[CANCER RESEARCH 60, 1111–1116, February 15, 2000]

Direct in Vitro Evidence andin Vivo Analysis of the Antiangiogenesis Effects ofInterleukin 121

Dan Gabriel Duda, Makoto Sunamura,2 Lucian Lozonschi, Tomohiro Kodama, Shin-ichi Egawa, Gaku Matsumoto,Hiromune Shimamura, Kazuhiko Shibuya, Kazunori Takeda, and Seiki MatsunoFirst Department of Surgery, Tohoku University Medical School, Aoba-ku, Sendai 980-8574, Japan

ABSTRACT

As an antitumor agent, interleukin-12 (IL-12) has been revealed to bea key regulator of the immune response, particularly that involving CTLand natural killer (NK) cells. We report herein the antiangiogenesis effectof IL-12 on human as well as murine tumors in NK-depleted severe-combined immunodeficient mice using fibroblasts genetically engineeredto secrete this cytokine. Although thein vitro growth of tumor cells was notaffected by the presence of IL-12, coinoculation of IL-12-secreting fibro-blasts strongly inhibited tumor growth in immunodeficient mice. Theneovascularization surrounding the tumor was remarkably inhibited inthe area in which the IL-12-secreting fibroblasts were implanted, resultingin the suppression of tumor growth. Lectin staining in tumor samplesections also showed a significant reduction in the number of vessels. TheRNA expression of IFN-g and its inducible antiangiogenic chemokine IFNg-inducible protein 10 was stimulated in endothelial cells cultured withIL-12. It was also found that IL-12 down-regulated the expression of theendothelial cell mitogens vascular endothelial growth factor and basicfibroblast growth factor. The antitumor effects of IL-12 were accompa-nied by interesting histological changes consisting of a high degree ofkeratinization and apoptosis and a decrease in the proliferation rate ofhuman tumors and extensive necrosis in the murine ones.

INTRODUCTION

IL-123 is a disulfide-linked heterodimeric cytokine composed of alight chain (p35) and a heavy chain (p40; Ref. 1). IL-12 is producedprimarily by antigen-presenting cells (APCs) and binds to receptorson T cells and NK cells, activates them, and promotes the induction ofthe T helper type 1 phenotype (Th1) responsein vitro as well asinvivo (2, 3). IL-12 is able to elicit a strong immune response, which isresponsible for its proven potent antitumor and antimetastatic activity.Several ongoing clinical trials have used IL-12 administered system-ically or locally (4, 5). More recently, it has also been reported thatIL-12 has an antiangiogenesis effect in several experimental settings(6, 7). This effect is exerted in an indirect manner by triggering thehigh secretion of IFN-g, which presumably induces the antiangiogen-esis effect through the secretion of IP-10 and MIG. NK cell involve-ment in this effect of IL-12 has also been documented (8). NeitherIL-12 nor IFN-g was reported to exhibit antiproliferative effectsinvitro. However, it is not easy to demonstrate that the antiangiogenesiseffect of IL-12 is responsible for its antitumor effect because theantitumor immunity and the antiangiogenesis are induced simulta-neously.

In the present study, IL-12 was locally delivered by coinoculationof tumor cells with fibroblasts genetically engineered to secrete IL-12(IL-12/3T3). This delivery method was used to estimate the directeffect of this cytokine on the tumor vasculature. We analyzed theprocess of human and murine tumor angiogenesis in a skinfold cham-ber installed in SCID mice pretreated with antiasialo GM-1 antibodyto block the strong nonspecific immune response induced by IL-12administration (9). NK depletion was tested by FACScan analysis. Toassess the involvement of IFN-g in the effects of IL-12, we admin-istered anti-IFN-g antibody in mice inoculated with IL-12/3T3. ByRT-PCR, a very useful semiquantitative method for RNA detection,we investigated the cytokine network in the tumor microenvironmentboth in vivo and in vitro. Several lines of evidence, including thehistopathological features in tumor tissue sections, indicated thatIL-12 exerts its antiangiogenesis effect through the expression ofIFN-g in endothelial cells and downstream of IP-10, but also by thedown-regulation of the endothelial cell mitogens VEGF and bFGF. Inaddition, using a vital microscopy system, we demonstrated the anti-angiogenesis effect induced by paracrine delivery of IL-12 on anestablished tumor.

MATERIALS AND METHODS

Mice. Male, 6-to-8-week-old SCID mice (Fox Chase C.B-17/Icr-SCID Jcl),were kept in pathogen-limited conditions, and skinfold chamber bearing micewere housed individually. We followed the NIH guidelines for the care and useof the research animals.

Cells and Culture Conditions. PK-1 human adenocarcinoma, establishedin our department (10), and C26 murine colon carcinoma cell lines weremaintained in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) sup-plemented with 10% FCS, 100 units/ml penicillin, and 0.1 mg/ml streptomycinin a humidified 5% CO2 atmosphere at 37°C. Wild-type fibroblasts (NIH3T3)were retrovirally transfected with IL-12 (IL-12/3T3). The construction andcharacterization of the retroviral vector have been described by Zitvogelet al.(11). The infection of NIH3T3 and the selection of high producing clones wereperformed as described previously (12). KOP 2.16 endothelial cells, describedelsewhere (13), were used for thein vitro experiments. Fibroblast and endo-thelial cells were cultured in DMEM low glucose, 10% FCS, and antibioticsand were maintained in the same conditions as the tumor cells.

Cytokine Level Measurement. The IL-12—secretedin vitro by fibro-blasts, endothelial, or tumor cells—was measured by enzyme-linked immu-nosorbent assay using an ELISA kit for murine IL-12 (Endogen Inc., Woburn,MA).

Antibodies. Rabbit antiasialo GM-1 antibody (Wako, Tokyo, Japan) wasadministered via the tail vein (100ml/mouse) twice a week, the first time 3days before the PK-1 inoculation. Anti-IFN-g antibody (a-mIFN-g R4-GA2),provided by Dr. Yagita (Juntendo University, Tokyo), was administered i.v.(200 ml/mouse) every 2 days.

In Vivo Tumor Growth. Five 3 106 PK-1 cells were inoculated s.c. in theSCID mice. In another group, the same number of tumor cells were inoculatedadmixed with IL-12/3T3, and anti-IFN-g antibody was administered to someof the mice (n5 5 in all three of the groups) The tumor volume was measuredtwice a week, in a blind fashion. Using a caliper, the diameters of the tumorswere measured, and the tumor volume was determined by the formula:V 5 D 3 d2 3 0.4, whereV 5 tumor volume,D 5 biggest dimension, andd 5 smallest dimension.

Received 8/31/99; accepted 12/15/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by two grants-in-aid for scientific research (09557094 and10470251) from the Ministry of Education, Science, Sports and Culture of Japan.

2 To whom requests for reprints should be addressed, at the First Department ofSurgery, Tohoku University Medical School, 1–1 Seiryomachi, Aoba-ku, Sendai 980-8574, Japan. Phone: 81-22-717-7205; Fax: 81-22-717-7209.

3 The abbreviations used are: IL-12, interleukin 12; bFGF, basic fibroblast growthfactor; VEGF, vascular endothelial growth factor; IP-10, interferong-inducible protein10; MIG, monokine induced byg-interferon; NK, natural killer (cell); IL-12/3T3, IL-12-transfected fibroblasts; SCID, severe combined immunodeficient/immunodeficiency; RT-PCR, reverse transcription PCR; TUNEL, Tdt-mediated nick end labeling; Tdt, terminaldeoxynucleotidyl transferase.

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In Vitro Growth Measurement. PK-1 cells were suspended in RPMI 1640and 0, 1, 2, or 3 ml of IL-12/3T3 48 h-culture supernatant were added. Also,we cocultured PK-1 (13 105 cells) with either IL-12/3T3 or NIH3T3 usingBIOCOAT Cell Culture inserts in 24-wells plates (Becton Dickinson Labware,Bedford, MA). The PK-1 cells were counted after 2 days.

In Vivo Microscopy. A simplified version of the dorsal skinfold chamberpreviously described (14) was designed in our laboratory and manufactured byAoba Science Ltd. (Sendai, Japan). Chamber implantation, cell inoculation,tumor observation, and off-line analysis were performed as described previ-ously (15). PK-1 or C26 (13 106 cells) were implanted on the skin muscularlayer. After 11 and 8 days, respectively, 53 105 IL-12/3T3 and/or NIH/3T3were also implanted on the tumor. We also used FITC-dextran staining forvessel contrast enhancement.

FACScan Analysis. NK depletion after antiasialo GM-1 antibody admin-istration was assessed using FACScan analysis for hepatocytes harvested fromtreated SCID mice.

RT-PCR. PK-1 (1 3 106 cells) and KOP 2.16 (53 105 cells) werecocultured in 6-cm dishes, and 1 ml of 48-h culture supernatant of 13 106

IL-12/3T3 was added. Cells were harvested after 30 min, 120 min, and 24 h.Total cellular RNA was extracted from these cells and also from PK-1 tumortissue samples using Rneasy Mini Kit (Qiagen KK, Tokyo, Japan). Ten-mlaliquots were reversed transcribed using a Rnease H Minus RT (TOYOBOCo., Tokyo, Japan) and 100 ng of the resultant cDNA was amplified usingprimers with the nucleotide sequences, annealing temperatures, and number ofamplification cycles shown in Table 1. Glycerol-3 phosphate dehydrogenasewas used as an internal control. Ten-ml aliquots from the amplification reac-tions were electrophoresed on 3% agarose (FMC BioProducts, Rockland, ME)and Tris-acetate EDTA gels and were autoradiographed. We recovered thePCR products from the gel using SpinBind System (FMC BioProducts) and

then inserted them into plasmid vectors using TA Cloning kit (InvitrogenCorporation, Carlsbad, CA). Plasmids were purified using Wizards MiniprepsPlus DNA Recovery System, and fragment sequencing was performed usingthe ALFexpress II DNA Analysis System (Amersham Pharmacia Biotech,Uppsala, Sweden).

Histology. Tumor samples were fixed in phospho-lysine-paraformalde-hide overnight, embedded in OCT compound (Sakura Finetechnical Co.,Ltd., Tokyo, Japan) and stored at280°C. We also used tumor samplesfixed in 10% formalin and then embedded in paraffin. Standard H&Estaining was performed on 3–5-mm sections from frozen and paraffin-embedded samples. Furthermore, lectin staining of endothelial cells onfrozen and paraffin-embedded sample cuts was performed using biotiny-lated wheat germ agglutinin-WGA-1 (Vector Laboratories, Burlingame,CA), as previously described (16). The proliferation index was assessed bystaining for human ki-67, an antigen expressed throughout the cell prolif-eration cycle but absent in quiescent (G0) cells. The staining protocol usingmouse anti-ki-67 monoclonal antibody (Immunotech, Marseille, France)was as previously described (17). Sections from lymph nodes with reactivegerminal centers were used as positive controls. Quantification of thestaining results was made by determining the percentage of stained cells inat least 10 high-power fields.

TUNEL. Tdt-labeling (Apoptosis Detection Kit, Wako, Tokyo, Japan) ofDNA fragmentation in frozen cut-tumor samples was used for the staining ofthe apoptotic cells within the tumor sections.

Statistical Analysis. All of the experiments were performed in duplicate ortriplicate. Two-tailed Student’st test was used for the statistical analysis ofdata. Values ofP # 0.05 were considered significant.

RESULTS

In Vitro Cytokine Production by Genetically Engineered Fibro-blasts. IL-12/3T3s were able to secrete 12.06 ng/106 cells/48 h,measured by ELISA in the culture supernatant, a level that was stableafter four passages. No IL-12 expression was detectable in the culturesupernatant of NIH3T3, KOP 2.16, PK-1 or C26.

NK Depletion Test. FACScan was performed for hepatocytesharvested from SCID mice 3 days after antiasialo GM1 antibodyadministration. The NK cell population was depleted from 5.38% inthe control group to 0.89% in the treated group.

In Vitro Effect of IL-12 on PK-1 Growth. IL-12 released byIL-12/3T3 had no effectin vitro on the proliferation of endothelialcells or tumor cells, irrespective of the IL-12 dose (Fig. 1). Moreover,PK-1 cells were inoculated s.c. after coculture for 4 days using insertswith either IL-12/3T3 or NIH3T3. Both of them were able to form

Fig. 1. PK-1 cell growthin vitro was not affected by thecoculture with IL-12-transfected fibroblasts, regardless ofthe ratio between the tumor and fibroblast cells used.

Table 1 RT-PCR conditions

Cytokine PrimersNo. ofcycles

Annealingtemperature

mIP-10a 59-GCCGTCATTTTCTGCCTCAT-39 30 57°C39-GGAAGATGGTGGTTAAGTTC-59

VEGF 59-ATGAACTTTCTGCTCTCTTG-39 30 55°C39-CACATCTGCAAGTACGTTC-59

bFGF 59-AAAACGGGGGCTTCTTCCT-39 30 55°C39-TGCCCAGTTCGTTTCAGTGC-59

mIFN-g 59-CTCGTGTTTCTAGCTGCATA-39 30 59°C39-ACATTCTGCTACGCTTGGAA-59

G3PDH 59-GCCACATCGCTAAGACACCATGGG-39 30 57°C39-CCTGGTGACCAGGCGCCCAAT-59

a mIP-10, murine IP-10; mIFN-g, murine IFN-g.

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tumors in SCID mice, as did the wild-type PK-1. No differences couldbe observed in the histological examinations of the resultant tumorsections (data not shown).

In Vivo Tumor Growth of PK-1 Admixed with IL-12/3T3 inSCID Mice. Tumor growth was strongly inhibited in NK-depletedSCID mice receiving s.c. inoculation of PK-1 admixed with IL-12 trans-duced fibroblasts; in contrast, growing tumors were observed in miceinoculated with wild-type PK-1 cells. Anti-IFN-g antibody (i.p. 200mlevery 2 days) completely nullified the antitumor effect of IL-12 (Fig. 2).

Tumor Angiogenesis Inhibition by IL-12 Observed in SkinfoldChamber. The transparent-chamber model, together with thein vivomicroscopy system, allowed accurate observation of the process oftumor angiogenesis. Tumor angiogenesis of PK-1 and C26 was ob-served in the implanted chambers, whereas vessel disruption wasobserved and no tumor was detected after the admixed inoculation ofPK-1 cells with IL-12/3T3 (Fig. 3A-F). To clarify the antiangiogen-esis effect of IL-12, we examined the interaction between tumorvessels and IL-12/3T3. Wild-type fibroblasts or IL-12-transfectedfibroblasts were coinoculated with tumor cells or implanted in estab-lished tumors (day 8 in C26 tumors and day 11 in PK-1 tumors). Anincrease in the vessel disruption was detected in the admixed implan-tation group and at the IL-12/3T3 implantation site in the established

Fig. 2. s.c. tumor growth after inoculation of 53 106 PK-1 cells (e) was significantly(p, P , 0.05) inhibited by the coinoculation of 53 105 IL-12/3T3 cells (M). Adminis-tration of anti-IFN-g- antibody completely reversed this effect (‚).

Fig. 3. PK-1 tumor angiogenesis in dorsal skinfold chamber of SCID mice. PK-1 cells (A) or a mixture of PK-1 and IL-12/3T3 (D) were implanted in the chamber. At day 14, tumorneovasculature was detected in mice receiving PK-1 cell implant (B, 34; C, 310), whereas tumors failed to develop and normal vasculature was damaged in the mice from coinoculationgroup (E,34; F, 310). In a second experiment, at day 11, we implanted IL-12/3T3 and NIH/3T3 on the established PK-1 tumor following the design inG. Total vessel disruptioncould be seen at day 17 in the IL-12/3T3 site compared with developing tumor angiogenesis in the wild-type fibroblast implantation site (H).

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tumors (Fig. 3,G andH and Fig. 4,A-C). On the other hand, tumorangiogenesis was promoted in the tumor cell implantation groups withor without wild type.

Molecular Biology. The cytokine network of the tumor microen-vironment was assessed by RT-PCR. The IP-10 RNA expression ofcultured tumor and endothelial cells was induced by the presence ofIL-12 as early as 30 min after stimulation, with a peak at 2 h anddetectable secretion after 24 h (Fig. 5A). A decrease in the endothelialcell mitogens VEGF and bFGF, but not in acidic FGF RNA levels,was detected in the tumor and endothelial cells (Fig. 5,C, D, andF).The elevated RNA expression of IFN-g and the expression of tumornecrosis factora RNA in the presence of IL-12 were confirmedinvitro as well asin vivo (Fig. 5B and data not shown). Of particularinterest, the IFN-g expression in endothelial cells after IL-12 stimu-lation could be detectedin vitro (Fig. 5B). IL-12 failed to change theexpression ofavb3 or FasL in vivo and in vitro (data not shown).G3PDH amplification was used as internal control (Fig. 5E).

Band DNA recovery from gel after electrophoresis followed by TAcloning, and plasmid sequencing confirmed the PCR findings.

Histopathological Aspects.PK-1 tumors from the IL-12/3T3 coi-noculation group presented completely different histological featurescompared with the PK-1 tumors. IL-12/3T3 induced the increase ofkeratinization (Fig. 6,A-C) and apoptosis in PK-1 tumors, assessed by

Tdt-labeling in frozen tumor sections (Fig. 6,H andI). In C26 tumorstreated with IL-12/3T3, there were larger areas of necrosis as com-pared with the wild-type tumor. These effects were diminished inmice treated with anti-IFN-g antibody. These features were paralleledby a diminished proliferation index in the tumor and endothelial cells.ki-67 staining (Fig. 6,J-L) revealed a significant decrease (P, 0.01)in the percentage of stained cells in PK-1 and IL-12/3T3 tumorsections (1.54 6 0.68%) as compared with PK-1 tumors(6.60 6 0.87%). However, the percentage was increased to7.65 6 0.99% in coinoculation tumors treated with anti-IFN-g anti-body.

Lectin (biotinylated WGA-1) staining of PK-1 tumor sample sec-tions demonstrated that IL-12 diminished the numbers and the diam-eters of the tumor vessels (Fig. 6,D and F). In C26 tumors treatedwith IL-12/3T3, there were large areas of necrosis, and fewer vesselswere observed by WGA-1 staining as compared with the wild-typetumor (Fig. 6,E andG).

DISCUSSION

IL-12 is well known as a key regulator of the immune system andhas received much attention as an antitumor and antimetastasis agent.Recent reports regarding the involvement of IL-12 in the antiangio-genesis process have drawn attention to its potential for cancer ther-apy as tumor growth and metastasis are dependent on aberrant neo-vascularization (18).

Fibroblasts genetically engineered to secrete IL-12 are able toexpress a relatively high and stable level of the cytokine. Moreover,culturing, selection, and transduction of the fibroblasts are easy, andthe probability of migration of these cells compared with tumor orlymphoid cells is low. We used a paracrine delivery system to provethe effect induced by IL-12 on the growth of a human adenocarci-noma, a poorly vascularized tumor in immune-deficient mice. Thesame setting was applied for a murine tumor, C26. The vital micro-scope system, together with the skinfold chamber model and geneti-cally engineered fibroblasts, enabled us to precisely analyze the in-hibitory effect of IL-12 on the tumor growth. Moreover, by using thissystem, we could analyze the antiangiogenesis effect of IL-12 on theestablished neovasculature that supplies blood to the growing tumor.

Despite the efficient blocking of the IL-12-induced nonspecificimmune response by the administration of antiasialo GM-1 antibody,IL-12 was able to inhibit the tumor growth. Our precise analysis of theneovascularization has revealed that this antitumor effect could beattributed to the antiangiogenesis effect of IL-12. Different ratiosbetween the tumor cells and IL-12/3T3 fibroblasts were used, and thiseffect was confirmed at the lowest dose used (53 105 cells). How-ever, wild-type fibroblasts did not exhibit any antitumor effects. Ourdata showed that the antiangiogenic effect of IL-12, which is secreted

Fig. 4. C26 tumor angiogenesis in skinfold chamber of SCID mice. After implantation of C26 cells (A), tumor angiogenesis could be observed (B) at day 8 (FITC enhancement).At this time, IL-12/3T3 cells were applied onto the C26 tumor, and at day 14, extensive vessel disruption was apparent by FITC enhancement (C).

Fig. 5. Results of RT-PCR. IP-10 (A) and IFN-g (B) were induced by IL-12 as earlyas 30 min, and VEGF and bFGF levels of expression were suppressed by IL-12 (C, D,andF). G3PDH amplification is shown inE.

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Fig. 6. Histology and immunohistochemistry examination of tumors grown in SCID mice. H&E staining (34) shows an increased degree of keratinization in tumors from the coinoculation(PK-11IL-12/3T3) group (A) compared with the control (PK-1) group (B) but not in tumors from the coinoculation group, which had received anti-IFN-g antibody treatment (C). Lectin stainingof tumor samples revealed an increase in the numbers and diameters of vessels in the coinoculation groups [PK-11IL-12/3T3 (D, 320) and C261IL-12/3T3 (E, 310)] sections as comparedwith wild-type tumors [PK-1 (F) and C26 (G)]. TUNEL for apoptosis shows an increased apoptotic rate in samples from the coinoculation group (H) compared with wild-type PK-1 tumorsamples (I; 340). Proliferation index using ki-67 staining (310) revealed that the presence of IL-12 decreased significantly the tumor proliferation rate in mice from the coinoculation group(J) but not in tumors from the mice treated with anti-IFN-g antibody (K). L, control PK-1 tumor sample staining.

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continuously by IL-12/3T3, occurred at levels that can also induce astrong immune response in immunocompetent mice (6, 19).

IL-12 has no effect on endothelial cell proliferation and migration(6). The inhibition of the tumor neovasculature development in oursystem was promoted by the local delivery of IL-12, which causedIFN-g release by endothelial cells. Although the antiangiogenesiseffect induced by IL-12 is IFN-g-dependent, IFN-g administrationcould not induce the same effect (20). After IL-12 stimulation, thelevels of VEGF and matrix metalloproteinases showed a decrease incontrast to the increase of their tissue inhibitor, tissue inhibitor ofmetalloproteinase (TIMP) (7). IFN-g was reported to induce therelease of two chemokines, IP-10 and MIG (21), which exert theirantiangiogenic properties by inhibiting endothelial cell chemotaxisand differentiation into tube-like structures (22). However, other un-known pathways are speculated to be involved as well. We foundinvitro a decrease of the RNA levels in endothelial cell mitogens VEGFand bFGF (but not acidic FGF) induced by the presence of IL-12.These results strongly suggest that IL-12 is able to act as an inhibitorat different stages of angiogenesis. The antiangiogenesis effect ofIL-12 did not seem to be tumor-specific inasmuch as normal vesseldamage was also observed at the site of the transduced fibroblastimplantation. Histological analysis after H&E staining showed char-acteristic findings, including a high degree of keratinization of tumorsin the coinoculation group, suggesting the involvement of IL-12 in thecell differentiation. IL-12 was able to decrease significantly the pro-liferation index, as shown by ki-67 staining of the tumor samplesections. Tdt-labeling showed an increased apoptotic rate in frozentumor sections from the coinoculation group, which indicated thatIL-12 induced apoptosis, as reported for other angiogenesis inhibitors(TNP-470, matrix metalloproteinase inhibitors,avb3 antagonists; Ref.15). The relationship between angiogenesis, apoptosis, and prolifera-tion in tumors is currently under investigation.

Although the antitumor mechanism of IL-12 is not yet completelyunderstood, we demonstrated the involvement of IFN-g secretion byendothelial cells as well as the down-regulation of VEGF and bFGFexpression. Emerging initially as a promising anticancer agent for itsability to boost the immune system, IL-12 proved also to be veryeffective in inducing antiangiogenesis in the absence of CTL and NKcells. The systemic administration of IL-12 in clinical trials has beenshown to lead to major side effects (23). The local delivery of thiscytokine using fibroblasts, adenoviruses, or tumor cells engineered tosecrete IL-12, could be a much safer intervention (24–26). Pancreaticadenocarcinoma is a malignancy with a very poor prognosis aftersurgical treatment. IL-12 gene therapy, using transduced autologousskin fibroblasts, may prove to be an attractive therapy approachbecause of the potent and multiple effects of this cytokine.

ACKNOWLEDGMENTS

We thank Drs. S. Haruo Ohtani and Hirofumi Hamada for useful discus-sions and helpful advice. We also appreciate the expert technical assistance ofEmiko Shibuya, Hiroko Fujimura, and Keiko Inabe.

REFERENCES

1. Wolf, S. F., Temple, P. A., Kobayashi, M., Young, D., Dicig, M., Lowe, L., Dzialo,R., Fitz, L., Ferenz, C., Hewick, R. M., Kelleher, K., Herrmann, S. H., Clark, S. C.,Azzoni, L., Chan, S. H., Trinchieri, G., and Perussia, B. Cloning of cDNA for naturalkiller cell stimulatory factor, a heterodimeric cytokine with multiple biologic effectson T and natural killer cells. J. Immunol.,146: 3074–3081, 1991.

2. McKnight, A. J., Zimmer, G. J., Fogelman, I., Wolf, S. F., and Abbas, A. K. Effectsof IL-12 on helper T cell-dependent immune responsesin vivo. J. Immunol.,152:2172–2179, 1994.

3. Trinchieri, G. Interleukin-12: a proinflammatory cytokine with immunoregulatoryfunctions that bridge innate resistance and antigen-specific adaptive immunity. Annu.Rev. Immunol.,13: 251–276, 1995.

4. Lotze, M. T., Hellerstedt, B., Stolinski, L., Tueting, T., Wilson, C., Kinzler, D., Vu,H., Rubin, J. T., Storkus, W., Tahara, H., Elder, E., and Whiteside, T. The role ofinterleukin-2, interleukin-12, and dendritic cells in cancer therapy. Cancer J. Sci. Am.,1 (Suppl.):S109–S114, 1997.

5. Ohe, Y., Kasai, T., Heike, Y., and Saijo, N. Clinical trial of IL-12 for cancer patients.Gan To Kagaku Ryoho,25: 177–184, 1998.

6. Voest, E. E., Kenyon, B. M., O’Reilly, M. S., Truitt, G., D’Amato, R J., and Folkman,J. Inhibition of angiogenesisin vivo by interleukin 12. Int. J. Cancer,78: 361–365,1998.

7. Dias, S., Boyd, R., and Balkwill, F. IL-12 regulates VEGF and MMPs in a murinebreast cancer model. J. Natl. Cancer Inst.,87: 581–586, 1995.

8. Yao, L., Sgadari, C., Furuke, K., Bloom, E. T., Teruya-Feldstein, J., and Tosato, G.Contribution of natural killer cells to inhibition of angiogenesis by Interleukin-12.Blood, 93: 1612–1621, 1999.

9. Habu, S., Fukui, H., Shimamura, K., Kasai, M., Nagai, Y., Okumura, K., andTamaoki, N. In vivo effects of anti-asialo GM1. I. Reduction of NK activity andenhancement of transplanted tumor growth in nude mice. J. Immunol.,127: 34–38,1981.

10. Kobari, M., Matsuno, S., Sato, T., Kan, M., and Tachibana, T. Establishment of ahuman pancreatic cancer cell line and detection of pancreatic cancer associatedantigen. Tohoku J. Exp. Med.,143: 33–46, 1984.

11. Zitvogel, L., Tahara, H., Cai, Q., Storkus, W. J., Muller, G., Wolf, S. F., Gately, M.,Robbins, P. D., and Lotze, M. T. Construction and characterization of retroviralvectors expressing biologically active human interleukin-12. Hum. Gene Ther.,5:1493–1506, 1994.

12. Matsumoto, G., Sunamura, M., Shimamura, H., Kodama, T., Hashimoto, W., Kobari,M., Kato, K., Takeda, K., Yagita, H., Okumura, K., Hamada, H., and Matsuno, S.Adjuvant immunotherapy using fibroblasts genetically engineered to secrete interleu-kin 12 prevents recurrence after surgical resection of established tumors in a murineadenocarcinoma model. Surgery,125: 257–264, 1999.

13. Toyama-Sorimachi, N., Miyake, K., and Miyasaka, M. Activation of CD44 inducesICAM-1/LFA-1-independent, Ca21, Mg21-independent adhesion pathway in lym-phocyte-endothelial cell interaction. Eur. J. Immunol.,23: 439–446, 1993.

14. Lehr, H. A., Leunig, M., Menger, M. D., Nolte, D., and Messmer, K. Dorsal skinfoldchamber technique for intravital microscopy in nude mice. Am. J. Pathol.,143:1055–1062, 1993.

15. Lozonschi, L., Sunamura, M., Kobari, M., Egawa, S., Ding, L., and Matsuno, S.Controlling tumor angiogenesis and metastasis of C26 murine colon adenocarcinomaby a new matrix metalloproteinase inhibitor, KB-R7785, in two tumor models. CancerRes.,59: 1252–1258, 1999.

16. Ohtani, H., and Sasano, N. Microvascular changes in the stroma of human colorectalcarcinomas: ultrastructural histochemical study. Jpn. J. Cancer Res.,80: 360–365,1989.

17. Sahin, A. A., Ro, J., Ro, J. Y., Blick, M. B., el-Naggar, A. K., Ordonez, N. G.,Fritsche, H. A., Smith, T. L., Hortobagyi, G. N., and Ayala, A. G. Ki-67 immuno-staining in node-negative stage I/II breast carcinoma. Significant correlation withprognosis. Cancer (Phila.),68: 549–557, 1991.

18. Folkman, J. Tumor angiogenesis. Adv. Cancer Res.,43: 175–203, 1985.19. Brunda, M. J., Luistro, L., Warrier, R. R., Wright, R. B., Hubbard, B. R., Murphy, M.,

Wolf, S. F., and Gately, M. K. Antitumor and antimetastatic activity of interleukin 12against murine tumors. J. Exp. Med.,178: 1223–1230, 1993.

20. Nastala, C. L., Edington, H. D., McKinney, T. G., Tahara, H., Nalesnik, M. A.,Brunda, M. J., Gately, M. K., Wolf, S. F., Schreiber, R. D., Storkus, W. J., and Lotze,M. T. Recombinant IL-12 administration induces tumor regression in association withIFN-g production. J. Immunol.,153: 1697–1706, 1994.

21. Kanegane, C., Sgadari, C., Kanegane, H., Teruya-Feldstein, J., Yao, L., Gupta, G.,Farber, J. M., Liao, F., Liu, L., and Tosato, G. Contribution of the CXCchemokines IP-10 and Mig to the antitumor effects of IL-12. Int. J. Cancer,78:361–365, 1998.

22. Sgadari, C., Angiolillo, A. L., and Tosato, G. Inhibition of angiogenesis by interleu-kin-12 is mediated by the interferon-inducible protein 10. Blood,87: 3877–3882,1996.

23. Coughlin, C. M., Salhany, K. E., Wysocka, M., Aruga, E., Kurzawa, H., Chang, A. E.,Hunter, C. A., Fox, J. C., Trinchieri, G., and Lee, W. M. F. Interleukin-12 andinterleukin-18 synergistically induce murine tumor regression which involves inhi-bition of angiogenesis. J. Clin. Investig.,101: 1441–1452, 1998.

24. Tahara, H., Zeh, H. J., III, Storkus, W. J., Pappo, I., Watkins, S. C., Gubler, U., Wolf,S. F., Robbins, P. D., and Lotze, M. T. Fibroblasts genetically engineered to secreteinterleukin 12 can suppress tumor growth and induce antitumor immunity to a murinemelanomain vivo. Cancer Res.,54: 182–189, 1994.

25. Caruso, M., Pham-Nguyen, K., Kwong, Y. L., Xu, B., Kosai, K. I., Finegold, M.,Woo, S. L., and Chen, S. H. Adenovirus-mediated interleukin-12 gene therapyfor metastatic colon carcinoma. Proc. Natl. Acad. Sci. USA,93: 11302–11306,1996.

26. Bramson, J. L., Hitt, M., Addison, C. L., Muller, W. J., Gauldie, J, and Graham, F. L.Direct intratumoral injection of an adenovirus expressing interleukin-12 inducesregression and long-lasting immunity that is associated with highly localized expres-sion of interleukin-12. Hum. Gene Ther.,7: 1995–2002, 1996.

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ANTIANGIOGENESIS EFFECTS OF IL-12

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2000;60:1111-1116. Cancer Res   Dan Gabriel Duda, Makoto Sunamura, Lucian Lozonschi, et al.   Antiangiogenesis Effects of Interleukin 12

Analysis of thein Vivo Evidence and in VitroDirect

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