| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
Biomedical Graduate Program [M. S. G., H. K. K.], Departments of Radiation Oncology [C. J. K., S. M. E., W. T. J.], Pathology and Laboratory Medicine [C. H. P., J. S. M.], and Medicine [W. M. F. L.], Cancer Center [J. S. M., W. M. F. L.], and Flow Cytometry and Cell Sorting Facility [C. H. P., J. S. M.], University of Pennsylvania, Philadelphia, Pennsylvania 19104; Department of Microbiology and Immunology and Cancer Center Immunology Program, University of Rochester, Rochester, New York 14642 [J. L., E. M. L.]; and The Wistar Institute, Philadelphia, Pennsylvania 19104 [G. T., W. M. F. L.].
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
produced in response to rmIL-12 administration. These studies reveal that rmIL-12 controls K1735 tumors initially by IFN--induced apoptosis and later by hypoxia-induced apoptosis. They also establish hypoxia as an expected result of tumor angiogenesis inhibition and a mediator of its therapeutic effect. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in response to IL-12 stimulation. Because IL-12 and IFN- together induce Th1 differentiation of CD4+ T cells, enhance CD8+ T cell maturation and activation, and functionally activate NK cells (7)
, the antitumor activity of IL-12 has been attributed to the induction of antitumor immunity. Whereas this may be important for long-term tumor control by IL-12, its role in acute control of tumor growth is less certain. Recent studies demonstrated that therapeutically effective regimens of rmIL-12 impaired T cell-mediated immune responses during and beyond the period of cytokine administration, even as tumor growth was controlled (8
, 9)
. Impairment was attributable to suppression of T-cell mitogenic responses by macrophage-derived nitric oxide produced in response to IFN-. Interestingly, the immunosuppression seen in rmIL-12-treated mice waned following cessation of therapy and was followed by enhanced antitumor immunity when this was tested at later time points.
Tumor control and regression during rmIL-12 administration may be better explained by its ability to inhibit angiogenesis. This activity has been demonstrated in several model assays of neovascularization to involve IFN- and IFN--dependent production of antiangiogenic chemokines such as IP-10 (10, 11, 12, 13)
. Production of these cytokines in turn leads to inhibition of angiogenesis by down-regulation of several proangiogenic factors, including tumor cell vascular endothelial growth factor production, matrix metalloproteinase activity (14)
, and expression of integrins involved in endothelial cell adhesion and survival (15)
. However, whereas rmIL-12 is known to inhibit angiogenesis in model assays, the issues of whether it inhibits angiogenesis within established tumors and what the consequences of tumor angiogenesis inhibition might be remain unaddressed. In this study, we assess the contribution of tumor angiogenesis inhibition to overall IL-12 antitumor efficacy.
How angiogenesis inhibition by IL-12 controls tumor growth is important to our general understanding of the effects of antiangiogenesis therapy because the mechanism by which angiogenesis inhibitors cause tumor regression is not currently known. The maintenance of tumor dormancy by angiogenesis inhibitor therapy has been previously shown to be associated with an increase in tumor cell apoptosis (16
, 17)
. Whereas no formal mechanism for this increased apoptosis has been proposed, it has been speculated that angiogenesis inhibition could restrict the supply of endothelial cell-derived paracrine factors required for tumor cell survival (16)
. Because effective inhibition of tumor angiogenesis by definition should result in tumor ischemia, we reasoned that the signal for tumor cell apoptosis could be a downstream consequence of severe ischemia. A reasonable candidate is hypoxia, which has been shown to be a potent inducer of apoptosis by a mechanism possibly involving HIF1
-mediated stabilization of p53 (18)
and inhibition of Bcl-2 (19)
, and hypoxia-induced tumor cell apoptosis may explain how angiogenesis inhibitors control tumor growth.
In this study, we examine K1735 murine melanomas undergoing rmIL-12 therapy to determine whether tumor hypoxia is induced by treatment and whether this plays a role in controlling tumor growth. Our results demonstrate that treatment results in severe tumor cell hypoxia, the appearance of which is delayed relative to the onset of angiogenesis inhibition. The development of hypoxia is selective, occurring only in tumor areas relatively removed from blood vessels, which reinforces its relationship with angiogenesis inhibition. The severe hypoxia that develops with therapy appears to mediate rmIL-12 antitumor activity by inducing widespread tumor cell apoptosis. Our results also reveal that, prior to the appearance of severe tumor hypoxia, rmIL-12 control of K1735 tumor growth is hypoxia independent and attributable to direct induction of tumor cell apoptosis by IFN-. Thus, rmIL-12 is a complex antitumor agent that uses a combination of mechanisms to suppress tumor growth, and angiogenesis inhibitor therapy, in general, may control tumor growth largely through induction of tumor cell hypoxia and hypoxic cell death.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
R1 were described previously (12)
.
In Vivo Studies.
For tumor growth studies, 106 trypan blue-excluding K1735 cells were injected s.c. into the lower left flank. Injected cells were derived from low-passage frozen stocks that had been established in culture <1 week prior to injection. rmIL-12 was administered i.p. on a five doses per week schedule (five daily injections of 125 ng, followed by 2 days of rest) for the durations indicated. Tumors were measured by calipers at regular intervals, and mice were euthanized according to guidelines established by the Institutional Animal Care and Use Committee. Serum IFN- levels were assayed by RIA using the polyclonal antibody AN18 as the capture antibody and the monoclonal antibody XMG6 as the detection antibody. For EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide] studies, the mice were given an i.v. injection of 0.25 ml of 10 mM EF5 in 0.9% saline 3 h prior to tumor excision. On the basis of the half-life of EF5 in circulation, this assures that virtually all free EF5 is cleared by the time tumors are excised and that no EF5 binding is due to hypoxia resulting from interruption of circulation associated with euthanasia or tumor excision.
Matrigel Assay for Angiogenesis.
In vivo Matrigel neovascularization assays were performed essentially as described previously (12)
. Briefly, C3H/HeN mice were injected s.c. along the ventral midline with 0.5 ml of Matrigel (Collaborative Biomedical Products, Bedford, MA) mixed with 100 µl of PBS, either alone or with an angiogenic stimulus [200 ng of recombinant basic fibroblast growth factor (Upstate Biotechnology, Lake Placid, NY) and 1 x 106 K1735 or K1735.N23 cells]. In treated mice, rmIL-12 (125 ng) was injected i.p. daily on days 0–4. Cytokine ablation was achieved by injecting 0.5 mg of anti-IFN- monoclonal antibody i.p. on days -1, 1, and 3. Matrigel pellets were harvested on day 6 and reliquefied by incubation at 4°C overnight in 300 µl of PBS. Matrigel neovascularization was quantitatively determined by measuring the hemoglobin content of the liquefied pellets (Drabkin’s method; Sigma Chemical Co., St. Louis, MO).
Flow Cytometry.
Tumor cell suspensions were prepared and stained for EF5 with the monoclonal antibody ELK 3-51 essentially as described (21)
. For single-color analysis, ELK 3-51 was conjugated to the fluorochrome Cy3, whereas for two-color analysis, it was conjugated to cyanine-5. Apoptosis was measured by TUNEL staining method according to the manufacturer’s instructions (Boehringer Mannheim, Indianapolis, IN). For dual EF5/TUNEL staining, cells were first TUNEL stained and then washed twice with PBS prior to overnight staining with ELK 3-51. Analysis of tumor cell proliferation was performed using a rapid propidium iodide-based staining protocol as described.4
Briefly, 2 x 106 tumor cells were incubated for 30 min at 37°C in 500 µl of staining solution [3% polyethylene glycol 6000, 50 µg/ml propidium iodide (Sigma), 1 mg/ml RNase A (Boehringer Mannheim), 0.1% Triton X-100, and 4 mM sodium citrate (pH 7.2)]. Five hundred µl of salt solution [3% polyethylene glycol 6000, 50 µg/ml propidium iodide, 0.1% Triton X-100, and 0.4 M NaCl (pH 7.2)] were then added, and the nuclei were stored at 4°C until analysis.
Flow Cytometer Type and Laser Specifications.
Flow cytometry was performed on a Becton Dickinson Immunocytometry Systems FACS Vantage and FACS Calibur. The FACS Vantage was equipped with a green laser (514 nm) to excite Cy3 fluorescence, whereas the FACS Calibur was equipped with blue (488 nm) and red (635 nm) lasers to excite fluorescein and cyanine-5 fluorescence, respectively. Standard collection optics were used to collect emitted fluorescence. Photomultiplier tube voltages were adjusted daily by placing standard beads in the same channel. Standardization of EF5 fluorescence intensity was achieved using calibration beads (Flow Cytometry Standards Corporation, Research Triangle Park, NC). Isolation of tumor cells in cell suspensions was achieved by establishing gate parameters around in vitro cultured K1735 cells, which exhibit detectably higher forward and side scattering than erythrocytes and lymphocytes. Flow cytometric analysis of hypoxia and apoptosis was performed using CellQuest (Becton Dickinson, Mountain View, CA), whereas analysis of proliferation was performed using ModFit Version 2.0 (Verity Software, Topsham, ME).
In Vitro Studies.
For IFN- stimulation studies, 5 x 105 K1735 cells were plated onto 10-cm dishes and incubated for 24 h prior to addition of rIFN- (R&D Systems, Minneapolis, MN). IFN- was replaced every 48 h. Hypoxia studies were performed essentially as described previously (21)
. Cells (7.5 x 105) were spot-plated onto 5-cm glass dishes in JRH 610 medium supplemented with penicillin/streptomycin and 10% FCS (complete JRH 610) and incubated overnight at 37°C. The following day, the dishes were placed in leak-proof aluminum chambers connected to a manifold in which the oxygen concentration was reduced to the appropriate level. For EF5 hypoxia studies, EF5 was added to the medium up to a final concentration of 0.10 mM immediately before induction of the appropriate level of hypoxia, and the cells were incubated for 3 h. For prolonged hypoxia studies, the medium was replaced with complete JRH610 supplemented with 20 mM HEPES, 0.1% glucose, and 0.008 N NaOH prior to hypoxic induction. pH and glucose measurements were taken at the beginning and end of the hypoxic period to ensure that adequate glucose was present in the medium and document any significant changes in acidity.
Immunohistochemistry.
Immunohistological staining for platelet/endothelial cell adhesion molecule (PECAM) and EF5 and imaging were performed essentially as described previously (22)
. Briefly, cryostat sections were incubated with 5 µg of rat antimouse PECAM-1 antibody (MEC 13.3; PharMingen, San Diego, CA), followed by 40 µg/ml peroxidase-conjugated mouse antirat IgG (H&L; Jackson Immunoresearch Laboratories, West Grove, PA). Following a PBS wash, sections were incubated with the chromagen 3-amino-9-ethylcarbazole (Vector Laboratories, Burlingame, CA). Sections were then stained with ELK 3-51-Cy3. Normal tissues from tumor-bearing mice were also stained for EF5 to guarantee staining specificity (liver central vein hepatocytes stain positive, whereas s.c. tissue stains negative). Stained sections were imaged using an epifluorescence equipped Nikon microscope (x20 objective), digitized, and image-analyzed using Image Pro software (Version 3.0; Media Cybernetics, Silver Spring, MD). Images from adjacent microscope fields were automatically acquired and digitally combined to form a 5 x 4 montage (corresponding to 2.9 x 1.7 mm of the section). Tumors in each treatment group were size-matched for comparison.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-dependent Manner.
. Control of tumor growth by rmIL-12 was ablated by administration of a neutralizing monoclonal antibody to IFN- (XMG6), indicating that it was dependent on IFN- (Fig. 1B)
. Tumor control also required tumor cells to respond to IFN-, shown by the unresponsiveness of N23 tumors to rmIL-12 treatment (N23 is a transfected K1735 clone overexpressing a dominant negative IFN-R1 receptor; Ref. 12
). Similarly, a 5-day course of rmIL-12 treatment (Fig. 2)
inhibited Matrigel neovascularization induced by K1735 cells or a purified angiogenic factor, basic fibroblast growth factor (bFGF). This antiangiogenic effect was inhibited by coadministration of neutralizing antibodies to IFN- (data not shown) and was not apparent when N23 cells were in the Matrigel (Fig. 2)
. Thus, rmIL-12 acts in an IFN--dependent manner to retard growth of established K1735 tumors and inhibit angiogenesis by these cells.
|
|
. Thus, the range of O2 concentrations best determined by EF5 binding to K1735 cells is physiologically relevant because half-maximal cellular hypoxic responses occur at an O2 concentration of 1.5% (24)
.
|
. Cells from untreated K1735 tumors varying from 2- to 16-mm diameter exhibited a uniform pattern of low EF5 staining similar to that seen in K1735 cells cultured at 10–20% O2; only 1–2% of cells in these tumors showed EF5 staining at levels equivalent to that of cells cultured at <1% O2 (Fig. 3)
. Tumors treated with rmIL-12 for 1 week also appeared to be uniformly oxygenated with minimal EF5 staining, although there was a shift toward a lower median O2 percentage. Tumors receiving 2 weeks of rmIL-12 treatment, however, exhibited a peak of EF5-staining cells (median O2, 
0.1%) not seen in either of the previous treatment groups. This hypoxic peak, which comprised 22% of total tumor cells after 2 weeks of treatment, grew to include the vast majority of tumor cells (80%) after 3 weeks of therapy (Fig. 4B)
. The progressive development of K1735 tumor hypoxia with rmIL-12 therapy is consistently and reproducibly observed (note the SDs displayed in Figs. 4B
and 6B
, in which n = 3 for each group). Hypoxia did not develop when anti-IFN- antibody was given with rmIL-12 therapy, suggesting that rmIL-12 induction of tumor hypoxia, like its ability to inhibit angiogenesis, is IFN- dependent. In addition, rmIL-12 failed to induce significant hypoxia in N23 tumors (Fig. 4B)
, just as it had failed to inhibit N23 tumor cell-induced Matrigel angiogenesis (Fig. 2)
.
|
|
). These results supported those of the flow studies, showing that untreated tumors and tumors treated with rmIL-12 for 1 week have few, if any, visible areas of EF5-stained cells and that tumors treated for 2 weeks contain large patches of EF5-stained cells. The close correlation in EF5 binding obtained by cell suspension and histological studies of rmIL-12-treated K1735 tumors suggests that EF5 binding of tumor cell suspensions accurately reflects events in vivo. Examination of microscopic fields from multiple tumors revealed that hypoxia selectively developed in regions of the tumor that were devoid of PECAM-staining vessels (Fig. 5B)
. This is expected because O2 delivery in these regions depends upon extravascular diffusion, a less efficient process than direct capillary exchange, making them most susceptible to changes in O2 supply. Together, these studies show that treatment with rmIL-12 leads to the development of tumor cell hypoxia in a manner consistent with its angiogenesis-inhibitory activity. Significantly, the appearance of severe tumor hypoxia was delayed relative to the ability of rmIL-12 to inhibit tumor cell-induced Matrigel angiogenesis (Fig. 2)
and suppress tumor growth (Fig. 1A)
, both of which occurred during the first week of therapy.
|
. Apoptosis was strongly associated with hypoxia (20% of normoxic cells were apoptotic, whereas >80% of hypoxic cells were apoptotic), so that, although normoxic cells predominated in these tumors, the majority of apoptotic cells were in the hypoxic population. This trend continued in tumors treated for 3 weeks with rmIL-12, in which 67% of tumor cells were apoptotic and, because hypoxic cells were predominant, the vast majority of apoptotic cells were hypoxic.
The relationship between hypoxia and apoptosis during rmIL-12 treatment was examined by testing whether hypoxia could induce apoptosis in K1735 cells in vitro. When cells were incubated under a range of O2 concentrations (10, 1, and 0.1% O2) for 16 or 32 h, marked apoptosis was seen in cells grown at 0.1% O2 (Table 1)
, which was the median O2 concentration of the hypoxic tumor cell population in vivo after 2 weeks of rmIL-12 therapy (Fig. 4A)
. By 32 h, >70% of cells in 0.1% O2 were apoptotic, consistent with the 70–80% of hypoxic cells observed to undergo apoptosis in vivo (Fig. 6B)
. In the final 8 h of incubation in 0.1% O2, the pH of the culture medium decreased from pH 7.1 to 6.8. However, acidosis was not the cause of apoptosis in these cells, because culturing K1735 cells at pH 6.8 in air for up to 16 h did not induce significant apoptosis (data not shown). These results strongly suggest that, in vivo, rmIL-12 treatment suppresses K1735 tumor growth via the development of severe hypoxia, which triggers tumor cell apoptosis.
|
-dependent Tumor Cell Apoptosis Independent of Hypoxia.
. However, this apoptosis occurred in the absence of hypoxia, indicating that rmIL-12 was inducing hypoxia-independent as well as hypoxia-dependent tumor cell apoptosis. Several observations led us to suspect that IFN- caused this hypoxia-independent apoptosis. (a) IFN- levels were highest in the first week of rmIL-12 treatment when this apoptosis was present. (b) This apoptosis was abrogated by antibody neutralization of IFN- (Fig. 6C)
, and non-IFN--responsive N23 tumors treated with rmIL-12 for 1 week did not exhibit significant apoptosis. (c) Previous studies had shown that IFN- can induce apoptosis in vitro in a signal transducers and activators of transcription (STAT)-depen-dent manner (25)
. To test this hypothesis, we cultured K1735 cells with different concentrations of murine rIFN- for 48 or 96 h (Table 2
; 96 h approximates the time that tumor cells are exposed to high IFN- levels in vivo during a 5-day course of rmIL-12 therapy). The percentage of apoptotic cells increased with dose and duration of rIFN- treatment to a high of 41% with 100 ng rIFN-/ml for 96 h, which approximates the percentage of apoptotic cells seen in vivo after a week of rmIL-12 treatment (Fig. 6B)
. That apoptosis was not induced in N23 cells under the same conditions indicates that this is likely a direct effect of IFN- on K1735 cells. Thus, hypoxia-independent apoptosis induced after 1 week of rmIL-12 therapy can be attributed to direct IFN- induction of K1735 tumor cell apoptosis.
|
induced by rmIL-12 after the first week (Fig. 6B)
. This reduction in hypoxia-independent, IFN--dependent apoptosis was offset, however, by the concomitant rise in hypoxia-dependent apoptosis, so that a significant fraction of tumor cells was apoptotic throughout the 3 weeks of rmIL-12 treatment. Both apoptosis mechanisms contribute to rmIL-12 control of K1735 tumor growth, but each predominates at a different time.
To be certain that apoptosis accounted for rmIL-12 suppression of tumor growth, we also evaluated changes in tumor cell proliferation by propidium iodide staining for DNA content. These studies revealed no increase in S-phase fraction with rmIL-12 treatment to offset the increases in apoptosis (data not shown). In fact, the fraction of cells in S-phase dropped by 35% in tumors treated for 1 or 2 weeks and by 65% in tumors treated for 3 weeks. These decreases can be explained by the rise in the percentage of apoptotic tumor cells (which should not be cycling) observed with rmIL-12 treatment and probably do not represent therapeutic suppression of proliferation by healthy tumor cells.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our results support the validity of this approach. Both rmIL-12 angiogenesis inhibition (measured in Matrigel neovascularization assays) and induction of tumor cell hypoxia were dependent on IFN- and required tumor cells to respond to IFN-. This latter requirement is likely related to IFN--induced tumor cell IP-10 production because K1735 cells produce IP-10 in response to IFN- stimulation, whereas N23 cells do not (data not shown). The observed regions of tumor cell hypoxia were localized to relatively avascular areas of the tumor. The only difference between the two parameters is that, whereas angiogenesis inhibition was seen in the first week of rmIL-12 treatment, the appearance of severe tumor cell hypoxia was delayed. However, we believe that this delay can be explained physiologically and is consistent with a causal relationship. In a well-oxygenated tumor such as K1735, there are no hypoxic regions, indicating that oxygen delivery to tumor cells is uniformly adequate. Curtailment of new vessel development will likely reduce tumor perfusion gradually, so that there will be a temporal lag before oxygen delivery is reduced to a critical level and becomes manifest as severe hypoxia. In support of this model, median K1735 tumor cell oxygenation decreased after the first week of rmIL-12 treatment (Fig. 4A)
, although severe hypoxia was not yet apparent. Whereas we are confident from Matrigel neovasularization assays that rmIL-12 inhibits new blood vessel development, we do not know at this time whether it also has effects on existing vessels.
Our studies suggest that angiogenesis inhibition leads to tumor cell apoptosis through the agency of hypoxia. Therapeutic efficacy by antiangiogenic agents is associated with increased tumor cell apoptosis (16 , 17) , but the inducer of this apoptosis has yet to be identified. On the basis of the results with rmIL-12, we propose that inhibition of tumor angiogenesis produces severe tumor hypoxia (or exacerbates it if tumor hypoxia exists prior to therapy) and that hypoxia is largely responsible for the tumor cell apoptosis and tumor growth arrest seen with angiogenesis inhibitor therapy. This does not preclude a contribution from other apoptosis stimuli to tumor stasis, but hypoxia-dependent apoptosis may be an inevitable consequence of tumor ischemia from angiogenesis inhibition, provided that the tumor cells are susceptible. Of course, ischemia from angiogenesis inhibition brings about physiological changes in addition to hypoxia, such as acidosis or hypoglycemia, that may also contribute to tumor cell apoptosis. For example, acidosis has been demonstrated to induce apoptosis in a hypoxia-independent manner in vitro (28) . In our tumor model, however, acidosis seems to contribute little to tumor cell apoptosis induced by hypoxia.
rmIL-12 controls K1735 tumor growth from the outset of therapy, unlike agents such as angiostatin and endostatin, which are suspected to act purely as angiogenesis inhibitors and which control tumor growth only after 1 or 2 weeks of therapy (29
, 30) . This was a clue that another antitumor mechanism is responsible for the initial phase of rmIL-12 tumor control, which we suggest is direct induction of tumor cell apoptosis by IFN-. An earlier study had demonstrated this to be an effect of high-dose IFN- stimulation in vitro (25)
, but the studies presented here suggest a role in suppressing tumor growth in vivo. IFN- has pleiotropic effects on cells, many of which are indirect and mediated by other factors, making it difficult to prove that hypoxia-independent, IFN--dependent tumor cell apoptosis observed in vivo results from the apoptosis-inducing activity of IFN-. However, the absence of this type of apoptosis in N23 tumor cells, which should resist direct effects of IFN- but not those of intermediary factors induced by IFN-, argues that the apoptosis results from the direct action of IFN- on tumor cells. We do not know whether the concentrations of IFN- necessary to stimulate apoptosis in vitro are present in the local tumor environment in vivo. However, we do know that other proinflammatory cytokines likely to be induced by rmIL-12 in vivo (e.g., tumor necrosis factor-) act synergistically in vitro with IFN- to induce K1735 tumor cell apoptosis (data not shown). The presence of these other cytokines reduces the concentration of IFN- needed to induce apoptosis, increasing the likelihood that effective induction of tumor cell apoptosis by IFN- occurs with cytokine concentrations that are achieved in vivo. This mechanism explains the immediate suppression of K1735 tumor growth seen with rmIL-12 administration and warrants consideration as a mechanism underlying the rapid response of other tumors to rmIL-12 therapy.
A question that arises is which host cells are responsible for the IFN- produced during the period of rmIL-12 administration. We believe that the best candidates for primary acute mediators of rmIL-12 antitumor activity and producers of IFN- are NK cells. These have been shown to be the first cells to infiltrate tumors in response to IL-12 therapy, where they appear to mediate both tumor cell killing and angiogenesis inhibition (31
, 32)
. In addition, administration of antibodies directed against NK cells during the first week of rmIL-12 therapy is associated with a reduction in systemic levels of IFN- and a loss of angiogenesis inhibitory activity (data not shown). Finally, rmIL-12 used in the manner that generally produces the greatest acute antitumor effect (high doses administered frequently) has been shown to transiently but profoundly inhibit T-cell mitogenesis and antigen-specific responses (8
, 9)
, making T cells unlikely mediators of rmIL-12 acute antitumor effects. However, further studies are required to define the role of different effector cell populations during IL-12 therapy more completely.
In summary, examination of the time course of rmIL-12 effects on the physiology of therapeutically responsive K1735 melanoma tumors reveals that two distinct apoptosis mechanisms suppress tumor growth at different periods during treatment. IFN--induced apoptosis is initially active but subsequently declines. This decline is accompanied by a corresponding rise in hypoxia-induced apoptosis, which results in a relatively stable level of tumor cell apoptosis that suppresses tumor expansion throughout treatment. Beyond rmIL-12 therapy, these studies suggest that hypoxia-induced tumor cell apoptosis may be a general mechanistic pathway whereby treatment with antiangiogenic agents leads to tumor stasis or regression. The involvement of hypoxia carries potentially important consequences and implications for this form of therapy. For example, hypoxia is known to induce a variety of cellular changes that can be adaptive for tumors, such as alterations in glucose metabolism and increased vascularity (33)
. In addition, hypoxia confers resistance to the cytotoxic effects of ionizing radiation (34)
, suggesting that care is warranted if angiogenesis inhibitors are to be used in combination with radiation therapy. Hypoxia has also been shown to select for cells with diminished apoptotic potential and p53 mutation (35)
, which could lead to expansion of tumor cell populations that are more therapeutically refractory (36
, 37)
. Although these considerations may only be applicable with chronic hypoxia, long-term maintenance therapy with angiogenesis inhibitors has been proposed (30
, 38)
. Additionally, tumors may vary in their susceptibility to the apoptotic effects of hypoxia, which may be a basis for variation in tumor response to antiangiogenesis therapy. Finally, if severe tumor hypoxia results from effective inhibition of tumor angiogenesis and mediates its antitumor effect, measurements of tumor hypoxia may be a sensitive and meaningful way to monitor clinical therapy with antiangiogenic agents.
|
ACKNOWLEDGMENTS |
|---|
RIA reagents, and the Genetics Institute (Andover, MA) for rmIL-12. We thank Newman Yeilding and Denise LaTemple for critical reading of this manuscript. |
FOOTNOTES |
|---|
1 This work is supported by a NIH Medical Scientist Training Program grant (to M. S. G.), NIH Training Grant T32 AI-07285 (to J. L.), a grant from the Lucille B. Markey Trust (to J. S. M. and C. H. P.), and the following NIH grants: CA-74071 (to C. J. K., S. M. E. and W. T. J.); CA-28332 (to E. M. L.); CA-16520 (to J. S. M. and C. H. P.); AI-34412, CA-32898, and CA-20833 (to G. T.); and CA-77581 (to W. M. F. L.). 
2 To whom requests for reprints should be addressed, at University of Pennsylvania, BRB II/III Room 330, 421 Curie Boulevard, Philadelphia, PA 19104. Phone: (215) 898-0258; Fax: (215) 573-7912; E-mail: leemingf{at}mail.med.upenn.edu
3 The abbreviations used are: IL-12, interleukin 12; NK, natural killer; rmIL-12, recombinant murine IL-12; IP-10, IFN-inducible protein 10; Cy3, cyanine-3; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; rIFN-, recombinant IFN-.
4 K. D. Bauer, DNA analysis of tissue samples. Presented at the 1996 Annual Course in Flow Cytometry, Brunswick, ME, June 10–14, 1996.
Received 3/31/99. Accepted 8/ 9/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect. J. Exp. Med., 188: 1603-1610, 1998. affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity, 9: 25-34, 1998.[Medline]
Vß3 in the disruption of the tumor vasculature induced by TNF and IFN-. Nat. Med., 4: 408-414, 1998.[Medline]
This article has been cited by other articles:
|
|
 |
|
  J. H. Tsai and W. M.F. Lee Tie2 in Tumor Endothelial Signaling and Survival: Implications for Antiangiogenic Therapy Mol. Cancer Res., March 1, 2009; 7(3): 300 - 310. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-W. Yang, B. Shu, Y. Zhu, and H.-T. Yang E2F6 Inhibits Cobalt Chloride-Mimetic Hypoxia-induced Apoptosis through E2F1 Mol. Biol. Cell, September 1, 2008; 19(9): 3691 - 3700. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Eliopoulos, M. Francois, M.-N. Boivin, D. Martineau, and J. Galipeau Neo-Organoid of Marrow Mesenchymal Stromal Cells Secreting Interleukin-12 for Breast Cancer Therapy Cancer Res., June 15, 2008; 68(12): 4810 - 4818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Pisati, M. Belicchi, F. Acerbi, C. Marchesi, C. Giussani, M. Gavina, S. Javerzat, M. Hagedorn, G. Carrabba, V. Lucini, et al. Effect of Human Skin-Derived Stem Cells on Vessel Architecture, Tumor Growth, and Tumor Invasion in Brain Tumor Animal Models Cancer Res., April 1, 2007; 67(7): 3054 - 3063. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. S. Dace, P. W. Chen, H. Alizadeh, and J. Y. Niederkorn Ocular immune privilege is circumvented by CD4+ T cells, leading to the rejection of intraocular tumors in an IFN-{gamma}-dependent manner J. Leukoc. Biol., February 1, 2007; 81(2): 421 - 429. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Murphy, S. Makonnen, W. Lassoued, M. D. Feldman, C. Carter, and W. M.F. Lee Inhibition of Tumor Endothelial ERK Activation, Angiogenesis, and Tumor Growth by Sorafenib (BAY43-9006) Am. J. Pathol., November 1, 2006; 169(5): 1875 - 1885. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Ziemer, W. M. F. Lee, S. A. Vinogradov, C. Sehgal, and D. F. Wilson Oxygen distribution in murine tumors: characterization using oxygen-dependent quenching of phosphorescence J Appl Physiol, April 1, 2005; 98(4): 1503 - 1510. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Sabel, J. Skitzki, L. Stoolman, N. K. Egilmez, E. Mathiowitz, N. Bailey, W.-J. Chang, and A. E. Chang Intratumoral IL-12 and TNF-{alpha}-Loaded Microspheres Lead To Regression of Breast Cancer and Systemic Antitumor Immunity Ann. Surg. Oncol., February 1, 2004; 11(2): 147 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Gee, S. Makonnen, K. al-Kofahi, B. Roysam, F. Payvandi, H.-W. Man, G. W. Muller, and W. M. F. Lee Selective Cytokine Inhibitory Drugs with Enhanced Antiangiogenic Activity Control Tumor Growth through Vascular Inhibition Cancer Res., December 1, 2003; 63(23): 8073 - 8078. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mitola, M. Strasly, M. Prato, P. Ghia, and F. Bussolino IL-12 Regulates an Endothelial Cell-Lymphocyte Network: Effect on Metalloproteinase-9 Production J. Immunol., October 1, 2003; 171(7): 3725 - 3733. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. A. Portielje, C. H. J. Lamers, W. H. J. Kruit, A. Sparreboom, R. L. H. Bolhuis, G. Stoter, C. Huber, and J. W. Gratama Repeated Administrations of Interleukin (IL)-12 Are Associated with Persistently Elevated Plasma Levels of IL-10 and Declining IFN-{gamma}, Tumor Necrosis Factor-{alpha}, IL-6, and IL-8 Responses Clin. Cancer Res., January 1, 2003; 9(1): 76 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Gee, W. N. Procopio, S. Makonnen, M. D. Feldman, N. M. Yeilding, and W. M. F. Lee Tumor Vessel Development and Maturation Impose Limits on the Effectiveness of Anti-Vascular Therapy Am. J. Pathol., January 1, 2003; 162(1): 183 - 193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. C. Hill, T. F. Conway Jr., M. S. Sabel, Y. S. Jong, E. Mathiowitz, R. B. Bankert, and N. K. Egilmez Cancer Immunotherapy with Interleukin 12 and Granulocyte-Macrophage Colony-stimulating Factor-encapsulated Microspheres: Coinduction of Innate and Adaptive Antitumor Immunity and Cure of Disseminated Disease Cancer Res., December 15, 2002; 62(24): 7254 - 7263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Segal, N. C. Lee, Y. L. Tsung, J. A. Norton, and K. Tsung The Role of IFN-{gamma} in Rejection of Established Tumors by IL-12 : Source of Production and Target Cancer Res., August 15, 2002; 62(16): 4696 - 4703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Y. Crowe, M. J. Smyth, and D. I. Godfrey A Critical Role for Natural Killer T Cells in Immunosurveillance of Methylcholanthrene-induced Sarcomas J. Exp. Med., July 1, 2002; 196(1): 119 - 127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Smyth, N. Y. Crowe, D. G. Pellicci, K. Kyparissoudis, J. M. Kelly, K. Takeda, H. Yagita, and D. I. Godfrey Sequential production of interferon-gamma by NK1.1+ T cells and natural killer cells is essential for the antimetastatic effect of alpha -galactosylceramide Blood, February 15, 2002; 99(4): 1259 - 1266. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Lee, D. C. Kim, M. S. Gee, H. M. Saunders, C. M. Sehgal, M. D. Feldman, S. R. Ross, and W. M. F. Lee Interleukin-12 Inhibits Angiogenesis and Growth of Transplanted but not in Situ Mouse Mammary Tumor Virus-induced Mammary Carcinomas Cancer Res., February 1, 2002; 62(3): 747 - 755. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Saio, S. Radoja, M. Marino, and A. B. Frey Tumor-Infiltrating Macrophages Induce Apoptosis in Activated CD8+ T Cells by a Mechanism Requiring Cell Contact and Mediated by Both the Cell-Associated Form of TNF and Nitric Oxide J. Immunol., November 15, 2001; 167(10): 5583 - 5593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Hunter, D. Yu, M. Gee, C. V. Ngo, C. Sevignani, M. Goldschmidt, T. V. Golovkina, S. Evans, W. F. Lee, and A. Thomas-Tikhonenko Cutting Edge: Systemic Inhibition of Angiogenesis Underlies Resistance to Tumors During Acute Toxoplasmosis J. Immunol., May 15, 2001; 166(10): 5878 - 5881. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Gee, H. M. Saunders, J. C. Lee, J. F. Sanzo, W. T. Jenkins, S. M. Evans, G. Trinchieri, C. M. Sehgal, M. D. Feldman, and W. M. F. Lee Doppler Ultrasound Imaging Detects Changes in Tumor Perfusion during Antivascular Therapy Associated with Vascular Anatomic Alterations Cancer Res., April 1, 2001; 61(7): 2974 - 2982. [Abstract] [Full Text]
|
|
|
|
|
|
M. Strasly, F. Cavallo, M. Geuna, S. Mitola, M. P. Colombo, G. Forni, and F. Bussolino IL-12 Inhibition of Endothelial Cell Functions and Angiogenesis Depends on Lymphocyte-Endothelial Cell Cross-Talk J. Immunol., March 15, 2001; 166(6): 3890 - 3899. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. L. Beatty and Y. Paterson IFN-{{gamma}}-Dependent Inhibition of Tumor Angiogenesis by Tumor-Infiltrating CD4+ T Cells Requires Tumor Responsiveness to IFN-{{gamma}} J. Immunol., February 15, 2001; 166(4): 2276 - 2282. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Adris, E. Chuluyan, A. Bravo, M. Berenstein, S. Klein, M. Jasnis, C. Carbone, Y. Chernajovsky, and O. L. Podhajcer Mice Vaccination with Interleukin 12-transduced Colon Cancer Cells Potentiates Rejection of Syngeneic Non-Organ-related Tumor Cells Cancer Res., December 1, 2000; 60(23): 6696 - 6703. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. H. Shoemaker Genetic and Epigenetic Factors in Anticancer Drug Resistance J Natl Cancer Inst, January 5, 2000; 92(1): 4 - 5. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
d3/6, where d refers to the dimensions; tumors generally had the same perpendicular and bidirectional dimensions). A, the increase in tumor volume (log10 scale) over time of individual PBS-treated tumors (solid black lines) and rmIL-12-treated tumors (dashed gray lines; n = 3 for all groups, except controls, which had n = 4). Arrowheads, days of PBS or rmIL-12 injection. B, data points, increase in tumor volume as a multiple of the starting tumor volume is graphed as a function of time (n = 3); bars, SD. Top, the effect of giving anti-IFN-
and % hypoxic (<1% O2; 
) tumor cells from K1735 and N23 tumors treated for the indicated periods with PBS or rmIL-12 (n = 3).
