients with glioblastoma, which is one of BT4C-tk-expressing cells among wt BT4C glioma cells the most lethal human cancers. Gene therapy may offer are required for a beneficial GCV treatment effect. The a new strategy for the treatment of malignant glioma. In results are in line with previously published findings this study we used the syngeneic BT4C glioma model in from 9L and C6 glioma models5,26–30 and indicate that

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Animals in group VI (100% BT4C-tk-positive cells) were followed for up to 6 months without tumor recurrence. Significantly prolonged survival in animals with 10–50% HSV-tk-positive cells was also observed. The results correlate well with histological findings from animals sacrificed after the GCV treatment. It is note- worthy that in group V (50% BT4C-tk-positive cells), one animal still had some cancer cells in the treated area; in addition, residual tumors at the brain cortex were present in two group IV (10% BT4C-tk-positive cells) animals. It is likely that tumor recurrence will occur in some HSV-tk/GCV-treated animals even if the gene transfer efficiency is high. Thus, despite the effective short-term antitumoral effects, HSV-tk/GCV treat- Figure 5. Characterization of HLA-DR immunoreactivity and apo- ment may not solve the problem of tumor recurrence. It ptosis in BT4C gliomas in vivo. Representative micrographs from is possible that the long-term survival could be improved group I (A–C) and group III (D) animals are shown. A: Immunostain- by stimulation of an immune response toward inoculated ing for HLA-DR (arrows). B: Nonimmune control for (A) (first Ab glioma cells; in other glioma models that have shown a omitted); C: apoptotic cells in an end-stage tumor (arrows). D: better treatment effect, such as 9L, rich cellular infil- Increased frequency of apoptotic cells in an HSV-tk/GCV-treated trates containing microglia, macrophages, and T cells ϫ animal (arrows). Original magnification is 200. have been reported after HSV-tk/GCV treatment.34 Our results show that tissue reactions against stably trans- the bystander effect also occurs in the BT4C cells. Our fected BT4C-tk glioma cells are almost exclusively de- results confirm the important role of the bystander effect rived from astroglia. Apoptosis seems to be an important in the treatment of experimental gliomas.31 9L and C6 mechanism for cell death, as also demonstrated by glioma cells that express HSV-tk have been eliminated others.35–38 However, only limited CD68, HLA-DR, or in nude mice or rat brains after the GCV treatment, and OX-42 immunoreactivity (microglia or macrophages) complete remissions have been reported in 50–78% of was detected in the treated area, which is a normal the treated animals.5,26–30 However, there are also re- feature for BT4C tumors. A better understanding of ports indicating that HSV-tk gene transfer followed by tissue reactions, of the time course of the immunological GCV treatment does not lead to a complete eradication responses, and of patterns of gene expression in cells of glioma in the rat brain.32,33 The reason for the surrounding the tumor may prove essential for the discrepancy in HSV-tk/GCV results is currently unclear, improvement of the treatment effect. but may involve different cell lines or conditions used for With current gene transfer techniques, Ն10% trans- the gene transfer and GCV treatment. Also, differences fection efficiency can be obtained in the vicinity of the in tissue reactions in various experimental glioma mod- gene transfer site in human glioma tumors in vivo.20 els probably affect the final outcome.15 However, the overall transfection efficiency in human glioma tumors is still very low because the number of cells in the vicinity of the transfection site is low compared with the total tumor mass.20 According to our results and evidence published elsewhere,7,32,33 it is likely that in human clinical trials based on HSV-tk/ GCV treatment, residual glioma cells will remain in the brain. Because astrogliosis and apoptosis are the major responses in the BDIX rat brain after the cytotoxic effect of GCV treatment, it is unlikely that effective immune responses will develop toward malignant cells. Accord- ingly, stimulation of microglia-related inflammatory and immunological reactions by combined gene transfer of HSV-tk and cytokines such as interleukin-2 (IL-2), IL-3, IL-12, or macrophage colony-stimulating factor might improve the effectiveness of the HSV-tk/GCV treatment, especially when only low transfection efficiencies Figure 6. Kaplan-Meier survival plots of the treated animals. Per- can be obtained. centage figures refer to the amount of HSV-tk-positive tumor cells at the time of tumor inoculation. Group I versus group II: not significant; group I versus group III: not significant; group I versus group IV, P Ͻ .01; group I versus group V, P Ͻ .01; group I versus group ACKNOWLEDGMENTS VI, P Ͻ .01 (log rank test for Kaplan-Meier survival plots). Number of animals studied in each group: I ϭ 6, II ϭ 3, III ϭ 6, IV ϭ 7, V ϭ 6, We thank Dr. A. Dusty Miller for pLXSN vector; Drs. Jukka and VI ϭ 11. Luoma, Pauliina Lehtolainen, Leo Palja¨rvi, and Asla Pitka¨nen

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