Purpose: We determined whether chronic administration of IFN-α at optimal biological dose inhibits angiogenesis of human pancreatic carcinoma growing in the pancreas of nude mice.

Experimental Design: Cells of the human pancreatic cancer cell line L3.6pl were implanted into the pancreas of nude mice. Seven days later, groups of mice received s.c. injection with IFN-α alone (50,000 units biweekly or 10,000 units daily), i.p. injection with gemcitabine alone (125 mg/kg biweekly), or injection with both daily IFN-α and biweekly gemcitabine for 35 days. In a survival study, the mice were treated until they became moribund.

Results: Biweekly treatments with 50,000 units of IFN-α alone were ineffective. In contrast, daily injections of IFN-α (10,000 units/day) alone, biweekly injections of gemcitabine alone, or the combination of IFN-α and gemcitabine reduced tumor volume by 53%, 70%, and 87%, respectively. Immunohistochemical analysis revealed that treatment with IFN-α alone or with IFN-α plus gemcitabine inhibited expression of the proangiogenic molecules basic fibroblast growth factor and matrix metalloproteinase 9 more than did treatment with gemcitabine alone. These treatments also decreased the staining of proliferating cell nuclear antigen within the tumor and induced apoptosis in tumor-associated mouse endothelial cells (staining with CD31/terminal deoxynucleotidyl transferase-mediated nick end labeling), leading to a decrease in microvessel density.

Conclusions: These data show that administration of IFN-α at optimal biological dose and schedule in combination with gemcitabine induced apoptosis in tumor-associated endothelial cells and decreased growth of human pancreatic cancer cells in the pancreas, leading to a significant increase in survival.

Extensive local invasion and early lymphatic and hematogenous metastasis characterize cancer of the exocrine pancreas. At the time of diagnosis, >80% of patients present with either locally advanced or metastatic disease (1, 2). The inability to detect pancreatic cancer at an early stage, its aggressiveness, and the lack of effective systemic therapy are all responsible for the high mortality of this disease. Only 3–4% of all patients with adenocarcinoma of the pancreas survive 5 years after diagnosis (3). Not even the recent introduction of the deoxycytidine analogue, gemcitabine, has extended median survival beyond 6 months for patients with advanced pancreatic cancer (4, 5).

IFNs are a family of natural glycoproteins first discovered in the 1950s because of their antiviral activity (6). IFN-α, for example, has been shown to directly inhibit the proliferation of tumor cells of different histological origins (7, 8, 9). More recently, IFN-α has been shown to down-regulate the expression of the proangiogenic molecules bFGF,3 MMP-2 and MMP-9 (10, 11, 12), and IL-8 (13, 14) and to activate host effector cells (9).

Unfortunately, several clinical trials of recombinant IFN-α combined with standard chemotherapy (15, 16, 17) in advanced pancreatic cancer were associated with significant toxicity, failing to prove of any benefit to the patients. More encouraging, a recent Phase II trial using IFN-α/cisplatin/5-fluorouracil-based adjuvant chemoradiation after pancreatic duodenectomy reported the best-published 2-year survival for patients with resected pancreatic cancer (18). It should be noted, however, that in these trials, IFN-α was typically administered once weekly or 3×/week, and none of these trials used IFN-α in combination with gemcitabine (the most active cytotoxic agent against advanced pancreatic cancer; Ref. 5). These schedules may well be suboptimal, given that the reported half-life of IFN-α ranges from 4–8 h, with peak serum concentrations occurring at 3–8 h after i.v. or s.c. administration. Twenty-four h after i.v. or s.c. administration, little or no IFN-α is present in the serum (19). Previous reports from our laboratory demonstrated that down-regulation of angiogenesis-related genes (20) and therapy of solid tumors are dependent on optimization of biological dose and schedule of IFN-α (21).

Using a murine orthotopic model of pancreatic cancer (22), we set out to determine the most effective of two IFN-α schedules plus a combined regimen of IFN-α and gemcitabine. We show that daily s.c. injections of IFN-α at the dose of 10,000 units in combination with i.p. injections of gemcitabine significantly inhibited the growth of human pancreatic carcinoma by directly down-regulating the expression of bFGF, MMP-9, IL-8, and VEGF/vascular permeability factor; by inducing apoptosis in tumor-associated endothelial cells; and by inhibiting tumor cell proliferation.

Pancreatic Cancer Cell Lines and Culture Conditions.

The metastatic human pancreatic cancer cell line L3.6pl (22) was maintained in DMEM supplemented with 5% fetal bovine serum, sodium pyruvate, nonessential amino acids, l-glutamine, a 2-fold vitamin solution (Life Technologies, Inc., Grand Island, NY), and penicillin-streptomycin mixture (Flow Laboratories, Rockville, MD). Adherent monolayer cultures were maintained on plastic and incubated at 37°C in a mixture of 5% CO2 and 95% air. The cultures were free of Mycoplasma and the following pathogenic murine viruses: reovirus type 3; pneumonia virus; K virus; Theiler’s encephalitis virus; Sendai virus; minute virus; mouse adenovirus; mouse hepatitis virus; lymphocytic choriomeningitis virus; ectromelia virus; and lactate dehydrogenase virus (assayed by M. A. Bioproducts, Walkersville, MD). The cultures were maintained for no longer than 12 weeks after recovery from frozen stock. All reagents used in tissue culture were free of endotoxins as determined by the Limulus amebocyte lysate assay (sensitivity limit of 0.125 ng/ml) purchased from Associates of Cape Cod (Falmouth, MA).

Reagents.

Recombinant human IFN-α-2a (IFN-α; Roferon; Hoffman-LaRoche, Nutley, NJ) was kept at 4°C and diluted in HBSS as necessary at the time of use. Gemcitabine (GENZAR; Eli Lilly Co., Indianapolis, IN) was kept at room temperature and made fresh in 0.9% NaCl on the day of use. Antibodies purchased were as follows: polyclonal rabbit anti-VEGF/vascular permeability factor (Santa Cruz Biotechnology, Santa Cruz, CA); peroxidase-conjugated goat antirabbit (IgG) F(ab′)2 (Jackson Research Laboratories, West Grove, CA); monoclonal rat antimouse CD31/PECAM-1 (PharMingen, San Diego, CA); peroxidase-conjugated goat antirat IgG (H&L; Jackson Research Laboratories); monoclonal mouse anti-PCNA clone PC-10 (DAKO A/S); peroxidase-conjugated rat antimouse IgG2a heavy chain (Serotec; Harlan Bioproducts for Science, Inc., Indianapolis, IN); polyclonal rabbit antihuman IL-8 (Biosource International, Camarillo, CA); rabbit antihuman bFGF (Sigma); peroxidase-conjugated rat antimouse IgG1 (PharMingen); mouse antihuman MMP-9 (Oncogene, Boston, MA); and Texas Red-conjugated goat antirat IgG (Jackson Research Laboratories).

Hoechst Dye 3342 Mr 615.9 was purchased from Hoechst (Warrington, PA), stable 3,3′-diaminobenzidine was purchased from Research Genetics (Huntsville, AL), 3-amino-9-ethylcarbazole was purchased from Biogases Laboratories (San Ramon, CA), and Gill’s hematoxylin was purchased from Sigma. Prolong solution was purchased from Molecular Probes (Eugene, OR), and pepsin was purchased from Biomeda (Foster City, CA).

MTT (M2128) was purchased from Sigma, and a stock solution was prepared by dissolving 5 mg of MTT in 1 ml of PBS and filtering the solution to remove particulates. The solution was protected from light and stored at 4°C. The TUNEL assay was performed using a commercial apoptosis detection kit (Promega, Madison, WI) with modifications.

In Vitro Cytotoxicity Assay.

Tumor cells (1 × 103) were seeded into 38-mm2 wells of flat-bottomed 96-well plates in triplicate and allowed to adhere overnight. The spent medium was then removed, and the cultures were refed with new medium (negative control) or medium containing different concentrations of human IFN-α. After 4 days (control cultures did not reach confluence), the number of metabolically active cells was determined by MTT assay (23).

Animals and Orthotopic Implantation of Tumor Cells.

Male athymic nude mice (NCI-nu) were purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD). The mice were maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture, United States Department of Health and Human Services, and the NIH. The mice were used in accordance with institutional guidelines when they were 8–10 weeks old.

To produce pancreatic tumors, L3.6pl cells were harvested from subconfluent cultures by a brief exposure to 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped with medium containing 10% fetal bovine serum, and the cells were washed once in serum-free medium and resuspended in HBSS. Only suspensions consisting of single cells with >90% viability were used for the injections. Cells were injected into the pancreas as described previously (22). The mice were killed when moribund. The size and weight of the primary pancreatic tumors, the incidence of regional (celiac and para-aortic) lymph node metastasis, and the incidence of liver metastasis were recorded. Histopathology confirmed the nature of the disease. For histology and immunohistochemistry, one part of the tumor tissue was fixed in formalin and embedded in paraffin. Another part of the tumor was embedded in OCT compound (Miles, Inc., Elkhart, IN), snap frozen in liquid nitrogen, and stored at −70°C.

Therapy of Established Human Pancreatic Carcinoma Tumors Growing in the Pancreas of Athymic Nude Mice.

Seven days after the implantation of tumor cells into the pancreas, five mice were killed, and the presence of tumor lesions was determined. At this time, the median tumor volume was 18 mm3. Histological examination confirmed the lesions to be actively growing pancreatic cancer. For the first experiment, the mice were randomized into the following five treatment groups: (a) daily s.c. administration of human IFN-α (10,000 units); (b) biweekly s.c. administrations of IFN-α (50,000 units); (c) twice a week i.p. injections of gemcitabine (125 mg/kg); (d) daily s.c. administrations of IFN-α (10,000 units) and twice a week i.p. injections of gemcitabine (125 mg/kg); and (e) biweekly s.c. administrations of IFN-α (50,000 units) and twice a week i.p. injections of gemcitabine (125 mg/kg). In the sixth group, control mice received s.c. and i.p. HBSS (vehicle). The mice were necropsied on day 35, and the volume of pancreatic tumors and incidence of lymph node and liver metastasis were recorded.

In a second experiment, the most effective dosage of IFN-α as determined in the first experiment was used. Three treatment groups were used as follows: (a) daily s.c. injections of human IFN-α (10,000 units) alone; (b) twice per week i.p. injections of gemcitabine (125 mg/kg) alone; and (c) daily s.c. injections of IFN-α (10,000 units) and twice per week i.p. injections of gemcitabine. The fourth group of control mice were treated s.c. and i.p. with HBSS. Mice were killed and autopsied on day 35, and the results were analyzed as described above. In a third therapy experiment, mice were randomized to the above-described four groups (n = 15), and five mice in each treatment group were killed after 14, 21, and 28 days of treatment. A final experiment determined overall survival of treated mice. Mice received implant of L3.6pl cells in the pancreas and were randomized on day 7 to the four groups (n = 10). The mice were killed and necropsied only when they became moribund. The volume of pancreatic tumors and the incidence of lymph node and liver metastases were recorded.

Necropsy Procedures.

Mice were killed, and body weight was determined. Primary tumors in the pancreas were excised, measured, and weighed. Visible liver metastases were counted with the aid of a dissecting microscope and processed for H&E staining. All macroscopically enlarged regional (celiac and para-aortal) lymph nodes were harvested, and the presence of metastatic disease was confirmed by histology.

IHC Determination of PCNA, CD31/PECAM-1, bFGF, MMP-9, IL-8, and VEGF.

Paraffin-embedded tissues were used for IHC identification of PCNA, CD31/PCAM-1, bFGF, MMP-9, IL-8, and VEGF. Sections (4–6-μm thick) were mounted on positively charged Superfrost slides (Fisher Scientific, Houston, TX) and dried overnight. Sections were deparaffinized in xylene, followed by treatment with a graded series of alcohol [100%, 95%, and 80% ethanol/double-distilled H2O (v/v)] and rehydrated in PBS (pH 7.5). Sections analyzed for PCNA were microwaved for 5 min for “antigen retrieval.” All other paraffin-embedded tissues were treated with pepsin (Biomeda) for 15 min at 37°C and washed with PBS. Frozen tissues used for identification of CD31/PECAM-1 were sectioned (8–10 μm), mounted on positively charged Plus slides (Fisher Scientific), and air dried for 30 min. Frozen sections were fixed in cold acetone (5 min), acetone/chloroform (v/v; 5 min), and acetone (5 min) and washed with PBS. IHC procedures were performed as described previously (24). Positive reactions were visualized by incubating the slides with stable 3,3′-diaminobenzidine for 10–20 min. The sections were rinsed with distilled water, counterstained with Gill’s hematoxylin for 30 s, and mounted with Universal Mount (Research Genetics). Control samples exposed to secondary antibody alone showed no specific staining.

Double Immunofluorescence Staining for CD31/PECAM-1 (Endothelial Cells) and TUNEL (Apoptotic Cells).

Frozen tissues were sectioned (8–10 μm), mounted on positively charged slides, air dried for 30 min, and fixed in cold acetone for 5 min, acetone/chloroform (1:1) for 5 min, and acetone for 5 min. Samples were washed three times with PBS, incubated with protein blocking solution containing 5% normal horse serum and 1% normal goat serum in PBS for 20 min at room temperature, and then incubated with the appropriate dilution (1:400) of rat monoclonal antimouse CD31 antibody (human cross-reactive) for 18 h at 4°C. After the samples were rinsed four times (3 min/each) with PBS, the slides were incubated with the appropriate dilution (1:200) of secondary goat antirat conjugated to Texas Red for 1 h at room temperature while avoiding light exposure. Samples were washed twice with PBS containing 0.1% Brij and washed with PBS for 5 min (25). TUNEL was performed using a commercially available apo-ptosis detection kit with the following modifications: samples were fixed with 4% paraformaldehyde (methanol-free) for 10 min at room temperature; washed twice with PBS for 5 min; and then incubated with 0.2% Triton X-100 for 15 min at room temperature. After being washed twice (5 min/each) with PBS, the samples were incubated with equilibration buffer for 10 min at room temperature. The equilibration buffer was drained, and reaction buffer containing equilibration buffer, nucleotide mix, and terminal deoxynucleotidyl transferase enzyme was added to the tissue sections and incubated in a humidified chamber at 37°C for 1 h in the dark. Immersing the samples in 2× SSC for 15 min terminated the reaction, and samples were washed 3 times for 5 min to remove unincorporated fluorescein-dUTP. For quantification of endothelial cells, the samples were incubated with 300 μg/ml Hoechst stain for 10 min at room temperature. Fluorescent bleaching was minimized by treating slides with an enhancing reagent (Prolong solution). Immunofluorescence microscopy was performed using a ×40 objective (Zeiss Plan-Neofluar) on an epifluorescence microscope equipped with narrow bandpass excitation filters mounted in a filter wheel (Ludl Electronic Products, Hawthorne, NY) to individually select for green, red, and blue fluorescence. Images were captured using a chilled cooled charge-coupled device camera (Photometrics, Tucson, AZ) and SmartCapture software (Digital Scientific, Cambridge, United Kingdom) on a Macintosh computer. Images were further processed using Adobe Photoshop software (Adobe Systems, Mountain View, CA). Endothelial cells were identified by red fluorescence, and DNA fragmentation was detected by localized green and yellow fluorescence within the nucleus (visualized by Hoechst stain) of apoptotic cells. Quantification of apoptotic endothelial cells was expressed as a percentage of TUNEL+ endothelial cells (that made up the total endothelial cell population/field) in 5–10 random 0.011-mm2 fields at ×400 magnification. For the quantification of total TUNEL expression, the number of apoptotic events was counted in 10 random 0.159-mm2 fields at ×100 magnification (25).

Quantification of MVD and PCNA.

For the quantification of MVD, 10 random 0.159-mm2 fields at ×100 magnification were captured for each tumor, and microvessels were quantified according to the method described previously (26, 27). For the quantification of PCNA expression, the number of positive cells was quantified in 10 random 0.159-mm2 fields at ×100 magnification.

Statistical Analysis.

Pancreatic tumor volume was compared by the Mann-Whitney t test, and the incidence of metastasis was evaluated by using Fisher’s exact test. Quantification of PCNA, TUNEL, CD31, and the number of apoptotic endothelial cells was compared by unpaired Student’s t test. Survival analysis was computed by the Kaplan-Meier method and compared by the log-rank test.

In Vitro IC50 of IFN-α.

L3.6pl cells were incubated for 4 days in medium containing increasing concentrations of IFN-α (0–10,000 units). The cells were relatively resistant to the antiproliferative effects of IFN-α, whose IC50 was 5,200 units/ml.

Inhibition of Pancreatic Cancer Growth and Metastasis.

L3.6pl cells were injected into the pancreas of athymic nude mice. Seven days later, the mice were randomized into six groups of 10 mice each. The first group received twice weekly i.p. injections of gemcitabine at 125 mg/kg, the second group received daily human IFN-α at 10,000 units s.c., the third group received twice weekly IFN-α at 50,000 units s.c., the fourth group received twice weekly i.p. gemcitabine and daily s.c. IFN-α (10,000 units), and the fifth group received twice weekly i.p. gemcitabine and twice weekly s.c. IFN-α (50,000 units); the sixth group (control) received HBSS i.p. and s.c. All mice were killed on day 35. All mice had tumors in the pancreas. The data summarized in Table 1 show that daily s.c. injections of IFN-α (10,000 units) or twice weekly i.p. injections of gemcitabine significantly decreased median tumor volume as compared with control mice (P < 0.001). Treatment with gemcitabine and daily IFN-α (10,000 units, s.c.) produced the greatest decrease in median volume of pancreatic tumor (P < 0.001). Administration of biweekly IFN-α (50,000 units, s.c.) was ineffective. The experiment was repeated using daily IFN-α (10,000 units, s.c.), gemcitabine, and daily IFN-α plus gemcitabine. Visible liver metastases (enumerated with the aid of a dissecting microscope) were present in 40% of the control mice and 0–30% of the treatment groups (Tables 1 and 2). Histologically positive regional lymph node metastases were found in 70–100% of control- and gemcitabine-treated mice, whereas in mice receiving both gemcitabine and IFN-α (10,000 units), the incidence of regional lymph node metastasis was 40% (P < 0.05; Table 1). Treatments with IFN-α alone or in combination with gemcita-bine were well tolerated as determined by maintenance of body weight (Tables 1 and 2).

A final experiment was carried out to determine survival. Mice were killed and necropsied only when they became moribund (Fig. 1). The median survival time for the control group was 38 days. After treatment with gemcitabine alone, daily IFN-α alone, and a combination of daily IFN-α and gemcita-bine, the median survival time was 44, 38, and 78 days, respectively (control versus gemcitabine, P < 0.05; control versus IFN-α, P = 0.06; control versus IFN-α and gemcitabine, P < 0.001).

Histology and IHC Analyses.

We next determined the in vivo effects of IFN-α (alone or in combination with gemcitabine). L36pl cells were injected into the pancreas of mice, and the mice were randomized into the above-mentioned four treatment groups. After 14, 21, and 28 days of treatment, five mice from each treatment group were killed and necropsied, and tumors were harvested and processed for routine histology and IHC analyses of cell proliferation and apoptosis using anti-PCNA antibodies and the TUNEL method, respectively (Fig. 2). At 28 days of treatment, the mean number of PCNA+ tumor cells in control tumors was 586 ± 125. After therapy with gemcitabine or IFN-α, the mean number was 400 ± 71 or 407 ± 65, respectively (Table 3). The lowest number of PCNA+ cells (361 ± 72) was found in tumors of mice treated with both IFN-α and gemcitabine (P < 0.05 versus other treatment groups).

The mean number of TUNEL+ cells was inversely related to PCNA positivity. In control tumors, the mean number of TUNEL+ cells was 9 ± 2, in gemcitabine-treated tumors, it was 60 ± 2, in IFN-α-treated tumors, it was 58 ± 20, and in IFN-α + gemcitabine-treated tumors, it was 84 ± 19 (control versus gemcitabine, P < 0.001; control versus IFN-α, P < 0.004; control versus IFN-α and gemcitabine, P < 0.00001). There was a significant difference in the number of TUNEL+ tumor cells in tumors treated with gemcitabine or IFN-α alone as compared with tumors in the combination therapy group (P < 0.05). In control mice and in mice treated with gemcitabine alone, IFN-α alone, and the combination of the two, the ratio of PCNA+:TUNEL+ cells was 65, 7, 7, and 4, respectively.

There was a significant reduction in tumor MVD (measured by staining with antibodies against CD31) per field after treatment with IFN-α or combination therapy as compared with control tumors or gemcitabine-treated tumors (Fig. 2; Table 3). There was no significant difference between MVD of tumors treated with IFN-α and MVD of tumors from mice given combination therapy (P = 0.3). IHC staining of tumors after 28 days of treatment with IFN-α and gemcitabine revealed a significant decrease in expression of bFGF, MMP-9, IL-8, and VEGF (P < 0.05 versus control, Fig. 3).

The CD31/TUNEL fluorescent double-labeling technique (25) revealed that many endothelial cells in tumors from mice treated with IFN-α or with IFN-α and gemcitabine underwent apoptosis (Fig. 4, yellow reaction). The number of apoptotic endothelial cells significantly increased in pancreatic tumors harvested 28 days after the initiation of treatment with IFN-α or IFN-α + gemcitabine as compared with tumors from control mice or from mice treated with gemcitabine (P < 0.001; Table 3; Figs. 4 and 5 A).

Apoptosis of tumor-associated endothelial cells (CD31/TUNEL+ cells, Fig. 5,A) was found as early as day 14 of treatment (Fig. 5). Endothelial cell apoptosis was higher in tumors from mice treated with IFN-α or IFN-α and gemcitabine than in tumors from control mice or those treated with gemcitabine alone (control versus IFN-α, P < 0.05; gemcitabine versus IFN-α and gemcitabine, P < 0.01; gemcitabine versus IFN-α, P < 0.05). There was no significant difference in the number of apoptotic endothelial cells between tumors from mice treated with IFN-α alone and tumors from mice treated with combination therapy (P = 0.07). In all treatment groups, overall cell death (TUNEL+ cells) increased with the duration of therapy (Fig. 5 B).

We report that daily s.c. injections of 10,000 units of human IFN-α combined with twice a week i.p. injections of gemcitabine significantly inhibited the growth and metastasis of human pancreatic carcinoma cells implanted into the pancreas of nude mice. Twice weekly s.c. injections of 50,000 units of IFN-α were ineffective. As reported previously by our laboratory (20, 21), to produce maximal therapeutic effects, IFN-α must be administered daily at an optimal biological dose. Treatment with daily IFN-α alone or gemcitabine alone reduced the growth of primary neoplasms by 53% and 70%, respectively. Combination therapy with IFN-α and gemcitabine further decreased tumor growth by 87% and decreased the incidence of lymph node metastasis, significantly prolonging survival. Daily s.c. doses of IFN-α were well tolerated by nude mice. IHC analyses of the pancreatic cancers demonstrated a significant decrease in PCNA+ cells, an associated increase in TUNEL+ cells (apoptotic cells), and a significant decrease in MVD in the IFN-α-treated or IFN-α + gemcitabine-treated tumors. The reduction in MVD was due to a significant increase in endothelial cell apoptosis.

The reduction in MVD is noteworthy because the progressive growth and metastasis of malignant neoplasms depend on adequate neovascularization. The balance between the proangiogenic and antiangiogenic molecules released by tumor cells and surrounding host cells (28, 29) determines the extent of angiogenesis. Among the major proangiogenic molecules are bFGF, MMP-9, IL-8, and VEGF (20, 21, 30, 31, 32, 33). IFN-α has been shown to inhibit transcription and protein production of many of these proangiogenic molecules (11, 13, 20, 21, 28). This regulation, however, is associated with induction of a feedback mechanism involving a family of proteins called SOCS that negatively regulate cytokine signaling. The induction of SOCS directly correlates with the concentration of a given cytokine (34, 35, 36). At daily doses exceeding 10,000 units of IFN-α, the induction of SOCS may have been responsible for the failure of IFN-α to down-regulate the target genes (37).

In this study, the daily administration of human IFN-α at 10,000 units/dose decreased the expression of bFGF, MMP-9, IL-8, and VEGF in human pancreatic cancer cells. These are important survival factors for human and mouse endothelial cells (38, 39, 40). This decreased expression correlated with an increased apoptosis of endothelial cells. The administration of gemcitabine and the decrease in survival factor receptor signaling can clearly enhance apoptosis in tumor-associated endothelial cells. Decreased MVD, i.e., vascularization, leads to apoptosis of tumor cells and the surrounding stroma (41).

In summary, we show that a 10,000-unit dose of human IFN-α, which we consider the optimal biological dose, given daily in combination with biweekly gemcitabine significantly reduces the growth of human pancreatic carcinoma in nude mice. The inhibition of primary tumor growth and lymph node metastasis is mediated by both direct antitumor effects (gemcitabine) and antiangiogenic effects (IFN-α). This combination therapy may provide a new approach to the treatment of a devastating disease.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported in part by Cancer Center Support Core Grant CA16672, Specialized Programs of Research Excellence (SPORE) in Prostate Cancer Grant CA90270, SPORE in Ovarian Cancer Grant CA93639, and SPORE in Head and Neck Cancer Grant CA97007 from the National Cancer Institute, NIH.

3

The abbreviations used are: bFGF, basic fibroblast growth factor; IHC, immunohistochemical; IL, interleukin; MMP, matrix metalloproteinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MVD, microvessel density; PCNA, proliferating cell nuclear antigen; VEGF, vascular endothelial growth factor; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; PECAM-1, platelet/endothelial cell adhesion molecule 1.

Fig. 1.

Therapeutic effects of IFN-α and gemcitabine. L3.6pl cells were injected in the pancreas of nude mice. Seven days later, the mice were randomized into four treatment groups (n = 10). Mice were killed when moribund. Survival was analyzed by the Kaplan-Meier method and compared by the log-rank test. Control group, •; gemcitabine alone, ▿ (P < 0.05 compared with control); IFN-α alone, ○ (P = 0.06); combination of gemcitabine and IFN-α, □ (P < 0.001 compared with control).

Fig. 1.

Therapeutic effects of IFN-α and gemcitabine. L3.6pl cells were injected in the pancreas of nude mice. Seven days later, the mice were randomized into four treatment groups (n = 10). Mice were killed when moribund. Survival was analyzed by the Kaplan-Meier method and compared by the log-rank test. Control group, •; gemcitabine alone, ▿ (P < 0.05 compared with control); IFN-α alone, ○ (P = 0.06); combination of gemcitabine and IFN-α, □ (P < 0.001 compared with control).

Close modal
Fig. 2.

IHC analyses. Tumors were harvested from control mice or mice treated with gemcitabine, human IFN-α, or both IFN-α and gemcitabine. The sections were immunostained for expression of PCNA (to show cell proliferation), TUNEL (FITC; to show cell death), and CD31/PECAM-1 (to show MVD). Tumors from mice treated with IFN-α plus gemcitabine had a decrease in PCNA+ cells, an increase in TUNEL+ cells, and a decrease in MVD (CD31+ cells). Representative samples (×100) are shown.

Fig. 2.

IHC analyses. Tumors were harvested from control mice or mice treated with gemcitabine, human IFN-α, or both IFN-α and gemcitabine. The sections were immunostained for expression of PCNA (to show cell proliferation), TUNEL (FITC; to show cell death), and CD31/PECAM-1 (to show MVD). Tumors from mice treated with IFN-α plus gemcitabine had a decrease in PCNA+ cells, an increase in TUNEL+ cells, and a decrease in MVD (CD31+ cells). Representative samples (×100) are shown.

Close modal
Fig. 3.

IHC analyses. Tumors were harvested from control mice and mice treated with gemcitabine, human IFN-α, or both IFN-α and gemcitabine. The sections were immunostained for expression of bFGF, MMP-9, IL-8, and VEGF. Tumors from mice treated with IFN-α + gemcitabine had a significant decrease in the expression of bFGF, MMP-9, IL-8, and VEGF. Representative samples (×100) are shown.

Fig. 3.

IHC analyses. Tumors were harvested from control mice and mice treated with gemcitabine, human IFN-α, or both IFN-α and gemcitabine. The sections were immunostained for expression of bFGF, MMP-9, IL-8, and VEGF. Tumors from mice treated with IFN-α + gemcitabine had a significant decrease in the expression of bFGF, MMP-9, IL-8, and VEGF. Representative samples (×100) are shown.

Close modal
Fig. 4.

Immunofluorescent CD31 (endothelial cells), TUNEL (apoptosis) double labeling of human pancreatic cancer growing in the pancreas of nude mice. Pancreatic tumors (L3.6pl cells) from mice treated with HBSS (control), gemcitabine, IFN-α, or gemcitabine + IFN-α were harvested on day 28 of the treatment. The tissues were processed for immunohistochemistry using anti-CD31 antibodies (Texas Red) and TUNEL (FITC-green). A representative sample (×400) of this CD31/TUNEL fluorescent double staining is shown. Fluorescent red, CD31+ endothelial cells; fluorescent green, TUNEL+ cells; yellow, TUNEL+ endothelial cells.

Fig. 4.

Immunofluorescent CD31 (endothelial cells), TUNEL (apoptosis) double labeling of human pancreatic cancer growing in the pancreas of nude mice. Pancreatic tumors (L3.6pl cells) from mice treated with HBSS (control), gemcitabine, IFN-α, or gemcitabine + IFN-α were harvested on day 28 of the treatment. The tissues were processed for immunohistochemistry using anti-CD31 antibodies (Texas Red) and TUNEL (FITC-green). A representative sample (×400) of this CD31/TUNEL fluorescent double staining is shown. Fluorescent red, CD31+ endothelial cells; fluorescent green, TUNEL+ cells; yellow, TUNEL+ endothelial cells.

Close modal
Fig. 5.

A, apoptosis of tumor-associated endothelial cells. Tumors were harvested from control mice and mice treated with gemcitabine, IFN-α, or both IFN-α and gemcitabine after 14, 21, and 28 days of treatment. The sections were double-stained with CD31/TUNEL fluorescent antibodies as described in “Materials and Methods.” Quantification of apoptotic endothelial cells was expressed as the percentage of total endothelial cells per field that were TUNEL+ in 5–10 random 0.011-mm2 fields at ×400 magnification. Numbers represent mean ± SD. ∗, P < 0.05 versus gemcitabine and control. ∗∗, P < 0.01 versus gemcitabine and control. B, induction of apoptosis. Tumors were harvested from control mice and mice treated with gemcitabine, IFN-α, or both IFN-α and gemcitabine after 14, 21, and 28 days of treatment. The sections were stained using the TUNEL technique as described in “Materials and Methods.” The number of TUNEL+ cells/field (mean ± SD) was determined from measuring 10 random 0.159-mm2 fields at ×100 magnification. ∗, P < 0.05 versus control. ∗∗, P < 0.05 versus others.

Fig. 5.

A, apoptosis of tumor-associated endothelial cells. Tumors were harvested from control mice and mice treated with gemcitabine, IFN-α, or both IFN-α and gemcitabine after 14, 21, and 28 days of treatment. The sections were double-stained with CD31/TUNEL fluorescent antibodies as described in “Materials and Methods.” Quantification of apoptotic endothelial cells was expressed as the percentage of total endothelial cells per field that were TUNEL+ in 5–10 random 0.011-mm2 fields at ×400 magnification. Numbers represent mean ± SD. ∗, P < 0.05 versus gemcitabine and control. ∗∗, P < 0.01 versus gemcitabine and control. B, induction of apoptosis. Tumors were harvested from control mice and mice treated with gemcitabine, IFN-α, or both IFN-α and gemcitabine after 14, 21, and 28 days of treatment. The sections were stained using the TUNEL technique as described in “Materials and Methods.” The number of TUNEL+ cells/field (mean ± SD) was determined from measuring 10 random 0.159-mm2 fields at ×100 magnification. ∗, P < 0.05 versus control. ∗∗, P < 0.05 versus others.

Close modal
Table 1

Therapy of human pancreatic carcinoma growing in the pancreas of nude mice

Treatment groupaPancreatic tumorsMetastasis
TumorigenicityTumor volume (mm3)LiverLymph nodeBody weight (g)
MedianRangeMedianRange
Saline control 10/10b 435 216–660 3/10b 10/10b 25 18–28 
Gemcitabine 10/10 134c 80–294 3/10 10/10 26 18–27 
IFN-α (10,000 units) 10/10 111c 29–196 2/10 10/10 22 18–25 
IFN-α (50,000 units) 10/10 550 306–1041 3/10 10/10 23 17–30 
IFN-α (10,000 units)+ gemcitabine 10/10 33d 10–82 2/10 4/10e 25 18–28 
IFNα (50,000 units)+ gemcitabine 10/10 100c 14–445 2/10 9/10 23 19–29 
Treatment groupaPancreatic tumorsMetastasis
TumorigenicityTumor volume (mm3)LiverLymph nodeBody weight (g)
MedianRangeMedianRange
Saline control 10/10b 435 216–660 3/10b 10/10b 25 18–28 
Gemcitabine 10/10 134c 80–294 3/10 10/10 26 18–27 
IFN-α (10,000 units) 10/10 111c 29–196 2/10 10/10 22 18–25 
IFN-α (50,000 units) 10/10 550 306–1041 3/10 10/10 23 17–30 
IFN-α (10,000 units)+ gemcitabine 10/10 33d 10–82 2/10 4/10e 25 18–28 
IFNα (50,000 units)+ gemcitabine 10/10 100c 14–445 2/10 9/10 23 19–29 
a

L3.6pl human pancreatic cancer cells (1 × 106) were injected into the pancreas of nude mice. Seven days later, groups of mice were treated with biweekly i.p. injections of gemcitabine (125 mg/kg) alone, daily s.c. injections of human IFN-α (10,000 units) alone, or with gemcitabine, biweekly s.c. injections of human IFN-α (50,000 units) alone or with gemcitabine, or saline (control). All mice were killed on day 35.

b

Number of positive mice/number of mice injected.

c

P < 0.001 versus control.

d

P < 0.001 versus control and P < 0.05 versus single-agent therapy.

e

P < 0.05 versus control.

Table 2

Therapy of human pancreatic carcinoma growing in the pancreas of nude mice

Treatment groupaPancreatic tumorsMetastasis
TumorigenicityTumor volume (mm3)LiverLymph nodeBody weight (g)
MedianRangeMedianRange
Saline control 10/10b 749 343–1837 4/10b 10/10b 23 19–28 
Gemcitabine 10/10 226c 80–352 3/10 7/10 27 18–28 
IFN-α (10,000 units) 10/10 352c 198–1352 2/10 8/10 23 16–29 
IFN-α (10,000 units)+ gemcitabine 10/10 100d 72–225 0/10 4/10c 24 16–28 
Treatment groupaPancreatic tumorsMetastasis
TumorigenicityTumor volume (mm3)LiverLymph nodeBody weight (g)
MedianRangeMedianRange
Saline control 10/10b 749 343–1837 4/10b 10/10b 23 19–28 
Gemcitabine 10/10 226c 80–352 3/10 7/10 27 18–28 
IFN-α (10,000 units) 10/10 352c 198–1352 2/10 8/10 23 16–29 
IFN-α (10,000 units)+ gemcitabine 10/10 100d 72–225 0/10 4/10c 24 16–28 
a

L3.6pl human pancreatic cancer cells (1 × 106) were injected into the pancreas of nude mice. Seven days later, groups of mice were treated with biweekly i.p. injections of gemcitabine (125 mg/kg) alone, daily s.c. injections of human IFN-α (10,000 units) alone, or with gemcitabine or saline (control). All mice were killed on day 35.

b

Number of positive mice/number of mice injected.

c

P < 0.05 versus control.

d

P < 0.001 versus control and P < 0.01 versus single-agent therapy.

Table 3

Immunohistochemical analysis of human pancreatic carcinoma in the pancreas of control and treated nude mice

Treatment groupaHuman tumor cellsMouse endothelial cells
PCNA+TUNEL+CD31+TUNEL+
Saline control 586 ± 125b 9 ± 2b 70 ± 1b 0.5 ± 0.6c 
Gemcitabine 400 ± 71 60 ± 2d 80 ± 1.5 2 ± 1 
IFN-α (10,000 units) 407 ± 65d 58 ± 20d 44 ± 1.2d 12 ± 6d 
IFN-α (10,000 units)+ gemcitabine 361 ± 72e 84 ± 19e 30 ± 1.8d 19 ± 5d 
Treatment groupaHuman tumor cellsMouse endothelial cells
PCNA+TUNEL+CD31+TUNEL+
Saline control 586 ± 125b 9 ± 2b 70 ± 1b 0.5 ± 0.6c 
Gemcitabine 400 ± 71 60 ± 2d 80 ± 1.5 2 ± 1 
IFN-α (10,000 units) 407 ± 65d 58 ± 20d 44 ± 1.2d 12 ± 6d 
IFN-α (10,000 units)+ gemcitabine 361 ± 72e 84 ± 19e 30 ± 1.8d 19 ± 5d 
a

L3.6pl human pancreatic cancer cells (1 × 106) were injected into the pancreas of nude mice. Seven days later, groups of mice were treated with biweekly i.p. injections of gemcitabine (125 mg/kg) alone, daily s.c. injections of human IFN-α (10,000 units) alone, or with gemcitabine or saline (control). Data after 28 days of treatment are shown.

b

Number (mean ± SD) positive cells/field determined from measurement of 10 random 0.159-mm2 fields at ×100 magnification.

c

Number (mean ± SD) of CD31/TUNEL-positive cells in 10 random 0.011-mm2 fields at ×400 magnification. Fluorescent double labeling was performed on frozen tissue sections.

d

P < 0.05 as compared with controls.

e

P < 0.05 as compared with single-agent therapy.

We thank Walter Pagel for critical editorial review and Lola López for expert preparation of the manuscript.

1
Warshaw A. L., Fernandez-del Castillo C. Pancreatic carcinoma.
N. Eng. J. Med.
,
326
:
455
-465,  
1992
.
2
Evans D. B., Abbruzzese J. L., Willet C. G. Cancer of the pancreas deVita V. Hellman S. Rosenberg S. A. eds. .
Principles and Practice of Oncology
,
1126
-1160, J. B. Lippincott Philadelphia  
2000
.
3
Jemal A., Thomas A., Murray T., Thun M. Cancer statistics, 2002.
CA Cancer J. Clin.
,
52
:
23
-47,  
2002
.
4
Burris H. A., Moore M. J., Andersen J., Green M. R., Rothenberg M. L., Modiano M. R., Cripps M. C., Portenoy R. K., Storniolo A. M., Tarassoff P., Nelson R., Dorr F. A., Stephens C. D., Von Hoff D. D. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial.
J. Clin. Oncol.
,
15
:
2403
-2413,  
1997
.
5
Moore M. Activity of gemcitabine in patients with advanced pancreatic carcinoma: a review.
Cancer (Phila.)
,
78
:
633
-638,  
1996
.
6
Isaacs A., Lindenmann J. Virus interference.
J. Interferon Res.
,
7
:
429
-438,  
1987
.
7
Hertzog P. J., Hwang S. Y., Kola I. Role of interferons in the regulation of cell proliferation, differentiation, and development.
Mol. Reprod. Dev.
,
39
:
226
-232,  
1994
.
8
Gutterman J. U. Cytokine therapeutics: lessons from interferon-α.
Proc. Natl. Acad. Sci. USA
,
91
:
1198
-1205,  
1994
.
9
Thomas H., Balkwill F. R. Effects of interferons and other cytokines on tumors in animals: a review.
Pharmacol. Ther.
,
52
:
307
-330,  
1991
.
10
Fabra A., Nakajima M., Bucana C. D., Fidler I. J. Modulation of the invasive phenotype of human colon carcinoma cells by organ specific fibroblasts of nude mice.
Differentiation
,
52
:
101
-110,  
1992
.
11
Gohji K., Fidler I. J., Tsan R., Radinsky R., von Eschenbach A. C., Tsuruo T., Nakajima M. Human recombinant interferons-β and -γ decrease gelatinase production and invasion by human KG-2 renal carcinoma cells.
Int. J. Cancer
,
58
:
380
-384,  
1994
.
12
Kato N., Nawa A., Tamakoshi K., Kikkawa F., Suganuma N., Okamoto T., Goto S., Tomoda Y., Hamaguchi M., Nakajima M. Suppression of gelatinase production with decreased invasiveness of choriocarcinoma cells by human recombinant interferon-β.
Am. J. Obstet. Gynecol.
,
172
:
601
-606,  
1995
.
13
Singh R. K., Gutman M., Llansa N., Fidler I. J. Interferon-β prevents the upregulation of interleukin-8 expression in human melanoma cells.
J. Interferon Cytokine Res.
,
16
:
577
-584,  
1996
.
14
Oliveira I. C., Sciavolino P. J., Lee T. H., Vilcek J. Downregulation of interleukin-8 gene expression in human fibroblasts: unique mechanism of transcriptional inhibition by interferon.
Proc. Natl. Acad. Sci. USA
,
89
:
9049
-9053,  
1992
.
15
Sparano J. A., Lipsitz S., Wadler S., Hansen R., Bushunow P. W., Kirkwood J., Flynn P. J., Dutcher J. P., Benson A. B. Phase II trial of prolonged continuous infusion of 5-fluorouracil and interferon-α in patients with advanced pancreatic cancer. Eastern Cooperative Oncology Group Protocol 3292.
Am. J. Clin. Oncol.
,
19
:
546
-551,  
1996
.
16
Papapetropoulos A., Piccardoni P., Cirino G., Bucci M., Sorrentino R., Cicala C., Johnson K., Zachariou V., Sessa W. C., Altieri D. C. Hypotension and inflammatory cytokine gene expression triggered by factor Xa-nitric oxide signaling.
Proc. Natl. Acad. Sci. USA
,
95
:
4738
-4742,  
1998
.
17
Wagener D. J., Wils J. A., Kok T. C., Planting A., Couvreur M. L., Baron B. Results of a randomised phase II study of cisplatin plus 5-fluorouracil versus cisplatin plus 5-fluorouracil with α-interferon in metastatic pancreatic cancer: an EORTC gastrointestinal tract cancer group trial.
Eur. J. Cancer
,
38
:
648
-653,  
2002
.
18
Nukui Y., Picozzi V. J., Traverso L. W. Interferon-based adjuvant chemoradiation therapy improves survival after pancreaticoduodenectomy for pancreatic adenocarcinoma.
Am. J. Surg.
,
179
:
367
-371,  
2000
.
19
Wills R. J. Clinical pharmacokinetics of interferons.
Clin. Pharmacokinet.
,
19
:
390
-399,  
1990
.
20
Singh R. K., Gutman M., Bucana C. D., Sanchez R., Llansa N., Fidler I. J. Interferons α and β downregulate the expression of basic fibroblast growth factor in human carcinomas.
Proc. Natl. Acad. Sci. USA
,
92
:
4562
-4566,  
1995
.
21
Slaton J. W., Perrotte P., Inoue K., Dinney C. P., Fidler I. J. Interferon-α-mediated downregulation of angiogenesis-related genes and therapy of bladder cancer are dependent on optimization of biological dose and schedule.
Clin. Cancer Res.
,
5
:
2726
-2734,  
1999
.
22
Bruns C. J., Harbison M. T., Kuniyasu H., Eue I., Fidler I. J. In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice.
Neoplasia
,
1
:
50
-62,  
1999
.
23
Fan D., Poste G., Seid C., Earnest L. E., Bull T., Clyne R. K., Fidler I. J. Reversal of multidrug resistance in murine fibrosarcoma cells by thioxanthene flupentixol.
Investig. New Drugs
,
12
:
185
-195,  
1994
.
24
Vecchi A., Garlanda C., Lampugnani M. G., Resnati M., Matteucci C., Stoppacciaro A., Schnurch H., Risau W., Ruco L., Mantovani A. Monoclonal antibodies specific for endothelial cells of mouse blood vessels: their application in the identification of adult and embryonic endothelium.
Eur. J. Cell Biol.
,
63
:
247
-254,  
1994
.
25
Bruns C. J., Solorzano C. C., Harbison M. T., Ozawa S., Tsan R., Fan D., Abbruzzese J., Traxler P., Buchdunger E., Radinsky R., Fidler I. J. Blockade of the epidermal growth factor receptor signaling by a novel tyrosine inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma.
Cancer Res.
,
60
:
2926
-2935,  
2000
.
26
Weidner N., Semple J. P., Welch W. R., Folkman J. Tumor angiogenesis and metastasis: correlation in invasive breast carcinoma.
N. Engl. J. Med.
,
324
:
1
-8,  
1991
.
27
Shi S. R., Key M. E., Kalra K. L. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.
J. Histochem. Cytochem.
,
39
:
741
-748,  
1991
.
28
Fidler I. J., Ellis L. M. The implications of angiogenesis for the biology and therapy of cancer metastasis.
Cell
,
79
:
185
-188,  
1994
.
29
Hanahan D., Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.
Cell
,
86
:
353
-364,  
1996
.
30
Singh R. K., Llansa N., Bucana C. D., Sanchez R., Koura A., Fidler I. J. Cell density-dependent regulation of basic fibroblast growth factor expression in human renal cell carcinoma cells.
Cell Growth Differ.
,
7
:
397
-404,  
1996
.
31
Vermeulen P. B., Dirix L. Y., Martin M., Lemmens J., Van Oosterom A. T. Serum basic fibroblast growth factor and vascular endothelial growth factor in metastatic renal cell carcinoma treated with interferon-α-2b.
J. Natl. Cancer Inst. (Bethesda)
,
89
:
1316
-1317,  
1997
.
32
Dinney C. P., Bielenberg D. R., Perrotte P., Reich R., Eve B. Y., Bucana C. D., Fidler I. J. Inhibition of basic fibroblast growth factor expression, angiogenesis, and growth of human bladder carcinoma in mice by systemic interferon-α administration.
Cancer Res.
,
58
:
808
-814,  
1998
.
33
Ellis L. M., Fidler I. J. Angiogenesis and metastasis.
Eur. J. Cancer
,
32A
:
2451
-2460,  
1996
.
34
Nicholson S. E., Hilton D. J. The SOCS proteins: a new family of negative regulators of signal transduction.
J. Leukocyte Biol.
,
63
:
665
-668,  
1998
.
35
Nicola N. A., Nicholson S. E., Metcalf D., Zhang J. G., Baca M., Farley A., Willson T. A., Starr R., Alexander W., Hilton D. J. Negative regulation of cytokine signaling by the SOCS proteins.
Cold Spring Harbor Symp. Quant. Biol.
,
64
:
397
-404,  
1999
.
36
Narazaki M., Fujimoto M., Matsumoto T., Morita Y., Saito H., Kajita T., Yoshizaki K., Naka T., Kishimoto T. Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin-6 signaling.
Proc. Natl. Acad. Sci. USA
,
95
:
13130
-13134,  
1998
.
37
Tedjariti S., Baker C. H., Apte S., Huang S., Wolf J. K., Killion J. J., Fidler I. J. Synergistic therapy of human ovarian carcinoma implanted orthotopically in nude mice by optimal biological dose of pegylated interferon-α combined with paclitaxel.
Clin. Cancer Res.
,
8
:
2412
-2422,  
2002
.
38
Yamazaki K., Nagao T., Yamaguchi T., Saisho H., Kondo Y. Expression of basic fibroblast growth factor (FGF-2)-associated with tumour proliferation in human pancreatic carcinoma.
Virchows Arch.
,
431
:
95
-101,  
1997
.
39
Pepper M. S., Ferrara N., Orci L., Montesano R. Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro..
Biochem. Biophys. Res. Commun.
,
189
:
824
-831,  
1992
.
40
Fujio Y., Walsh K. Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner.
J. Biol. Chem.
,
274
:
16349
-16354,  
1999
.
41
Fidler I. J. Angiogenic heterogeneity: regulation of neoplastic angiogenesis by the organ microenvironment.
J. Natl. Cancer Inst. (Bethesda)
,
93
:
1040
-1041,  
2001
.