- Email: [email protected]
Antitumor activity of interleukin 12 against interleukin 2-transduced mouse glioma cells
Cancer Letters 135 (1999) 47±51
Antitumor activity of interleukin 12 against interleukin 2transduced mouse glioma cells Tetsuro Kikuchi a, b,*, Tatsuhiro Joki b, Yasuharu Akasaki b, Toshiaki Abe b, Tsuneya Ohno a a
Division of Oncology, The Institute of DNA Medicine, Jikei University, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105, Japan b Department of Neurosurgery, Jikei University, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105, Japan Received 18 June 1998; received in revised form 18 August 1998; accepted 19 August 1998
Abstract We subcutaneously inoculated parental and glioma cells genetically engineered to express interleukin-2 (SR/IL-2) into syngeneic mice. The tumor growth of the transfectants was slower than that of the parental cells. We then stereotactically inoculated transfectants into the brains of mice. The survival of the mice injected with parental cells was shorter than that of the mice inoculated with transfectants. SR/IL-2 cells were inoculated subcutaneously into the ¯ank of mice, after which rmIL-12 was administered intraperitoneally (i.p.). The resultant transient tumor growth was followed by regression. rmIL-12 or saline were then injected i.p. into mice that had been inoculated in the brain with SR/IL-2 cells. There was no signi®cant difference in survival time between the treated and control groups. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Immunotherapy; Interleukin 2; Interleukin 12; Glioma; Gene transfection
1. Introduction Malignant astrocytoma is the most common primary brain tumor in adults. The median survival of patients with malignant astrocytomas (high-grade astrocytomas) is about 1±2 years, despite aggressive treatment that includes surgical resection, radiotherapy, and cytotoxic chemotherapy [1]. Therefore, to prolong this survival, novel therapeutic approaches must be pursued. Immunotherapy is one such approach, and has been investigated using different types of tumors including brain tumors. In the central nervous system (CNS), considered an immunologi* Corresponding author. Division of Oncology, The Institute of DNA Medicine, Jikei University, 3-25-8 Nishishinbashi, Minatoku, Tokyo 105, Japan. Fax: 1 81-3-3459-6412; e-mail: [email protected].
cally privileged site, vaccination with interleukin 4-, interleukin 7-, or granulocyte-macrophage colonystimulating factor-producing tumor cells has been effective in an experimental mouse model [2,3]. Interleukin 12 (IL-12) enhances the lytic activity of natural killer (NK)/lymphokine-activated killer (LAK) cells, facilitates speci®c cytotoxic T lymphocyte (CTL) responses, acts as a growth factor for activated T and NK cells, induces interferon-g from T and NK cells, and acts as an angiogenesis inhibitor [4,5]. Although IL-12 itself activates strong antitumor activity [6±9], the combination of vaccine therapy with IL-2-transduced tumor cells and systemic recombinant mouse IL-12 (rmIL-12) cured mice bearing wild-type parental tumors more effectively than did rmIL-12 and IL-2-transduced tumor vaccines alone [10±12].
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(98)00268-7
48
T. Kikuchi et al. / Cancer Letters 135 (1999) 47±51
In the present study, we analyzed the antitumor effects of rmIL-12 on glioma cells genetically engineered to produce IL-2. 2. Materials and methods 2.1. Cell lines and transfection
Dulbecco's phosphate-buffered saline (D-PBS; Cosmo Bio.), suspended in serum-free D-MEM at a density of 1 £ 107 /ml. Cells (1 £ 106 ) were inoculated subcutaneously (s.c.) into the ¯ank of B10.A mice (Day 0). rmIL-12 (0.5 mg/100 ml) or saline, as a control, was injected intraperitoneally (i.p.) every other day for 2 weeks starting on day 14 (total 3.5 mg/mouse). Tumor size was measured at the longest
The mouse glioma cell line, SR-B10.A, provided by Dr. K. Sakamoto, has been described in detail elsewhere [13]. This cell line was maintained in monolayer cultures in DMEM (Cosmo Bio Co., Tokyo, Japan) supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Gaithersburg, MD). Human IL-2 cDNA was ampli®ed using a polymerase chain reaction method and subcloned into a pcDNA3 expression vector (Invitrogen, NV Leek, The Netherlands; designated pcDNA/IL-2, provided by Dr. H. Suzuki). SR-B10.A cells were transfected with pcDNA/IL-2 using a lipofectin reagent (GIBCO) according to the manufacturer's instructions. All transfectants were selected in 1000 mg/ml G418 (Sigma Chemicals, St. Louis, MO, USA) and cloned by limiting dilution to obtain stable IL-2 expression. The IL-2 production from transfectants was determined by an enzyme-linked immunoabsorbent assay (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. The concentration of IL2 was 286 pg/ml in clone 21, designated SR/IL-2. This clone was selected for the following experiments. 2.2. Animals Female B10.A mice were purchased from Shizuoka Laboratory Inc. (Shizuoka, Japan), and nude and severe combined immuno-de®ciency (SCID) mice were purchased from CLEA Japan (Tokyo). The mice were maintained in a speci®c pathogen-free room at 25 ^ 38C. Female mice aged 6 weeks (body weight 25 ^ 2 g) were used in the experiments. 2.3. IL-12 administration Recombinant mouse IL-12 (rmIL-12) was provided by Genetics Institute (Cambridge, MA). A total of 1 £ 106 exponentially growing SR-B10.A or IL-2- transduced glioma cells were harvested, washed twice with
Fig. 1. Tumorigenesis of parental and transfectant glioma cells. (A) While parental cells formed tumors that progressively increased in size (W), tumor growth of IL-2 transfectants was slower (A). Tumor volume in surviving mice is shown (mean ^ SD, n 10). Mice inoculated with transfectants had smaller tumors than the control mice at each time point (P , 0:05). (B) All mice injected with parental cells into the brain died within 3±6 weeks (W), whereas all those inoculated with transfectants survived over 7 weeks and died within 14 weeks (A) (n 10 each).
T. Kikuchi et al. / Cancer Letters 135 (1999) 47±51
49 4
Subsequently, 1 £ 10 parental or transfectant cells were inoculated stereotactically into the right frontal lobe of the brain of syngeneic mice. A 0.5 mg aliquot of rmIL-12 was injected i.p. every day from days 5 to 11 (total 3.5 mg/mouse). As a control, saline was administered instead of rmIL-12. 2.4. Data analysis
Fig. 2. Regression of established subcutaneous tumors following intraperitoneal injection of rmIL-12. Tumor volume in surviving mice is shown (mean ^ SD, n 10). Parental (W) or transfectant cells (A) were inoculated subcutaneously into the ¯ank of syngeneic mice. Transient tumor growth was followed by regression within 6 weeks after inoculation with transfectants.
(a) and shortest (b) arms and expressed as (mm).
p
a £ b
Calculated tumor sizes were compared among groups using a two-sample t-test. Survival was evaluated by the generation of Kaplan±Meier cumulative hazard plots and a Wilcoxon analysis. Differences were considered signi®cant when P , 0:05. 3. Results 3.1. Tumorigenicity of glioma cells expressing IL-2 in T-cell-de®cient nude, SCID and syngeneic mice We examined whether or not tumorigenicity differs between parental and IL-2 transfectant cells. We subcutaneously inoculated both types of cells into Tcell-de®cient nude and SCID mice. There were no remarkable differences in tumorigenicity between the groups (data not shown), suggesting that the transfection of IL-2 molecules into glioma cells did not in¯uence tumorigenicity. We then subcutaneously inoculated parental and IL-2 transfectant cells into syngeneic mice. While the parental cells formed tumors that progressively increased, the tumor growth of IL-2 transfectants was slower (Fig. 1A). All mice inoculated subcutaneously with IL-2 transfectants formed tumors. All mice injected with parental cells into the brain died within 3±6 weeks, whereas all those inoculated with transfectants survived over 7 weeks and died within 14 weeks (Fig. 1B). 3.2. Treatment with recombinant IL-12
Fig. 3. Survival of mice following inoculation of transfectants and i.p. injection of rmIL-12 or saline. 1 £ 104 transfectants were inoculated stereotactically into the right frontal lobe of the brain of syngeneic mice (Day 0). rmIL-12 (A; 0.5 mg) (n 5) or saline (A; n 10) was injected i.p. every day for 7 days starting on day 5. There was no statistical difference in survival time between the treated and control groups. All mice, either treated or untreated, died within 14 weeks.
After the inoculation of SR/IL-2 cells subcutaneously, rmIL-12 or saline were injected i.p. Inoculated tumors became palpable in all mice within 14 days. As shown in Fig. 2, transient tumor growth was followed by regression within 6 weeks after the inoculation with tumor cells. Tumors in control mice started growing again after the cessation of treatment and killed the host within 6 weeks (unpublished data).
50
T. Kikuchi et al. / Cancer Letters 135 (1999) 47±51
All treated mice survived more than 10 weeks after inoculation (data not shown). The effect of IL-12 on glioma cells was not due to a direct cytotoxic effect of the cytokine, because the addition of rmIL-12 to cultures of parental and transfectant cells had no effect on their morphology or proliferation (data not shown). Subsequently, rmIL-12 or saline was injected i.p. into mice that had been inoculated in the brain. No signi®cant difference in survival time between the treated group and control group was observed (Fig. 3). All mice, treated or untreated, died within 14 weeks. 4. Discussion IL-2 exhibits antitumor activity in a range of murine tumor models, and the antitumor effects of tumor cells genetically engineered to produce IL-2 have been investigated [14,15]. In addition, IL-12 possesses signi®cant single-agent antitumor activity in a range of murine tumor models [6±9], and a treatment regimen consisting of IL-12 administered in combination with IL-2 exhibits antitumor activity superior to that of either agent alone [10±12]. Furthermore, we have found that in a mouse brain tumor model, combination immunotherapy with IL-2-producing cells and recombinant IL-12 results in the potentiation of antitumor effects (unpublished data). In the present study, the antitumor effects of rmIL-12 on glioma cells genetically engineered to produce IL-2 were investigated. The intraperitoneal administration of IL-12 inhibited the growth of subcutaneous IL-2transduced gliomas. This result is compatible with the report by Vagliani et al. [11], in which they reported that the combination of systemic IL-12 and local IL-2 increased the percentage of mice that rejected two different IL-2 gene-transduced colon cancer cell lines. However, in the present study, the administration of rmIL-12 did not prolong the survival of mice inoculated in the brain with IL-2-transduced glioma cells. The CNS is generally considered to be an immunologically privileged site due to the lack of lymphatic drainage and the nature of the blood± brain barrier, in which tight junctions between cerebral vascular endothelial cells form a physical barrier to the passage of cells and antibodies [16]. However, in tumor-bearing hosts, the possibility exists that the
brain is not completely immuno-privileged, because barriers to the immune system can be surmounted in the presence of some tumors, resulting in crosstalk between systemic and focal immunity. Tjuvajev et al. [17] reported that the attenuation of brain tumor growth by IL-2 vector producer cells does not result in a prolongation of animal survival because of the severe vasogenic brain edema due to IL-2 secretion, and suggested that the clinicians investigating the application of IL-2 gene transfer therapy for intracerebral tumors must consider the potential for severe vasogenic brain edema associated with the intracerebral production of IL-2. However, in the present study, the survival of the mice inoculated with IL-2transduced cells was longer than that of the mice inoculated with parental cells. The production of IL2 in our transfectant cells was much lower than that in the transfectant cells of Tjuvajev et al. (200±300 pg/ ml versus 5±35 ng/ml, respectively), suggesting that the IL-2 concentration is an important factor for the successful treatment of brain tumors. In summary, the results of this study suggest that the gene transfer of IL-2 into brain tumors may be a potential therapeutic approach, but that the systemic administration of rmIL-12 does not inhibit the growth of IL-2- transduced cells in the brain. However, the treatment schedule and method may be improved by injecting IL-12 more frequently, or by injecting glioma cells genetically engineered to express not only IL-2 but also IL-12. Our future research will focus on characterizing the in vivo antitumor activities of glioma cells that express IL-12 or both IL-2 and IL-12 and on identifying an appropriate delivery system. References [1] A. Brandes, M. Soesan, M. Fiorentino, Medical treatment of high grade malignant gliomas in adults: an overview, Anticancer Res. 11 (1991) 1988±1992. [2] T. Aoki, K. Tashiro, S. Miyatake, T. Kinashi, T. Nakano, Y. Oda, H. Kikuchi, T. Honjo, Expression of murine interleukin 7 in a murine glioma cell line results in reduced tumorigenicity in vivo, Proc. Natl. Acad. Sci. USA 89 (1992) 3850±3854. [3] H. Wakimoto, J. Abe, R. Tsunoda, M. Aoyagi, K. Hirakawa, H. Hamada, Intensi®ed antitumor immunity by a cancer vaccine that produces granulocyte-macrophage colony-stimulating factor plus interleukin 4, Cancer Res. 56 (1996) 1828± 1833.
T. Kikuchi et al. / Cancer Letters 135 (1999) 47±51 [4] H. Tahara, M.T. Lotze, Antitumor effects of interleukin-12 (IL-12): applications for the immunotherapy and gene therapy of cancer, Gene Ther. 2 (1995) 96±106. [5] M.J. Brunda, M.K. Gately, Interleukin-12: potential role in cancer therapy, Important Adv. Oncol. (1995) 3±18. [6] M.J. Brunda, L. Luistro, R.R. Warrier, R.B. Wright, B.R. Hubbard, M. Murphy, S.F. Wolf, M.K. Gately, Antitumor and antimetastatic activity of interleukin 12 against murine tumors, J. Exp. Med. 178 (1993) 1223±1230. [7] M.K. Gately, R.R. Warrier, S. Honasoge, et al., Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo, Int. Immunol. 6 (1994) 157±167. [8] C.L. Nastala, H.D. Edington, T.G. McKinney, H. Tahara, M.A. Nalesnik, M.J. Brunda, M.K. Gately, S.F. Wolf, R.D. Schreiber, W.J. Storkus, et al., Recombinant IL-12 administration induces tumor regression in association with IFNgamma production, J. Immunol. 153 (1994) 1697±1706. [9] H. Tahara, L. Zitvogel, W.J. Storkus, P.D. Robbins, M.T. Lotze, Murine models of cancer cytokine gene therapy using interleukin-12, Ann. N. Y. Acad. Sci. 795 (1996) 275±283. [10] J.M. Wigginton, K.L. Komschlies, T.C. Back, J.L. Franco, M.J. Brunda, R.H. Wiltrout, Administration of interleukin 12 with pulse interleukin 2 and the rapid and complete eradication of murine renal carcinoma, J. Natl. Cancer Inst. 88 (1996) 38±43. [11] M. Vagliani, M. Rodolfo, F. Cavallo, M. Parenza, C. Melani,
[12]
[13]
[14]
[15]
[16] [17]
51
G. Parmiani, G. Forni, M.P. Colombo, Interleukin 12 potentiates the curative effect of a vaccine based on interleukin 2transduced tumor cells, Cancer Res. 56 (1996) 467±470. I. Pappo, H. Tahara, P.D. Robbins, M.K. Gately, S.F. Wolf, A. Barnea, M.T. Lotze, Administration of systemic or local interleukin-2 enhances the anti-tumor effects of interleukin-12 gene therapy, J. Surg. Res. 58 (1995) 218±226. K. Sakamoto, H. Hoshino, Y. Kiuchi, G. Nakano, Y. Nagamachi, Potential usefulness of a cultured glioma cell line induced by rous sarcoma virus in B10.A mouse as an immunotherapy model, Jpn. J. Exp. Med. 59 (1989) 173±180. Y. Heike, M. Takahashi, Y. Kanegae, Y. Sato, I. Saito, N. Saijo, Interleukin-2 gene transduction into freshly isolated lung adenocarcinoma cells with adenoviral vectors, Hum. Gene Ther. 8 (1997) 1±14. S. Nagashima, Y. Kashii, T.E. Reichert, Y. Suminami, T. Suzuki, T.L. Whiteside, Human gastric carcinoma transduced with the IL-2 gene: increased sensitivity to immune effector cells in vitro and in vivo, Int. J. Cancer 72 (1997) 174±183. H.F. Cserr, P.M. Knopf, Cervical lymphatics, the blood-brain barrier and the immunoreactivity of the brain: a new view, Immunol. Today 13 (1992) 507±512. J. Tjuvajev, B. Gansbacher, R. Desai, B. Beattie, M. Kaplitt, C. Matei, J. Koutcher, E. Gilboa, R. Blasberg, RG-2 glioma growth attenuation and severe brain edema caused by local production of interleukin-2 and interferon-gamma, Cancer Res. 55 (1995) 1902±1910.
Antitumor activity of interleukin 12 against interleukin 2transduced mouse glioma cells Tetsuro Kikuchi a, b,*, Tatsuhiro Joki b, Yasuharu Akasaki b, Toshiaki Abe b, Tsuneya Ohno a a
Division of Oncology, The Institute of DNA Medicine, Jikei University, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105, Japan b Department of Neurosurgery, Jikei University, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105, Japan Received 18 June 1998; received in revised form 18 August 1998; accepted 19 August 1998
Abstract We subcutaneously inoculated parental and glioma cells genetically engineered to express interleukin-2 (SR/IL-2) into syngeneic mice. The tumor growth of the transfectants was slower than that of the parental cells. We then stereotactically inoculated transfectants into the brains of mice. The survival of the mice injected with parental cells was shorter than that of the mice inoculated with transfectants. SR/IL-2 cells were inoculated subcutaneously into the ¯ank of mice, after which rmIL-12 was administered intraperitoneally (i.p.). The resultant transient tumor growth was followed by regression. rmIL-12 or saline were then injected i.p. into mice that had been inoculated in the brain with SR/IL-2 cells. There was no signi®cant difference in survival time between the treated and control groups. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Immunotherapy; Interleukin 2; Interleukin 12; Glioma; Gene transfection
1. Introduction Malignant astrocytoma is the most common primary brain tumor in adults. The median survival of patients with malignant astrocytomas (high-grade astrocytomas) is about 1±2 years, despite aggressive treatment that includes surgical resection, radiotherapy, and cytotoxic chemotherapy [1]. Therefore, to prolong this survival, novel therapeutic approaches must be pursued. Immunotherapy is one such approach, and has been investigated using different types of tumors including brain tumors. In the central nervous system (CNS), considered an immunologi* Corresponding author. Division of Oncology, The Institute of DNA Medicine, Jikei University, 3-25-8 Nishishinbashi, Minatoku, Tokyo 105, Japan. Fax: 1 81-3-3459-6412; e-mail: [email protected].
cally privileged site, vaccination with interleukin 4-, interleukin 7-, or granulocyte-macrophage colonystimulating factor-producing tumor cells has been effective in an experimental mouse model [2,3]. Interleukin 12 (IL-12) enhances the lytic activity of natural killer (NK)/lymphokine-activated killer (LAK) cells, facilitates speci®c cytotoxic T lymphocyte (CTL) responses, acts as a growth factor for activated T and NK cells, induces interferon-g from T and NK cells, and acts as an angiogenesis inhibitor [4,5]. Although IL-12 itself activates strong antitumor activity [6±9], the combination of vaccine therapy with IL-2-transduced tumor cells and systemic recombinant mouse IL-12 (rmIL-12) cured mice bearing wild-type parental tumors more effectively than did rmIL-12 and IL-2-transduced tumor vaccines alone [10±12].
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(98)00268-7
48
T. Kikuchi et al. / Cancer Letters 135 (1999) 47±51
In the present study, we analyzed the antitumor effects of rmIL-12 on glioma cells genetically engineered to produce IL-2. 2. Materials and methods 2.1. Cell lines and transfection
Dulbecco's phosphate-buffered saline (D-PBS; Cosmo Bio.), suspended in serum-free D-MEM at a density of 1 £ 107 /ml. Cells (1 £ 106 ) were inoculated subcutaneously (s.c.) into the ¯ank of B10.A mice (Day 0). rmIL-12 (0.5 mg/100 ml) or saline, as a control, was injected intraperitoneally (i.p.) every other day for 2 weeks starting on day 14 (total 3.5 mg/mouse). Tumor size was measured at the longest
The mouse glioma cell line, SR-B10.A, provided by Dr. K. Sakamoto, has been described in detail elsewhere [13]. This cell line was maintained in monolayer cultures in DMEM (Cosmo Bio Co., Tokyo, Japan) supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Gaithersburg, MD). Human IL-2 cDNA was ampli®ed using a polymerase chain reaction method and subcloned into a pcDNA3 expression vector (Invitrogen, NV Leek, The Netherlands; designated pcDNA/IL-2, provided by Dr. H. Suzuki). SR-B10.A cells were transfected with pcDNA/IL-2 using a lipofectin reagent (GIBCO) according to the manufacturer's instructions. All transfectants were selected in 1000 mg/ml G418 (Sigma Chemicals, St. Louis, MO, USA) and cloned by limiting dilution to obtain stable IL-2 expression. The IL-2 production from transfectants was determined by an enzyme-linked immunoabsorbent assay (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. The concentration of IL2 was 286 pg/ml in clone 21, designated SR/IL-2. This clone was selected for the following experiments. 2.2. Animals Female B10.A mice were purchased from Shizuoka Laboratory Inc. (Shizuoka, Japan), and nude and severe combined immuno-de®ciency (SCID) mice were purchased from CLEA Japan (Tokyo). The mice were maintained in a speci®c pathogen-free room at 25 ^ 38C. Female mice aged 6 weeks (body weight 25 ^ 2 g) were used in the experiments. 2.3. IL-12 administration Recombinant mouse IL-12 (rmIL-12) was provided by Genetics Institute (Cambridge, MA). A total of 1 £ 106 exponentially growing SR-B10.A or IL-2- transduced glioma cells were harvested, washed twice with
Fig. 1. Tumorigenesis of parental and transfectant glioma cells. (A) While parental cells formed tumors that progressively increased in size (W), tumor growth of IL-2 transfectants was slower (A). Tumor volume in surviving mice is shown (mean ^ SD, n 10). Mice inoculated with transfectants had smaller tumors than the control mice at each time point (P , 0:05). (B) All mice injected with parental cells into the brain died within 3±6 weeks (W), whereas all those inoculated with transfectants survived over 7 weeks and died within 14 weeks (A) (n 10 each).
T. Kikuchi et al. / Cancer Letters 135 (1999) 47±51
49 4
Subsequently, 1 £ 10 parental or transfectant cells were inoculated stereotactically into the right frontal lobe of the brain of syngeneic mice. A 0.5 mg aliquot of rmIL-12 was injected i.p. every day from days 5 to 11 (total 3.5 mg/mouse). As a control, saline was administered instead of rmIL-12. 2.4. Data analysis
Fig. 2. Regression of established subcutaneous tumors following intraperitoneal injection of rmIL-12. Tumor volume in surviving mice is shown (mean ^ SD, n 10). Parental (W) or transfectant cells (A) were inoculated subcutaneously into the ¯ank of syngeneic mice. Transient tumor growth was followed by regression within 6 weeks after inoculation with transfectants.
(a) and shortest (b) arms and expressed as (mm).
p
a £ b
Calculated tumor sizes were compared among groups using a two-sample t-test. Survival was evaluated by the generation of Kaplan±Meier cumulative hazard plots and a Wilcoxon analysis. Differences were considered signi®cant when P , 0:05. 3. Results 3.1. Tumorigenicity of glioma cells expressing IL-2 in T-cell-de®cient nude, SCID and syngeneic mice We examined whether or not tumorigenicity differs between parental and IL-2 transfectant cells. We subcutaneously inoculated both types of cells into Tcell-de®cient nude and SCID mice. There were no remarkable differences in tumorigenicity between the groups (data not shown), suggesting that the transfection of IL-2 molecules into glioma cells did not in¯uence tumorigenicity. We then subcutaneously inoculated parental and IL-2 transfectant cells into syngeneic mice. While the parental cells formed tumors that progressively increased, the tumor growth of IL-2 transfectants was slower (Fig. 1A). All mice inoculated subcutaneously with IL-2 transfectants formed tumors. All mice injected with parental cells into the brain died within 3±6 weeks, whereas all those inoculated with transfectants survived over 7 weeks and died within 14 weeks (Fig. 1B). 3.2. Treatment with recombinant IL-12
Fig. 3. Survival of mice following inoculation of transfectants and i.p. injection of rmIL-12 or saline. 1 £ 104 transfectants were inoculated stereotactically into the right frontal lobe of the brain of syngeneic mice (Day 0). rmIL-12 (A; 0.5 mg) (n 5) or saline (A; n 10) was injected i.p. every day for 7 days starting on day 5. There was no statistical difference in survival time between the treated and control groups. All mice, either treated or untreated, died within 14 weeks.
After the inoculation of SR/IL-2 cells subcutaneously, rmIL-12 or saline were injected i.p. Inoculated tumors became palpable in all mice within 14 days. As shown in Fig. 2, transient tumor growth was followed by regression within 6 weeks after the inoculation with tumor cells. Tumors in control mice started growing again after the cessation of treatment and killed the host within 6 weeks (unpublished data).
50
T. Kikuchi et al. / Cancer Letters 135 (1999) 47±51
All treated mice survived more than 10 weeks after inoculation (data not shown). The effect of IL-12 on glioma cells was not due to a direct cytotoxic effect of the cytokine, because the addition of rmIL-12 to cultures of parental and transfectant cells had no effect on their morphology or proliferation (data not shown). Subsequently, rmIL-12 or saline was injected i.p. into mice that had been inoculated in the brain. No signi®cant difference in survival time between the treated group and control group was observed (Fig. 3). All mice, treated or untreated, died within 14 weeks. 4. Discussion IL-2 exhibits antitumor activity in a range of murine tumor models, and the antitumor effects of tumor cells genetically engineered to produce IL-2 have been investigated [14,15]. In addition, IL-12 possesses signi®cant single-agent antitumor activity in a range of murine tumor models [6±9], and a treatment regimen consisting of IL-12 administered in combination with IL-2 exhibits antitumor activity superior to that of either agent alone [10±12]. Furthermore, we have found that in a mouse brain tumor model, combination immunotherapy with IL-2-producing cells and recombinant IL-12 results in the potentiation of antitumor effects (unpublished data). In the present study, the antitumor effects of rmIL-12 on glioma cells genetically engineered to produce IL-2 were investigated. The intraperitoneal administration of IL-12 inhibited the growth of subcutaneous IL-2transduced gliomas. This result is compatible with the report by Vagliani et al. [11], in which they reported that the combination of systemic IL-12 and local IL-2 increased the percentage of mice that rejected two different IL-2 gene-transduced colon cancer cell lines. However, in the present study, the administration of rmIL-12 did not prolong the survival of mice inoculated in the brain with IL-2-transduced glioma cells. The CNS is generally considered to be an immunologically privileged site due to the lack of lymphatic drainage and the nature of the blood± brain barrier, in which tight junctions between cerebral vascular endothelial cells form a physical barrier to the passage of cells and antibodies [16]. However, in tumor-bearing hosts, the possibility exists that the
brain is not completely immuno-privileged, because barriers to the immune system can be surmounted in the presence of some tumors, resulting in crosstalk between systemic and focal immunity. Tjuvajev et al. [17] reported that the attenuation of brain tumor growth by IL-2 vector producer cells does not result in a prolongation of animal survival because of the severe vasogenic brain edema due to IL-2 secretion, and suggested that the clinicians investigating the application of IL-2 gene transfer therapy for intracerebral tumors must consider the potential for severe vasogenic brain edema associated with the intracerebral production of IL-2. However, in the present study, the survival of the mice inoculated with IL-2transduced cells was longer than that of the mice inoculated with parental cells. The production of IL2 in our transfectant cells was much lower than that in the transfectant cells of Tjuvajev et al. (200±300 pg/ ml versus 5±35 ng/ml, respectively), suggesting that the IL-2 concentration is an important factor for the successful treatment of brain tumors. In summary, the results of this study suggest that the gene transfer of IL-2 into brain tumors may be a potential therapeutic approach, but that the systemic administration of rmIL-12 does not inhibit the growth of IL-2- transduced cells in the brain. However, the treatment schedule and method may be improved by injecting IL-12 more frequently, or by injecting glioma cells genetically engineered to express not only IL-2 but also IL-12. Our future research will focus on characterizing the in vivo antitumor activities of glioma cells that express IL-12 or both IL-2 and IL-12 and on identifying an appropriate delivery system. References [1] A. Brandes, M. Soesan, M. Fiorentino, Medical treatment of high grade malignant gliomas in adults: an overview, Anticancer Res. 11 (1991) 1988±1992. [2] T. Aoki, K. Tashiro, S. Miyatake, T. Kinashi, T. Nakano, Y. Oda, H. Kikuchi, T. Honjo, Expression of murine interleukin 7 in a murine glioma cell line results in reduced tumorigenicity in vivo, Proc. Natl. Acad. Sci. USA 89 (1992) 3850±3854. [3] H. Wakimoto, J. Abe, R. Tsunoda, M. Aoyagi, K. Hirakawa, H. Hamada, Intensi®ed antitumor immunity by a cancer vaccine that produces granulocyte-macrophage colony-stimulating factor plus interleukin 4, Cancer Res. 56 (1996) 1828± 1833.
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