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Experimental Therapeutics |
Department of Molecular Medicine [J. R. K., R. K. K., S. P., G. W. K.] and Auckland Cancer Society Research Centre [L-M. C.], School of Medicine and Health Science, University of Auckland, Auckland, New Zealand
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We hypothesized that combined immunotherapy and antiangiogenic therapy would have several advantages over each modality administered as a monotherapy. Cancer therapy using natural inhibitors of angiogenesis, such as endostatin and angiostatin, reveals that such reagents cause even very large tumors (1% of body weight) to regress to dormant, microscopic nodules (4 , 5) . However, the potential for dormant nodules to "reawaken" remains a potential drawback of this type of therapy. Similarly, synthetic small molecules, such as xanthenone analogues of the drug FAA, have been found to cause dramatic hemorrhagic necrosis of tumors by causing vascular collapse (6, 7, 8, 9, 10) . Here we investigate whether immunotherapy by gene transfer of B7.1 (11, 12, 13) would synergize with FAA and with DMXAA, a more potent analogue of FAA, that also works, in part, by causing vascular collapse and reduced blood flow (reviewed in Ref. 14 ). The rationale was that DMXAA may aid B7.1-mediated antitumor immunity by inhibiting tumor growth, thereby allowing time for the antitumor immune response to develop. By preventing tumor growth, DMXAA would be expected to prevent the development of tumor escape mutants and at the same time might render the tumor vulnerable to immune attack as the integrity of the tumor mass begins to disintegrate.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Single Tumor Model.
Tumors were established by s.c. injection of 2 x 105 EL-4 and LLC cells into the left flank of mice, and growth was determined by measuring two perpendicular diameters. Animals were euthanized when tumors reached >1 cm in diameter, in accord with Animal Ethics Approval (University of Auckland). EL-4 and LLC tumors reached 0.6–0.9 cm in diameter after approximately 21 and 14 days, respectively. All experiments included five or six mice/treatment group, and each experiment was repeated at least once.
Administration FAA and DMXAA Analogues.
FAA was a gift from the Department of Health and Human Services (Drug Synthesis and Chemistry Laboratory, National Cancer Institute, Bethesda, MD). The sodium salt of DMXAA was synthesized in the Auckland Cancer Society Research Center (School of Medicine and Health Science, University of Auckland). Solutions of FAA and DMXAA in 5% (w/v) sodium bicarbonate and water, respectively, were prepared fresh for each experiment and protected from light. FAA and DMXAA were injected i.p. at 300 and 25 mg/kg of body weight in a volume of 0.01 ml/g of body weight, respectively.
Gene Transfer of B7.1.
cDNA encoding full-length human VCAM-1 was purchased from R&D Systems (Abingdon, United Kingdom); human B7.2 (CD86; Ref. 15
) was provided by Dr. G. Freeman (Dana-Farber Cancer Institute, Boston, MA); human ICAM-1 was donated by Dr. J. Ni (Human Genome Sciences, Inc., Rockville, MD); mouse B7.1 (CD80; Ref. 12
) was provided by Dr. P. Linsley (Bristol-Myers-Squibb, Seattle, WA). We have reported previously the cloning and characterization of human MAdCAM-1 cDNA (16)
. The CAM pCDM8 expression vectors were prepared by cesium chloride gradient centrifugation and diluted to 600 µg/ml in a solution of 5% glucose in 0.01% Triton X-100. They were mixed in a ratio of 1:3 (w/w) with DOTAP cationic liposomes (Boehringer Mannheim, Mannheim, Germany). Tumors were injected with 100 µl of DNA (60 µg)/liposome complex, unless otherwise stated.
Combining B7.1-mediated Immunotherapy with FAA/DMXAA-mediated Vasculature Attack.
Tumors, 0.6 to 0.9 cm in diameter, were injected at multiple sites with 100 µl of DNA (60 µg)/liposome complex. Twenty-four h later, DMXAA or FAA was administered i.p. as described above. Treated mice that remained tumor free were rechallenged 6 weeks after administration of FAA or DMXAA and B7.1, by s.c. injection of EL-4 cells and LLC cells (0.1 ml) in the opposing flank (right flank).
Multiple Tumor Model.
Five separate tumors were established in a single mouse by injecting 2 x 105 EL-4 cells s.c. into one flank and the same number of EL-4 cells at four different sites into the opposing flank 14 days later. After 28 days, the first tumor that had reached 0.5–0.6 cm in diameter was injected with 60 µg of B7.1 expression plasmid, and 48 h later DMXAA was administered systemically by i.p. injection. The four tumors on the opposing flank were left untreated. The growth of all five tumors was measured as above.
Measurement of the Generation of Antitumor CTL.
Splenocytes were harvested 21 and 42 days after initial gene transfer and 22 days after a parental tumor challenge. They were incubated at 37°C with EL-4 target cells in graded E:T ratios in 96-well, round-bottomed plates. After a 4-h incubation, 50 µl of supernatant were collected, and lysis was measured using the Cyto Tox 96 Assay kit (Promega Corp., Madison, WI). Background controls for nonspecific target and effector cell lysis were included. After background subtraction, the percentage of cell lysis was calculated using the formula: 100 x (experimental - spontaneous E:T spontaneous target/maximum target - spontaneous target).
Adoptive Transfer of Antitumor CTLs.
Adoptive transfer of antitumor splenocytes was as described previously (1)
. Briefly, splenocytes obtained from mice 21 days after therapy were resuspended in HBSS containing 1% FCS and stimulated with 5 µg/ml phytohemagglutinin and 100 units/ml recombinant mouse interleukin 2 for 4–5 days. Animals bearing tumors with a mean diameter of 0.6 cm received both intratumoral and i.p. injections of 2 x 108 cultured splenocytes.
Depletion of Leukocyte Subsets.
Mice were depleted of CD8+ and CD4+ T cells and NK cells by i.p. and i.v. injections 4 days before gene transfer and thereafter every alternate day with 300 µg (0.1 ml) of the 53-6.72 (anti-CD8), Gk1.5 (anti-CD4), and PK136 (anti-NK) mAbs. Rat IgG (Sigma Chemical Co., St. Louis, MO) was used as a control antibody. Antibodies were an ammonium sulfate fraction of ascites, which titered to at least 1:2000 by fluorescence-activated cell sorter (Becton Dickinson and Co., CA) staining of splenocytes. Depletion of individual leukocyte subsets was found to be >90% effective, as determined by FACScan analysis. Rat hybridomas secreting mAbs against mouse CD8 (53-6.72 mAb), CD4 (Gk1.5 mAb), and NK cells (PK136 mAb) were purchased from the American Type Culture Collection (Rockville, MD).
In Situ Detection of Apoptotic Cells.
Tumors were excised and immediately frozen in dry ice and stored at -70°C, and serial sections of 6-µm thickness were prepared. TUNEL staining was performed using an in situ apoptosis detection kit from Boehringer Mannheim. Briefly, frozen sections were fixed with paraformaldehyde solution (4% in PBS, pH 7.4) and permeabilized with a solution containing 0.1% Triton X-100 and 0.1% sodium citrate. After washing, they were incubated with 20 µl of TUNEL reagent for 60 min at 37°C and examined by fluorescence microscopy. Some slides were counterstained with propidium iodide (Sigma) to distinguish necrotic cells from those undergoing apoptosis. Adjacent sections were counterstained with H&E and mounted. The total number of apoptotic or necrotic cells were counted. The apoptotic index or necrotic index was calculated as follows: Apoptotic index or necrotic index = number of apoptotic or necrotic cells x 100/total number of nucleated cells.
Immunohistology.
Tumors were excised on the seventh day after administration of therapeutic agents and controls, immediately frozen in dry ice, stored at -70°C in isopentane, and sections of 10-µm thickness were prepared. Sections were mounted on poly-L-lysine-coated slides, and endogenous peroxidases were blocked by incubating slides for 30 min in 0.3% H2O2 in methanol. Sections were stained with mouse anti-hsp70 mAb (SPA-810 antibody; StressGen Biotechnologies Corp., Victoria, Canada) using a mouse-on-mouse immunodetection ABC Elite peroxidase kit (Vector Laboratories, Burlingame, CA). They were developed with Sigma Fast DAB (3,3'-diaminobenzidine tetrahydrochloride) with metal enhancer CoCl2 tablets and counterstained with H&E. As a control, the primary antibody was substituted with rat IgG (Sigma).
Statistical Analysis.
All experiments performed in vivo were repeated at least once, with similar results obtained. Results were expressed as mean values + SD, and a Student’s t test was used for evaluating statistical significance. P < 0.05 denotes statistical significance, whereas P < 0.001 denotes results that are highly significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, larger tumors (>0.5 cm) are refractory to treatment in response to B7.1 and several other costimulatory CAMs. Tumor growth is slowed, but ultimately the tumor grows unchecked.
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, accompanied by marked tumor necrosis (refer below). DMXAA was the more potent of the two reagents, causing tumors to shrink to 0.1–0.2 cm over a period of 3 weeks, whereas the tumors of FAA-treated animals were reduced to 0.2–0.4 cm in diameter. However, tumors began to grow unchecked by day 28, and animals had to be sacrificed during the sixth week.
Combined Therapy by Timed Delivery of B7.1 and DMXAA/FAA Eradicates Large Tumors.
We hypothesized that simultaneous administration of the B7.1 pCDM8 expression vector and DMXAA/FAA might impair CAM-mediated antitumor immunity, because dying and necrotic tumor cells would not be able to adequately express B7.1. This notion proved correct (data not shown), and hence established tumors (0.6–0.8 cm in diameter) were first treated with B7.1 to stimulate antitumor immunity, and DMXAA and FAA were administered 1 day later to retard tumor growth. Remarkably, tumors rapidly diminished in response to the combination of B7.1 and DMXAA accompanied by massive necrosis, such that by the third week of treatment, tumors had completely disappeared (Fig. 1B)
. The gross tumors of DMXAA-B7.1-treated animals took on the appearance of a wound that rapidly healed, forming a scab that eventually flaked off, leaving perfectly healed skin. Unlike B7.1 treatment, which leaves what appears to be a palpable fibrotic core, the tumor sites of DMXAA-B7.1-treated animals were completely healed (Fig. 1C)
. Similar results were obtained with the combination of FAA and B7.1, although tumors did not completely disappear until the sixth week (data not shown).
Combinational Therapy Generates Potent and Prolonged Tumor-specific CTL Activity.
The antitumor CTL activity of splenocytes obtained from treated mice, 21 days after gene transfer, was significantly (P < 0.001) augmented in animals treated with the combination of B7.1 and DMXAA and slightly enhanced with the combination of B7.1 and FAA versus those receiving B7.1 monotherapy (Fig. 2A)
. In contrast, DMXAA and FAA alone were very poor effectors, although they did enhance CTL production above that seen with empty vector and liposome controls.
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; Table 1
). Both DMXAA-B7.1 and FAA-B7.1 therapy provided complete protection, indicating that potent systemic antitumor immunity had been achieved. Antitumor CTL activity was still very strong 42 days after initial treatment with DMXAA-B7.1 and was stimulated further after the rechallenge with parental tumor cells (Fig. 2A)
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. Tumors >0.8 cm in diameter were refractory to such treatment (data not shown). In contrast, splenocytes from DMXAA- or empty vector-treated mice displayed no detectable antitumor activity.
Composition of the Leukocyte Effector Population.
In vivo antibody blocking studies revealed that the primary rejection of tumors in response to either combined DMXAA-B7.1 or FAA-B7.1 therapy was very dependent on the presence of CD8+ T cells and NK cells but only partially dependent on CD4+ T cells (Fig. 3, A–C)
. Simultaneous depletion of either CD8+ T cells and NK cells; CD4+ and CD8+ T cells; or CD4+/CD8+ T cells and NK cells severely impaired antitumor immunity, leading to rapid tumor growth (Fig. 3, D–F)
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. However, mice cured of EL-4 tumors were unable to resist a challenge of 1 x 104 LLC cells, and in contrast, mice cured of LLCs were not protected against a challenge with 1 x 104 EL-4 cells, demonstrating that antitumor immunity is tumor specific.
Gene Dosage Effect.
Both FAA and DMXAA have an unusual "threshold" behavior in which only a very narrow range of high doses (22.5–25 mg/kg body weight) are active and acceptable in terms of toxicity (17
, 18)
. Furthermore, we have reported previously that B7.1 and other costimulatory CAMs display a restrictive gene dosage effect, such that gene transfer of 60 µg of B7.1/pCDM8 expression plasmid is optimal, whereas lesser or greater amounts are much less effective (1)
. To investigate whether a high gene dosage would impair combination therapy, tumors were injected with varying amounts (90–180 µg) of B7.1/pCDM8 plasmid, followed by administration of an optimal dose of DMXAA (25 mg/kg; Fig. 4A
). All mice rapidly rejected their tumors and a rechallenge of 2 x 105 parental EL-4 cells. Thus, an identical outcome is achieved with a broad high dose range of the therapeutic gene and an optimal dose of DMXAA. In contrast, when the DMXAA concentration was suboptimal, a gene dosage effect is clearly evident, such that only large amounts (180 µg) of B7.1/pCDM8 expression plasmid could generate effective antitumor immunity (Fig. 4B)
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). In contrast, there was a predominance of necrotic cells in tumor sections from DMXAA-treated mice, where very few apoptotic cells were present (Fig. 5d)
. Surprisingly, combination therapy increased the number of apoptotic cells compared with B7.1 monotherapy (Fig. 5c)
while retaining the same degree of necrosis observed with DMXAA monotherapy. Untreated tumors showed no sign of necrosis, and apoptotic cells were absent (Fig. 5e)
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. Similarly, hsp70 was up-regulated in tumors treated with B7.1, although not necessarily in the vicinity of blood vessels. In contrast, there was no sign of heightened hsp70 expression in tumors treated with empty vector alone.
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. In contrast, DMXAA monotherapy slowed tumor growth, but all tumors eventually grew unchecked (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IFNs, IP-10, nitric oxide, and serotonin (14)
. There is strong evidence that FAA-induced tumor vasculature collapse is mediated by TNF- (25)
. TNF- appears to increase endothelial procoagulant activity, leading to platelet-mediated vascular occlusion (10)
. DMXAA strongly induces TNF- expression in both mouse and human cells (26
, 27)
, raising the hope that it will have antitumor activity in humans. Both FAA and DMXAA activate transcription of the IFN-
gene (14)
, which is interesting because administration of TNF and IFN- to melanoma patients causes selective disruption of the tumor vasculature by a mechanism involving suppression of the activation of the integrin vß3 (28)
.
The importance of the adaptive immune response involving T cells in FAA/DMXAA antitumor activity is debatable. FAA and DMXAA-induced hemorrhagic necrosis, growth delay, and regression of colon 38 tumors appear to be largely independent of T-cell reactivity (29)
. An interaction of FAA and DMXAA with the innate immune system might be sufficient to generate the levels of TNF- and IFN- required to starve tumors of a blood supply. Two other studies indicated that an intact T-cell immune response was essential for FAA-induced growth inhibition of human colon tumors and the C-26 adenocarcinoma (30
, 31)
. However, the latter studies do not rule out the possibility that the tumors themselves stimulate a weak T-cell immune response, which is further enhanced by FAA- and DMXAA-mediated activation of innate immunity involving secretion of TNF and IFN-, and that FAA and DMXAA are not activators of T-cell function per se. Alternatively, by starving tumors of a blood supply, DMXAA and FAA may induce the expression of heat shock proteins on stressed tumor cells and thereby increase tumor immunogenicity (32)
. In accord with this notion, hsp70 was consistently up-regulated on tumor cells, either surrounding or within the vicinity of blood vessels. Such tumor cells may be more sensitive to stress because they would not be expected to adapt to the rapid deprivation of nourishment because of loss of their blood supply. Similarly, potent antitumor immunity generated by the in vivo killing of tumor cells with the herpes simplex virus thymidine kinase/ganciclovir system is thought to be mediated in part via the induction of hsp70 acting as an immune stimulatory signpost or danger signal (33)
. hsp70 appears to target immature APCs, making them more efficient at capturing tumor antigens released from dying cells. Hsp70 is taken up directly into dendritic cells and may be involved in direct chaperoning of tumor antigens into dendritic cells (33)
. The results of the present study support the concept that DMXAA and FAA probably indirectly augment T cell-mediated antitumor immunity rather than directly activate T-cell function. Thus, both FAA and DMXAA are poor effectors of CTL production. Although they induce extensive hemorrhagic necrosis, the necrosis appears to be preceded by negligible programmed cell death of tumor cells, although more detailed analysis at early time points is needed to confirm this notion.
Antibody depletion of leukocyte subsets revealed that the antitumor immune response was largely mediated by CD8+ T cells and NK cells, which is a feature of CAM-mediated antitumor immunity (1)
. Nevertheless, combination therapy is accompanied by enhanced production of antitumor CTLs and by a marked increase in the number of tumor cells subjected to apoptosis. DMXAA and FAA may facilitate B7.1 immunotherapy by: (a) inhibiting tumor cell growth, and thereby preventing the outgrowth of escape variants; (b) causing extensive tumor cell necrosis and generating tumor fragments available to APCs that have been recruited to the tumor site in response to B7.1 immunotherapy; (c) amplifying the innate immune response; and (d) rendering the tumor accessible to immune cells as the tumor mass begins disintegrate (Fig. 8)
. The inability of adoptively transferred antitumor CTLs to eradicate or halt the growth of tumors larger in size than 0.8 cm in diameter is in accord with the latter notion. Combined therapy was able to cure 50% of mice bearing extremely large tumors (
1.0 cm in diameter; data not shown). When B7.1 and DMXAA were re-administered to mice not cured of their disease, they were able to halve the growth rate of tumors. In contrast, DMXAA monotherapy had no effect on tumor regression, unlike other antiangiogenic reagents that cause tumors to regress on each repeat administration.
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The major objective of cancer immunotherapy is to rid the body of not only the primary tumor but also tumors derived from the primary tumor that have become established at distant sites. Here we revealed that the combination of immunotherapy with antiangiogenic therapy is able to achieve this goal. Thus, treatment of a single tumor by B7.1-mediated CAM immunotherapy, followed by systemic administration of DMXAA, leads to the complete eradication of multiple untreated tumor nodules established at sites distant to the primary tumor. Hence in a clinical setting, treatment of the primary tumor by gene therapy to induce antitumor immunity, followed by systemic antiangiogenic therapy, could lead to the rejection of sizeable metastatic nodules. Ultimately, it would be preferable that the immunotherapeutic agent was also administered systemically, in the event that metastasized tumor nodules had evolved to become antigenically disparate from the primary tumor. We cannot totally exclude the possibility that localized intratumoral gene injection of B7.1 led to systemic leakage; however, enhanced B7.1 expression was not detectable in spleen, kidney, or liver. Systemic administration of T-cell costimulators may never be acceptable for the treatment of cancer in humans because of the risk of inducing autoimmunity. Nevertheless, systemic administration of B7.1 combined with antiangiogenic therapy warrants investigation, but this will require a separate detailed study to exclude the problem of autoimmunity.
Phase I clinical trials of DMXAA in the United Kingdom and New Zealand are under way. Whatever the outcome, the results of the present study have important implications for future cancer therapy trials. There is the implication that novel therapeutic approaches, which combine immunotherapy with vascular attack, may overcome many of the problems associated previously with immunotherapy. The goal now must be to determine whether other natural, less toxic, antiangiogenic factors can also synergize with B7.1 and to search for effector molecules responsible for the success of B7.1-DMXAA combination therapy so that these same molecules might be used in the fight against human cancers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Royal Society of New Zealand, the Cancer Society of New Zealand, The Lottery Grants Board, the Health Research Council of New Zealand, the Wellcome Trust, and the Maurice and Phyllis Paykel Trust. G. W. K. was supported by a James Cook Research Fellowship funded by the Royal Society of New Zealand. 
2 To whom requests for reprints should be addressed, at Department of Molecular Medicine, School of Medicine and Health Science, University of Auckland, Private Bag 92019, 85 Park Road, Grafton, Auckland, New Zealand. Phone: (64-9) 3737599, extension 6280; Fax: (64-9) 3737674; E-mail: gw.krissansen{at}auckland.ac.nz
3 The abbreviations used are: CAM, cell adhesion molecule; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; DMXAA, 5,6-dimethylxanthenone-4-acetic acid; FAA, flavone acetic acid; LLC, Lewis lung carcinoma; DOTAP, N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate; NK, natural killer; mAb, monoclonal antibody; TUNEL, terminal deoxynucleotidyltransferase-mediated nick end labeling; hsp70, heat shock protein 70; TNF, tumor necrosis factor; APC, antigen-presenting cell.
Received 6/22/00. Accepted 12/27/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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