Interferon-α-mediated Down-Regulation of Angiogenesis-related Genes and Therapy of Bladder Cancer Are Dependent on Optimization of Biological Dose and Schedule¹

The IFNs are a family of natural glycoproteins initially discovered in the 1950s on the basis of their antiviral activity (1). Subsequent studies revealed that the IFNs are multifunctional regulatory cytokines involved in control of cell function and replication (2, 3, 4). IFN-α and IFN-β directly inhibit the proliferation of tumor cells of different histological origins (2, 3, 4, 5, 6). Recent studies indicate that IFN-α and IFN-β can also down-regulate the expression of proangiogenic molecules, such as bFGF⁴(7, 8, 9, 10), IL-8 (11, 12) and MMP-2 and MMP-9 (13, 14, 15, 16), and activate host effector cells (6, 17). Although these data suggest a great potential for IFN-α and IFN-β in therapy of solid tumors, clinical trial results have been disappointing (2, 3, 4, 5, 6, 18, 19, 20, 21, 22). Extensive clinical trials have concluded that although IFNs can be therapeutic against many hematopoietic neoplasms, they are ineffective against most solid tumors (2, 3, 4, 5, 6, 18, 19, 20, 21, 22). Pharmacokinetic studies have demonstrated that the half-life of IFNs in the circulation of patients is on the order of minutes (23); therefore, the resulting lack of sustained therapeutic levels (19, 23) may have been responsible for the failure to inhibit or eradicate primary tumors or metastasis. To improve the duration of exposure, a variety of dosing schedules of IFN-α have also been assessed in clinical trials. Thus far, however, these trials have not shown the optimal schedule for direct antitumor effects, much less the best schedule to sustain the indirect antiangiogenic effects of IFN.

We have developed a reliable preclinical in vivo model to study the biology and therapy of human bladder tumor (24). Subsequent to implantation of 253J B-V cells into the bladder wall of nude mice, this highly metastatic human bladder cancer expresses high levels of bFGF, induces extensive vascularization, and then produces metastasis to regional lymph nodes and visceral organs (24). We have used this model to demonstrate that systemic administrations of human IFN-α can inhibit angiogenesis and hence tumorigenicity of the bladder cancers (25), but the mechanism remained unclear. In the present study, we determined the optimal antiangiogenic activity of IFN-α by altering the dose and schedule of its administration to nude mice implanted with 253J B-V cells that were preselected for resistance against the direct antiproliferative effects of IFN-α or IFN-β (25). We show that maximal inhibition of angiogenesis-regulating genes and tumorigenicity depends on the optimal biological dose (not maximal tolerated dose) and frequency of administration.

The highly metastatic 253J B-V human bladder cancer variant line was originally selected from the 253J line by sequential cycles of intrabladder wall implantation and isolation of lymph node metastases (24). The 253J B-V IFN^R human bladder cancer cell line was isolated from the 253J B-V cell line by prolonged culture with increasing concentrations of human IFN-α-2a (specific activity, 6 × 10⁶ IU/mg protein; Hoffman-LaRoche, Nutley, NJ; Refs. 24 and 25). This cell line is resistant to the antiproliferative effects of human IFN-α and human IFN-β at concentrations exceeding 10,000 units/ml (25). The 253J B-V IFN^R cells were grown as monolayer cultures in Eagle’s minimal essential medium supplemented with 5% fetal bovine serum, vitamins, sodium pyruvate, l-glutamine, and nonessential amino acids. The tumor cells were free of Mycoplasma, reovirus type 3, pneumonia virus of mice, mouse adenovirus, murine hepatitis virus, lymphocytic choriomeningitis virus, ectromelia virus, and lactate dehydrogenase virus (assayed by Microbiological Associates, Bethesda, MD).

Male NCr-nu/nu nude mice were obtained from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research Facility (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 according to institutional guidelines when they were 8–10 weeks of age.

To produce bladder tumors, cultures (50% confluence) of 253J B-V IFN^R cells were given fresh medium 24 h before harvest by a brief treatment with 0.25% trypsin and 0.02% EDTA. Only single-cell suspensions with a viability of >95% were used for in vivo studies. Mice anesthetized with methoxyflurane were placed in the supine position. A lower midline abdominal incision was made, the bladder was exteriorized, and tumor cells (1 × 10⁶/0.05 ml HBSS) were injected into the dome of the bladder. A well-localized bleb was the sign of a technically satisfactory injection. The abdominal incision was closed in one layer using metal clips (24, 25).

A fixed total weekly dose of 70,000 units of IFN-α-2a (Roeferon; Hoffman-LaRoche) was given to mice bearing the implants. Therapy started on day 3 or day 7 after tumor cell implantation. Groups of mice were injected s.c. with saline (control) or IFN-α-2a (specific activity, 6 × 10⁶ IU/mg protein) according to the schedule shown in Table 1. The treatment continued for 4 weeks. On day 35, the mice were euthanized. The presence of tumor in the bladder and at metastatic sites was evaluated. The bladders were removed, weighed, and processed for histological, immunohistochemical, and ISH analyses.

The level of bFGF protein in the serum was analyzed by the Quantikine ELISA kit (R&D Systems, Minneapolis, MN). The concentration of bFGF in the samples was determined by comparing their absorbance with a standard curve. The minimal detectable level of bFGF by this assay is 1 μg/ml (24).

Tumors harvested from the bladders of nude mice at autopsy were divided into fragments and placed in either 10% (v/v) neutral formalin or OCT compound (Miles Laboratories, Elkhart, IN) to be snap-frozen in liquid nitrogen. For histological studies, 5-μm-thick sections were prepared and stained with H&E. For immunohistochemical analysis, frozen tissues (8 μm thick) were fixed with cold acetone. (Samples were fixed and stored in PBS at 4°C if the procedure could not be finished on the same day.) Tissue sections (5 μm thick) of formalin-fixed, paraffin-embedded specimens were deparaffinized in xylene, rehydrated in graded alcohol, and transferred to PBS. The slides were rinsed twice with PBS, and endogenous peroxidase was blocked by use of 3% hydrogen peroxide in PBS for 12 min. The samples were washed three times with PBS and incubated for 20 min at room temperature with a protein-blocking solution consisting of PBS (pH 7.5) containing 5% normal horse serum and 1% normal goat serum. Excess blocking solution was drained, and the samples were incubated for 18 h at 4°C with the appropriate dilution (1:100) of monoclonal rat anti-CD31 antibody (PharMingen, San Diego, CA), a 1:50 dilution of a rabbit polyclonal anti-IL-8 antibody (Biosource International, Camarillo, CA), a 1:200 dilution of rabbit polyclonal anti-bFGF antibody (Sigma Chemical Co., St. Louis, MO), a 1:750 dilution of rabbit polyclonal anti-VEGF/VPF antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or a 1:50 dilution of rabbit polyclonal anti-MMP-9 antibody (Calbiochem, La Jolla, CA).

The samples were then rinsed four times with PBS and incubated for 60 min at room temperature with the appropriate dilution of peroxidase-conjugated anti-rabbit IgG or anti-rat IgG. The slides were rinsed with PBS and incubated for 5 min with diaminobenzidine (Research Genetics). The sections were then washed three times with distilled water, counterstained with Gill’s aqueous hematoxylin (Sigma), washed once with distilled water and once with PBS, and rinsed again with distilled water. The slides were mounted with a Universal mount (Research Genetics) and examined in a bright-field microscope. A positive reaction was indicated by a reddish-brown precipitate in the cytoplasm. The intensity of staining was quantitated in three different areas of each sample by an image analyzer using the Optimas software program (Bioscan, Edmonds, WA) to yield an average measurement (24).

Blood vessels in human bladder cancers growing in the bladder wall of nude mice were counted under a light microscope after immune staining of sections with anti-CD31 antibodies (26). Areas containing the highest number of capillaries and small venules were identified by scanning the tumor sections at low power. After the areas of high vascular density were identified, individual vessels were counted in ×100 fields [×10 objective and ×10 ocular (0.739-mm²/field)]. We classified structures as vessels on the basis of criteria described by Weidner et al. (27); therefore, observation of a vessel lumen was not required (27).

Specific antisense oligonucleotide cDNA probes complementary to the mRNA transcripts of four metastasis-related genes, bFGF, VEGF/VPF, IL-8, and MMP-9, identified on the basis of published reports of the cDNA sequences, were designed as described previously (28). All DNA probes were synthesized with six biotin molecules (hyperbiotinylated) at the 3′ end via direct coupling using standard phosphoramidine chemistry (Research Genetics; Refs. 29 and 30). The lyophilized probes were reconstituted to a 1 μg/μl stock solution in 10 mm Tris·HCl (pH 7.6) and 1 mm EDTA. The stock solution was diluted with probe diluent (Research Genetics) immediately before use.

ISH was performed as described previously (31, 32). The Microprobe manual staining system (Fisher Scientific, Pittsburgh, PA) was used to stain tissue sections (4 μm) of formalin-fixed, paraffin-embedded bladders. Two from each treatment group were mounted on Silane-coated ProbeOn slides (Fisher Scientific; Ref. 33). The slides were placed in the Microprobe slide holder, dewaxed, and dehydrated with Autodewaxer and Autoalcohol (Research Genetics), followed by enzymatic digestion with pepsin. Hybridization of the probe was carried out for 80 min at 45°C, and the samples were then washed three times with 2× SSC (0.15 m NaCl and 0.015 m sodium citrate) for 2 min at 45°C. The samples were incubated with alkaline phosphatase-linked enhancer (Biomeda Corp., Foster City, CA) for 1 min and finally incubated at 45°C for 40 min with chromogen substrate FastRed (Research Genetics). A positive reaction in this assay stained red. Control for endogenous alkaline phosphatase included treatment of the samples in the absence of biotinylated probe and use of chromogen in the absence of any oligonucleotide probes. The integrity of the mRNA in each sample was verified by using a poly d(T)₂₀ probe. All specimens analyzed produced an intense histochemical reaction, indicating that the mRNA was well preserved. To avoid variabilities in probe concentration, all specimens were stained in a single session for each probe.

Stained sections were examined in a Zeiss photomicroscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a three-chip CCD color camera (model DXC-960 MD; Sony Corp., Tokyo, Japan). The images were analyzed using Optimas image analysis software (version 5.2; Bothell, WA). The slides were prescreened to determine the range in staining intensity of the slides to be analyzed. Images covering the range of staining intensities were captured electronically, a color bar (montage) was created, and a threshold value was set in the red, green, and blue mode of the color camera. All subsequent images were quantified based on this threshold. The integrated absorbance of the selected fields was determined based on its equivalence to the mean log inverse gray scale value multiplied by the area of the field. The samples were not counterstained; therefore, the absorbance was attributable solely to the product of the ISH reaction. Measured areas of 1 mm² were located at the center or edge of the tumor. In each area, cytoplasmic staining of 20–30 tumor cells was measured. The level of cytoplasmic staining/area was quantified to derive an average value (34). The intensity of staining was compared by integrated absorbance of poly d(T)₂₀ and was standardized by comparison with the integrated absorbance of nonpathological transitional epithelium of normal bladder, which was set at 100.

The significance of the differences in the in vivo data were analyzed by the Mann-Whitney U test and Student’s t test (35).

In the first set of experiments, we evaluated the therapeutic efficacy of systemic administration of IFN-α-2a against human bladder cancer cells growing in the bladder wall of nude mice. Specifically, we determined whether the IFN-α-2a treatment was associated with the down-regulation of angiogenesis-regulating genes. To distinguish between the direct antiproliferative effects and indirect antiangiogenic effects of IFN-α, we used 253J B-V IFN^R cells, which are resistant to IFN-α or IFN-β antiproliferative effects. Therapy began on day 3 after tumor cell implantation. Control mice received daily s.c. injections of saline. IFN-α-2a was administered at a total dose per week of 70,000 units. Groups of mice (n = 10) received s.c. injections of IFN-α-2a once weekly (70,000 units/dose), twice weekly (35,000 units/dose), or once daily (10,000 units/dose) for 4 weeks. The mice were killed and necropsied 4 days after the last daily s.c. injection (day 35 of the study). Bladder tumors were resected, weighed, and prepared for immunohistochemistry. The data summarized in Table 1 demonstrate that significant inhibition in tumorigenicity occurred only in mice injected with 10,000 units of IFN-α-2a on a daily basis, i.e., the tumor weight was significantly reduced from a median of 581 mg (range, 414–928 mg) in control mice to 105 mg (range, 68–360; P < 0.001).

The inhibition in tumor growth observed in tumors of mice treated with daily injections of 10,000 units of IFN-α-2a was accompanied by a significant reduction in tumor vascularization as revealed by immunohistochemical analysis using antibodies against CD31. The number of blood vessels (100× high-power fields) was reduced from 95 ± 15 in tumors of untreated control mice to 40 ± 6 in tumors growing in mice receiving daily s.c. injections of 10,000 units of IFN-α-2a (P < 0.001). The intensity of bFGF and MMP-9 immunostaining in tumor tissues was also significantly reduced in the mice treated with daily injections of IFN-α-2a (P < 0.001). The intensity of IL-8 or VEGF/VPF immunostaining in the bladder cancer cells was not significantly reduced in any of the treated groups (Table 1).

The therapy experiment was repeated to compare the antiangiogenic activity of IFN-α-2a administered once daily (10,000 units/dose) and three times per week (23,000 units/dose). Control mice received daily s.c. injections of saline. The IFN-α-2a treatment continued for 4 weeks, and the mice were killed and necropsied on day 35 of the study. Bladder tumors were resected, weighed (Table 1), and prepared for ISH analysis (Fig. 1 and Table 2) and immunohistochemistry (Fig. 2 and Table 1). Tumor weight was significantly reduced from the median weight of 529 mg (range, 325–759) in saline-treated mice to 390 mg (range, 135–752) in mice injected three times per week (P < 0.01). In mice treated daily, the median tumor weight was reduced to 211 mg (range, 51–475; P < 0.001).

The inhibition of tumor growth inversely correlated with expression of bFGF and MMP-9 mRNA (Fig. 1 and Table 2). Specifically, the mRNA level (as measured by intensity of ISH staining) for bFGF was reduced from a mean of 240 ± 22 in the saline-treated mice to 70 ± 13 in mice receiving daily injections (P < 0.01). For MMP-9, the mRNA intensity level was reduced from a mean of 208 ± 25 in saline-treated mice to 95 ± 15 in mice treated with daily injections (P < 0.01). The expression levels of bFGF and MMP-9 were not reduced in bladder tumors of mice treated three times per week. No discernible differences in expression of VEGF/VPF or IL-8 were found among the groups.

Immunohistochemical analysis (Fig. 2) agreed with results of the ISH analysis (Fig. 1). The intensities of bFGF and MMP-9 immunostaining in the bladder tumors was significantly reduced in mice treated daily (Fig. 2 and Table 1). Specifically, bFGF intensity was reduced from a mean of 200 ± 19 in control mice to 91 ± 12 in control mice treated daily (P < 0.01), and MMP-9 intensity was reduced from a mean of 257 ± 29 in control mice to 135 ± 20 in mice treated with daily injections of 10,000 units of IFN-α-2a (P < 0.01).

The above experiments demonstrated that IFN-α-2a administered daily produced superior antiangiogenic and antitumorigenic activities as compared with an equivalent weekly dose of IFN-α-2a administered in one, two, or three doses/week. In the next set of experiments, we determined whether increasing the daily dose of IFN-α could improve antiangiogenic effects. To produce bladder tumors, 5 × 10⁶ viable 253J B-V IFN^R cells were implanted into the bladder wall of nude mice. Seven days later, groups of mice (n = 10) received daily s.c. injections of saline or daily injections of IFN-α-2a at 2,500 units, 5,000 units, 10,000 units, 25,000 units, or 50,000 units per dose. The daily s.c. treatments continued for 4 weeks. On day 35 of the study, the mice were bled, serum was collected, and the mice were euthanized and necropsied. The bladders containing tumors were resected, weighed, and prepared for immunostaining with antibodies against CD31 (to show endothelial cells). The serum was examined by ELISA for level of bFGF. The data of one representative experiment of two are shown in Fig. 3. Treatment with 2,500 units of IFN-α-2a did not significantly inhibit tumor weight (Fig. 3,A) or vascularization (Fig. 3,B). Serum level of bFGF (Fig. 3 C) was inhibited (P < 0.01). Maximal inhibition in tumor weight (P < 0.01), vascular density (P < 0.01), and serum level of bFGF (P < 0.001) was observed in mice treated by daily s.c. injections of 5,000 or 10,000 units of IFN-α-2a. Surprisingly, increasing the daily dose of IFN-α-2a to 25,000 units or 50,000 units was associated with loss of the inhibition in tumor weight and tumor vascularity, although the serum level of bFGF was significantly inhibited (P < 0.01).

Our results demonstrate that the systemic administration of human IFN-α-2a to nude mice bearing bladder wall implants of human 253J B-V-IFN^R bladder cancer cells inhibits angiogenesis and tumor growth. Altering the dose of IFN-α and schedule of administration profoundly influenced therapeutic outcome; daily administration of IFN-α-2a at far below maximal tolerated doses produced maximal antiangiogenic effects. Because the human bladder cancer cells we used are resistant to the antiproliferative effects of IFN-α, the data clearly show that the indirect antiangiogenic activity of IFN-α requires a different approach than that designed to inhibit tumor cell proliferation directly.

The progressive growth and metastasis of malignant neoplasms depend on adequate neovascularization, i.e., angiogenesis, the extent of which is determined by the balance between the proangiogenic and antiangiogenic molecules that are released by both the tumor cells and surrounding normal cells (36, 37, 38). Among the major proangiogenic molecules are bFGF, VEGF/VPF, IL-8, and MMP-2/9 (38, 39, 40, 41). The major endogenous inhibitors of angiogenesis include angiostatin (42, 43, 44), endostatin (45), thrombospondin (46), and IFN-α and IFN-β (25, 47, 48, 49). Studies from our laboratory have shown that the angiogenic phenotype of rodent and human carcinomas can be modulated by cytokines released by host cells in specific organ microenvironments (50, 51, 52). For example, human renal cancer cells implanted into the kidney of nude mice express high levels of bFGF (mRNA and protein), whereas the same cells growing s.c. do not (7). Human melanoma cells growing in the subcutis of nude mice express high levels of IL-8, whereas the same cells growing in the liver do not (52). The expression of bFGF and IL-8 within the tumors inversely correlated with the expression of IFN-β in the surrounding stroma (7, 52).

IFN-α has been widely used alone or in combination with other agents to treat a variety of neoplasms including melanoma, renal cell carcinoma, and transitional cell carcinoma. Various doses and schedules have been assessed; however, response rates have been modest, and the optimal dosing schedule has yet to be determined (53, 54, 55, 56, 57, 58, 59, 60). Traditionally, IFN-α has been used as an antiproliferative cytokine, and thus, investigators have used high doses (5–50 million units /m²) given two to three times per week, producing response rates that varied between 5 and 20% (4, 19, 52, 53, 54, 55, 56, 57, 58, 59, 60). Pharmacokinetic studies have shown that 1 h after an i.v. injection of 6 million units of IFN, serum levels drop to <8 units/ml (23, 61). These serum concentrations are well below those necessary to inhibit expression of bFGF (8, 9, 25, 47) or MMPs (14, 15, 16, 25), which are required in the process of angiogenesis.

Most studies with IFN-α have attributed its antitumor effects to inhibition of cell proliferation or induction of cell differentiation (62). More recently, it has become apparent that IFN-α also has potent antiangiogenic effects. The treatment of patients with Kaposi’s sarcoma with IFN-α demonstrated that 40% of patients experienced significant regression (63, 64, 65, 66). Chronic administration of IFN-α has also produced regression of life-threatening hemangiomas of infancy (67, 68, 69, 70, 71), especially when given at low dose (3 million units /m² daily s.c.) over a period of 9–12 months (67, 71). The inhibition of angiogenesis by IFN-α (and IFN-β) is independent of its well-documented antiproliferative effects. Several years ago, Sidky and Borden (72) showed that IFN-β inhibited the in vivo growth of mouse leukemia cells resistant to the antiproliferative effects of IFN by inhibiting the proliferation of endothelial cells instead of the tumor cells themselves (73). Similarly, treatment with IFN-α or IFN-β was also shown to inhibit the motility of endothelial cells (73) and inhibit transcription and protein production of bFGF (9, 12, 25, 47) and MMPs (14, 15, 16) in different human carcinoma cells in a manner independent of its antiproliferative effects. These data are indeed encouraging because of a recent report showing that daily administrations of IFN-α-2a to a child with rapidly growing giant cell tumor inhibited expression of bFGF, suppressed angiogenesis, and induced involution of the large lesion (74).

The present study suggests that the antiangiogenic activity of IFN-α, which requires continuous low levels (47), can be achieved in vivo by increasing the frequency of administration. Our data show that the schedule of IFN administration is critical to the down-regulation of expression and inhibition of tumor growth by angiogenic genes. Daily s.c. injections of IFN-α-2a significantly inhibited the protein and mRNA expression of bFGF and MMP-9 and tumor-induced neovascularization, whereas once, twice, or three weekly administrations did not. The down-regulation of angiogenesis in the tumors directly correlated with inhibition of growth.

The use of IFN-α as a direct antiproliferative agent requires the use of maximal tolerated doses (53, 54, 55, 56, 57, 58, 59). In contrast, the antiangiogenic effects of IFN-α were dependent on daily administrations of optimal biological dose. The systemic administration of IFN-α-2a at doses exceeding 10,000 units failed to inhibit tumorigenicity or decrease vascularization. The mechanisms for this reversal of beneficial effects (observed with lower doses of IFN-α) are unclear. The administration of maximal tolerated dose of cytokines is associated with induction of a feedback mechanism, perhaps related to a new family of proteins that negatively regulate cytokine signaling (75, 76, 77). Whether the administration of high doses of IFN-α (>25,000 units/day) induces the expression of proteins that suppress cytokine signaling (SCOS) or STAT-1 activity is unclear and is under active study.

In summary, optimizing both dose and schedule of IFN-α is necessary for maximal inhibition of angiogenesis. This conclusion is intuitive because the half-life of IFN-α is measured in hours; consistent exposure of the tumor and host to IFN could thus be presumed to require frequent administration. Also of interest was the reduction in efficacy of the drug as daily doses exceeded 10,000 units. This phenomenon may reflect a feedback mechanism countering high doses of the cytokine. Thus, efforts to treat a patient with maximal tolerable doses of IFN-α may be counterproductive. In addition, given the experience with IFN-α therapy of hemangiomas (67, 68, 69, 70, 71), it appears that the maximal response to the antiangiogenic effect of IFN-α requires chronic frequent administration of biologically optimal doses and not maximal tolerated doses.

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