Cancer Research Cancer Health Disparities Conference 2009
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
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cuadros, C.
Right arrow Articles by Lustgarten, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cuadros, C.
Right arrow Articles by Lustgarten, J.
[Cancer Research 63, 5895-5901, September 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Cooperative Effect between Immunotherapy and Antiangiogenic Therapy Leads to Effective Tumor Rejection in Tolerant Her-2/neu Mice1

Camilo Cuadros, Ana Lucia Dominguez, Gregory I. Frost, Per Borgstrom and Joseph Lustgarten2

Sidney Kimmel Cancer Center, San Diego, California 92121


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immunotherapy is an attractive strategy for cancer treatment. However, self-tolerance is one of the major mechanisms that dampen immune responses against self-tumor antigens. We have demonstrated that Her-2/neu transgenic mice (neu mice) are tolerant to neu antigens and contain only a low avidity repertoire for neu. However, this repertoire has antitumor activity. Immunizations of neu mice are capable of activating the low-avidity T cells that, at best, retard the tumor growth. To increase the efficacy of the antitumor responses in neu mice, we hypothesized that immunotherapy in combination with antiangiogenic therapy would be a more efficient strategy for tumor eradication. The rationale for using this combination was that by decreasing the growth rate of the tumor with an antiangiogenic therapy, the low-avidity repertoire of neu mice stimulated by immunotherapeutic intervention would be more effective in destroying the slow growing tumor. To test this hypothesis, we stably expressed a soluble form of the Flt-1 vascular endothelial growth factor receptor (sFlt-1) on N202.1A cells, using a retrovirus vector. Expression of sFlt-1 on N202.1A (N202-Flt) cells significantly inhibited the tumor growth compared with N202.1A parental cells. In contrast to the application of immunotherapy alone or antiangiogenic therapy alone, which delayed the tumor growth, the combination of the two therapies provided complete inhibition of tumor growth in Her-2/neu mice. These results indicate that the use of tumor targeting with immunotherapy in simultaneous combination with antiangiogenic therapy provides a more efficient strategy for the treatment of solid tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is believed that effective T-cell immunity is a critical component of the immune response to a growing tumor. With the identification of TAAs3 (1 , 2) , immunotherapeutic strategies designed to induce cellular immune responses have received much attention as a promising approach for the treatment of many types of cancer. However, the induction of protective antitumor immunity with the use of known TAAs is often disappointing. Considering that the majority of TAAs are in fact aberrantly or overly expressed normal gene products (3 , 4) , mechanisms responsible for self-tolerance have dampened immune responses against these self-tumor antigens. We have evaluated the T-cell responses against self-tumor antigens, using Her-2/neu transgenic mice (5 , 6) . We demonstrated that Her-2/neu mice were functionally tolerant to neu antigens and that, despite such tolerances, neu mice contain a residual low-avidity T-cell repertoire against neu with antitumor activity.4 Multiple rounds of immunizations with DCs pulsed with Her-2/neu antigens in conjunction with IL-2 and anti-OX40 mAb significantly enhanced the antitumor responses in these mice and led to substantial tumor reduction.4 These data demonstrate that it is possible to manipulate immune responses in tolerant hosts. However, the effect of the antitumor immune response is not sufficient for complete eradication of tumors.

Angiogenesis is the ability of preexisting vasculature to send out capillary sprouts leading to the formation of new vasculature (7 , 8) . It is now well accepted that progression of solid tumors is intrinsically dependent on angiogenesis for growth of both the primary tumor and the metastatic lesions (9) . The angiogenic process is promoted by several growth factors, including the VEGF family (10) , the angiopoietin family (11) , and the ephrin family (12) . Among these factors, VEGF maintains its position as the most critical factor for angiogenesis. Tumor cells secrete VEGF, initiating the neovascularization process (13) . VEGF regulates vessel formation by signaling through Flt-1 and Flk-1 kinase receptors (14) . With the identification of specific biological mediators of angiogenesis, it is now possible to develop antiangiogenic therapies in which molecules can be administered to inhibit the angiogenesis process.

It has been hypothesized that inhibition of tumor angiogenesis blocks tumor growth and decreases the potential for tumor metastases. To disrupt the effect of VEGF, anti-VEGF antibodies have been developed that are able to reduce the growth of a variety of tumors in nude animal models (15) . In addition, anti-Flk-1 antibodies (16) , soluble Flk-1 (17) , and Flt-1 (18) have been demonstrated to inhibit tumor growth. We tested whether the expression of a soluble form of Flt-1 receptor (sFlt-1) in tumor cells would be sufficient for tumor eradication. The sFlt-1 was expressed on N202.1A cells [a cell line generated from a spontaneous tumor from neu mice (19) ], using a retroviral vector. At best, inoculation of these cells on neu mice inhibits tumor growth. These results are in accordance with reports from other laboratories (20 , 21) showing that antiangiogenic therapy alone is not sufficient to eliminate tumors completely. We hypothesized that the combination of immunotherapy with antiangiogenic therapy could be synergistic, providing a more effective strategy to eliminate the tumor. The rationale for this combination therapy was that decreasing the growth rate of the tumor with an antiangiogenic therapy would make the low-avidity repertoire of neu mice stimulated by immunotherapeutic intervention more effective in destroying the slow-growing tumor. There are a few reports demonstrating that the combination of immunotherapy and antiangiogenic therapy induces stronger antitumor responses (22 , 23) , validating our rationale for using this combination. However, in these studies we show for the first time that although neu mice are tolerant to the tumor antigen, the combination of immunotherapy and antiangiogenic complemented each other and was far superior than each monotherapy alone, producing a dramatic inhibition of tumor growth.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and Cell Lines.
The Her-2/neu transgenic mice (line 202) were obtained commercially from The Jackson Laboratory and maintained homozygously. The N202.1A mammary cell line derived from a tumor from the Her-2/neu transgenic mice was obtained from Dr. P-L. Lollini (University of Bologna, Bologna, Italy). These cells express H2Dq/H-2Lq and high levels of Her-2/neu molecules; they form tumors in neu mice but not in conventional FVB mice. The N202 cells form tumors that are histologically similar to the spontaneous tumors.

The N202-H2B-GFP cell line was generated by infecting the N202.1A cell line with the H2B-GFP-LXRN vector as described previously (24) . The N202-Flt cell line was derived by expressing the murine Flt-1 gene in the N202.1A cells. The Flt-1 gene was amplified by reverse transcription-PCR from RNA prepared from kidneys. The amplified product was inserted in the TOPO vector (Invitrogen), and the sequence was verified and then inserted in the LXRN expression vector. The resultant vector was used to produce retroviral supernatant to transduce the N202.1A cell line. Expression of the Flt-1 gene on N202.1A cells was confirmed by reverse transcription-PCR. All cell lines were maintained in complete RPMI medium (RPMI 1640) supplemented with 10% FCS, 2 mM glutamine, 5 x 10-5 2-mercaptoethanol, and 50 µg/ml gentamicin. All infected cells were selected in the presence of 1 mg/ml G418. The embryonic 3T3-FVB fibroblast cell line was generated in our laboratory in the same manner as other embryonic 3T3 fibroblast cell lines that had developed previously (25) . Recombinant human IL-2 was obtained from the Biological Resource Branch, National Cancer Institute (Bethesda, MD). Anti-OX40 (OX86) mAb was obtained from the European Cell Culture Collection (Wiltshire, United Kingdom).

Cell Proliferation.
To evaluate whether expression of H2B-GFP or sFlt-1 affected the growth of the N202.1A cells in vitro, we compared proliferation among N202.1A, N202-H2B-GFP, and N202-Flt cells. We plated 1 x 104 N202.1A, N202-Flt, and N202-H2B-GFP cells in 96-well plates in set of triplicates and assessed cell proliferation at 24, 48, and 72 h post-plating by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (26) .

Generation of DCs and Induction of Apoptosis of Tumor Cells.
DCs were derived from bone marrow as described by Inaba et al. (27) . Briefly, bone marrow cells were depleted of lymphocytes by use of magnetic beads conjugated with antibodies against CD4, CD8, B220, and class II molecules. The remaining cells were cultured in complete RPMI medium containing 3% GM-CSF (supernatant from J558L cells transfected with murine GM-CSF gene; obtained from Dr. R. Steinman, Rockefeller University, New York, New York). Medium was changed every second day; each time fresh complete RPMI medium containing 3% GM-CSF was added. On day 8, DCs were collected and tested for cell surface expression of class I and II, B7-1-2, and CD11b. The N202.1A cells were exposed to UVB irradiation (2 mJ/cm2/s) and incubated for 18 h in medium without serum at 37°C for the induction of apoptosis. DCs and apoptotic cells were collected and cocultured in complete RPMI medium containing 3% GM-CSF overnight at a ratio of two apoptotic cells per one DC (5 x 105 DCs/well). Maturation of pulsed DCs was confirmed by FACS analysis evaluating the levels of B7-1-2 and class I and II. Phagocytosis of apoptotic cells was confirmed by FACS analysis. The N202.1A cells were labeled with the aliphatic green fluorochrome PKH2-GL before apoptosis was induced by UV irradiation and serum deprivation. On day 7, cultured DCs were cocultured with the fluorescent-apoptotic-N202.1A cells. After 18 h, cocultured cells were harvested and stained with phycoerythrin-labeled antibodies against class II and B7.2 (CD86; data not shown).

Analysis of Tumor Growth Inhibition by Intravital Microscopy.
Dorsal skin chambers in the mouse were prepared as described previously (25) . Female Her-2/neu mice (25–30 g body weight; around 10–12 weeks of age) were anesthetized (7.3 mg ketamine hydrochloride and 2.3 mg xylazine/100 g body weight; i.p.) and placed on a heating pad. Two symmetrical titanium frames were implanted into a dorsal skinfold so as to sandwich the extended double layer of skin. A 15-mm full-thickness layer was excised. The underlying muscle (M. cutaneous max) and s.c. tissues were covered with a glass coverslip incorporated in one of the frames. After a recovery period of 3–4 days, tumor spheroids were carefully placed in the chamber. For spheroid formation, N202-H2B-GFP or a mixture of N202-H2B-GFP and N202-Flt cells at 1:1 ratio were plated at 50 x 104 cells/well in 100 µl/well in 96-well round-bottomed plates coated with 50 µl of 1% agarose. The spheroids were allowed to compact for 48 h and then were washed in serum-free medium for implantation into the chambers. Vaccination with pulsed DCs was performed as described above.

Fluorescence microscopy was performed using a Mikron Instrument Microscope (Mikron Instrument, San Diego, CA) equipped with an epiilluminator and video-triggered stroboscopic illumination from a xenon arc (MV-7600; EG&G, Salem, MA). A silicon-intensified camera (SIT68; Dage-MTI, Michigan City, IN) was attached to the microscope. A Hamamatsu image processor (Argus 20) with firmware version 2.5 (Hamamatsu Photonic System, USA) was used for image enhancement and for capturing images to a computer. A Leitz PL1/0.04 objective was used to obtain an overview of the chamber and to determine tumor size. A Zeiss long-distance objective 10/0.22 was used to capture images for calculation of vascular parameters. The tumor spheroid was analyzed off-line from the captured images with use of photodensitometric computer software to calculate tumor area and relative photometric density of the tumor (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). For each spheroid, video recordings were used to calculate vascular density of the neovasculature being induced by the implanted spheroids (Image-Pro Plus,).

In Vivo Tumor Model.
To evaluate the antitumor effect of N202-Flt or the combination of immunotherapy and antiangiogenic therapy in vivo, neu mice were implanted with the transplantable N202-Flt cell line. On day 0, animals received s.c. implants of 106 tumor cells. Tumors were allowed to grow for 7 days before treatment was initiated. On day 7 after tumor inoculation, animals were randomly divided into groups of five and vaccinated three times s.c. with DCs pulsed with apoptotic N202.1A cell (with intervals of 10 days between each vaccination) in the presence of rIL-2 and anti-OX40 mAb. The rIL-2 was injected daily for 3 weeks (104 IU/day), and anti-OX40 mAb was injected once a week for 3 weeks (100 µg/injection). Tumor growth was monitored every 5 days, and growth rates were determined by caliper measurements in two diameters. Tumor volume was expressed as: (minor diameter)2 x major diameter/2. Statistical analysis was performed with Student’s t test. In each experiment at least five animals were included per group.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro and in Vivo Analysis of Cell Growth of Transduced N202.1A Cells.
To determine whether the expression of sFlt-1 or H2B-GFP affected the growth of stably transduced N202.1A cells, we first analyzed the cell growth in vitro. These cell clones are microscopically indistinguishable from one and another and, as shown in Fig. 1Citation , N202-Flt and N202-H2B-GFP had similar growth rates compared with N202.1A parental cells. To examine the effect of sFlt-1 expression in vivo, we used a dorsal skinfold chamber tumor model and a s.c. tumor model. The dorsal skinfold chamber tumor model allowed us to (a) micro implant tumors (5 x 104 cells) and study tumor growth in real time, making it possible to do a kinetic analysis; (b) evaluate, in a short period of time, the effect of therapeutic intervention by measuring the tumor size; and (c) observe the angiogenic process. The s.c. tumor model allowed us to test therapies with a larger tumor burden, implanting 106 cells.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1. In vitro proliferation of transduced N202.1A cells. N202.1A cells transduced with the sFlt-1 and H2B-GFP genes and N202.1A cells were plated at 1 x 104 cells/well in three sets of triplicates in 96-flat-well plates. Cell proliferation was measured 24, 48, and 72 h after plating by assaying with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. N202-Flt and N202-H2B-GFP clones grew with equivalent rates compared with the N202.1A parent cell line. Data are representative of one of three experiments. Bars, SE.

 
To examine the effect of sFlt-1 expressed on N202.1A cells at the level of angiogenesis and tumor growth, we implanted N202-Flt tumor spheroids in dorsal skinfold chambers in neu mice. Because the N202-Flt cells are not fluorescently labeled, to visualize the tumor in the chamber, we mixed N202-H2B-GFP and N202-Flt cells at a 1:1 ratio to form the spheroids. As illustrated by the photomicrographs in Fig. 2ACitation , tumor spheroids that secreted the sFlt-1 (mixture of N202-H2B-GFP and N202-Flt cells) grew more slowly than the tumor spheroids composed of the N202-H2B-GFP cells. Measurements of the tumor growth (Fig. 2B)Citation indicated that there was a significant delay in tumor growth in animals implanted with the mixture of N202-H2B-GFP and N202-Flt cells compared with animals implanted with only the N202-H2B-GFP cells. In addition, vascular density measurements (Fig. 2C)Citation demonstrated that the sFlt-1 secreted by the tumor substantially inhibited the neovascularization of the tumor. Although new vasculature was generated in the presence of the N202-Flt cells, the angiogenic activity was significantly lower compared with N202-H2B-GFP tumors. We confirmed the antiangiogenic activity of the sFlt-1 secreted by the transduced N202.1A cells by use of a recombinant sFlt-1 protein prepared in a baculovirus expression vector. This recombinant Flt-1 protein was able to inhibit the neovascularization of the tumor and delayed tumor growth in the same manner that tumor cells secreting the Flt-1 did (data not shown). In addition, our data indicated that sFlt-1 secreted from N202.1A or the recombinant sFlt-1 did not affect the existing vasculature, and no overt effects such as hemorrhage or disruption of the already formed vasculature in the chamber were observed. These data indicate that the sFlt-1 secreted by the N202-Flt cells is bioactive with antiangiogenic activity inhibiting the neovascularization process of the tumor.



View larger version (83K):
[in this window]
[in a new window]
  		Fig. 2. Evaluation in the chamber model of the effect of sFlt expressed on N202.1A tumor cells. A, photomicrographs illustrate the effect of sFlt expressed on N202.1A cells. A mixture of 25 x 103 N202-Flt and 25 x 103 N202-H2B-GFP cells were implanted in the chamber and compared with 50 x 103 N202-H2B-GFP cells. Animals were evaluated every second or third day by intravital microscopy. Panels represent fluorescent (tumor cells) and light images (neovascularization) taken at the indicated times. Fluorescent pictures were taken with a x1 objective, and light pictures were taken with a x3.5 objective. B, tumor growth was analyzed from video recordings as described in "Materials and Methods." Data are means of five animals per group ± SE (bars). A significant (P < 0.001, Student’s t test) difference was found between the mice that had received an injection of N202-H2B-GFP cells and the mice that had received an injection of N202-H2B-GFP+N202-Flt cells. C, relative tumor vascular density. Data are means of five animals per group ± SE (bars). A significant (P < 0.001, Student’s t test) difference was found between the mice that had received an injection of N202-H2B-GFP cells and the mice that had received an injection of N202-H2B-GFP+N202-Flt cells. Data are representative of one of three experiments.

 
In a second series of experiments, we performed a dose titration by mixing N202.1A and N202-Flt cells at different ratios. We implanted 106 N202.1A or N202-Flt cells or a mixture of N202.1A and N202-Flt cells at 1:1 and 9:1 ratios, respectively, s.c. in the neu mice. The N202-Flt cells grew at a much slower rate (~65% growth inhibition) than the N202.1A parental cells, and the mixed cells demonstrated dose-dependent growth rates (~10% growth inhibition at 9:1 ratio; ~45% growth inhibition at 1:1 ratio; Fig. 3Citation ). These data indicate that higher levels of sFlt-1 in the tumor microenvironment lead to slower tumor growth.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3. In vivo growth of N202-Flt cells. neu mice received s.c. injections of 106 N202.1A or N202-Flt cells or a mixture of N202.1A and N202-Flt cells at 1:1 and 9:1 ratios, respectively. Tumor volume was measured every 5 days by caliper in two dimensions. Data are means of five animals per group ± SE (bars). Data are representative of one of two experiments. A significant (P < 0.001, Student’s t test) difference was found between the mice that received N202.1A cells and mice that received N202-Flt cells.

 
Effectiveness of Combining Immunotherapy and Antiangiogenic Therapy for Tumor Elimination.
We previously demonstrated that neu mice were tolerant to neu antigens. However, vaccination with DCs pulsed with apoptotic N202.1A tumors in conjunction with IL-2 and anti-OX40 mAb retarded the tumor growth in these mice.4 Our data showed that expression of sFlt-1 in N202.1A cells significantly delayed tumor growth. Taken together, these data suggested that neither immunotherapy nor antiangiogenic therapy alone was sufficient to completely eliminate the tumor. The combination therapy was evaluated in both the dorsal skinfold chamber and s.c. tumor models. The neu mice with implanted dorsal skinfold chambers were inoculated with a mixture of N202-H2B-GFP and N202-Flt cells at a 1:1 ratio or with N202-H2B-GFP cells alone. Four days later animals were vaccinated with DCs pulsed with N202.1A apoptotic tumors plus IL-2 and anti-OX40 mAb. As a control for the specificity of the vaccine, we included animals vaccinated with DCs pulsed with apoptotic 3T3-FVB cells plus rIL-2 and anti-OX40 mAb. As illustrated in the photomicrographs in Fig. 4ACitation , the combination of specific immunotherapy (DCs pulsed with N202.1A cells) and antiangiogenic therapy led to complete elimination of the tumors, indicating that there was cooperation between these therapies. At day 22, when no tumor cells were detected, no more neovasculature was observed. Measurements of tumor growth demonstrated the effectiveness of combining immunotherapy and antiangiogenic therapy for elimination of solid tumors (Fig. 4B)Citation . As demonstrated previously, the application of immunotherapy or antiangiogenic therapy alone delayed tumor growth, although to a much lesser extent than the combination therapy.



View larger version (66K):
[in this window]
[in a new window]
  		Fig. 4. Effect of antiangiogenic therapy and immunotherapy in the chamber model. A, photomicrographs illustrating the effect of combining immunotherapy and antiangiogenesis therapy. The neu mice with surgically implanted chambers were inoculated with N202-H2B-GFP or a mixture of N202-Flt/N202-H2B-GFP spheroids (5 x 104 cells total) on day 0. On day 4 animals were immunized with 106 DCs pulsed with apoptotic N202.1A cells or DCs pulsed with apoptotic 3T3 FVB cells. Animals that received rIL-2 (104 IU/injection) received injections from day 5 to day 15. Animal that received anti-OX40 mAb (100 µg/injection) received injections on days 6 and 13. Animals were evaluated every second or third day by intravital microscopy. Panels represent fluorescent and light images taken at the indicated times. B, percentiles of tumor growth of all animals per group. One hundred percent represents tumor size at the time that tumor measurements were initiated (day 4 after implantation). Data are means of percentile of five animals per group ± SE (bars). A significant (P < 0.001, Student’s t test) difference was found between mice receiving N202-H2B-GFP cells and mice receiving N202-H2B-GFP+N202-Flt cells + Vacc-DCs-Apop-N202.1A. Data are representative of one of two experiments.

 
Having demonstrated that the combination therapy in the dorsal skinfold chambers inhibited the tumor growth, it was important to evaluate whether the same immunotherapeutic intervention was effective with a larger tumor burden. On day 0, we implanted 106 N202-Flt cells s.c. in female neu mice and allowed the tumor to grow for 7 days. On day 7, animals were randomly divided and started on the immunotherapeutic treatment. We have shown that for the induction of an effective antitumor immune response in the s.c. tumor model, neu mice need to receive multiple vaccinations of DCs plus continuous treatment with IL-2 and anti-OX40 mAb.4 Animals were immunized a total of three times, each time with 106 DCs pulsed with apoptotic N202.1A or apoptotic 3T3-FVB cells every 10 days; they also received daily injections of IL-2 and a weekly injection of anti-OX40 mAb. In addition, we included another group of animals that received four vaccinations with DCs pulsed with apoptotic N202.1A plus IL-2 and anti-OX40 mAb. As shown in Fig. 5Citation , the combination of immunotherapy and antiangiogenic therapy produced a 90% inhibition of tumor growth. Animals receiving four doses of DC vaccination had a more profound growth inhibition (~90% inhibition of tumor growth) compared with the animals that received three doses of DC vaccination (~80% inhibition of tumor growth). We confirmed that the antitumor immune response was CD4+ and CD8+ T-cell mediated because treatment with anti-CD4 or anti-CD8 mAb abrogated the immune protection (data not shown). Mice vaccinated with DCs pulsed with apoptotic 3T3-FVB cells plus IL-2 and anti-OX40 showed no additional protection beyond that conferred by antiangiogenic therapy alone, indicating that vaccination with DCs pulsed with apoptotic N202.1A tumors induced a specific T-cell antitumor response. Taken together, our data show that there is a cooperative effect between immunotherapy and antiangiogenic therapy that provides complete inhibition of tumor growth.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 5. Combination of immunotherapy and antiangiostatic therapy is synergistic. The neu mice received s.c. inoculations on day 0 (106 N202-Flt or N202.1A parental cells). On day 7 when tumors were ~50 mm3, animals began receiving immunotherapeutic treatment. Animals were immunized three times (3) or four times (4). Animals were immunized s.c. with 106 DCs pulsed with apoptotic N202.1A cells or DCs pulsed with apoptotic 3T3 FVB cells on days 7, 17, and 27. Animals that were immunized four times received an additional immunization on day 37. Animals that were immunized three times received i.p. injections of rIL-2 (104 IU/injection) from day 7 to day 37, and animals that were immunized four times received rIL-2 until day 47. Anti-OX40 mAb (100 µg/injection) was injected on days 9, 19, and 29 into animals immunized three times, with an additional injection on day 39 for animals that received four immunizations. Tumors were measured every 5 days with a caliper in two dimensions. Data are means of five animals per group ± SE (bars). A significant (P < 0.001, Student’s t test) difference was found between mice treated with immunotherapy or antiangiogenic therapy alone and mice treated with the combination of immunotherapy and antiangiogenic therapy.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Antiangiogenic therapy is a promising approach to controlling tumor growth by inhibiting the formation of tumor vasculature. VEGF regulates vessel formation by signaling through Flt-1 and Flk-1 receptors (20 , 21) . In these studies we established a tumor model using Her-2/neu transgenic mice in which the syngeneic N202.1A tumor cell line stably expressed the soluble form of Flt-1 (sFlt-1). It has been demonstrated that sFlt-1 binds VEGF with high affinity and selectively sequestered VEGF, impeding tumor growth (7) . Our data showed that animals inoculated with N202.1A cells expressing sFlt-1 had significantly slower tumor growth and prolonged survival times compared with animals that had received implanted parental cells. With the use of intravital microscopy, we demonstrated that animals inoculated with N202-Flt cells displayed substantially lower tumor neovascularization compared with animals with implanted parental cells, indicating that the sFlt-1 secreted by the tumor cells was the factor responsible for the inhibition of tumor vascularization. It is important to note that the presence of sFlt-1 secreted by the tumor cells did not have any adverse effects on the existing vasculature. Experiments performed with recombinant sFlt-1 also showed the capability to inhibit the tumor neovascularization, and we did not observe any adverse effects on the existing vasculature. Therefore, the use of sFlt-1 shows potential as a therapy to control tumor growth. In this regard, Kuo et al. (23) demonstrated that adenoviruses expressing different angiostatic molecules, such as angiostatin, endostatin, Flk-1, Flt-1, and neuropilin, could inhibit 50–70% of preexisting tumor growth in vivo. Interestingly, adenovirus expressing Flt-1 had the strongest antitumor effect.

We showed that higher levels of sFlt-1 in the tumor microenvironment contributed to a stronger tumor growth inhibition. Although the inoculation of N202-Flt cells prolonged the survival times of the animals compared with control animals, these animals eventually developed tumors with a slow growth rate. It is possible that the levels of sFlt-1 produced by the tumor cells were not sufficient to completely sequester all of the VEGF produced. In addition, we should not discard the possibility that other angiogenic factors were secreted that might contribute to the neovascularization of the tumor. Our data and data from others (28, 29, 30) suggest that antiangiogenic therapy is capable of retarding tumor growth; however, it is not sufficient to completely eliminate the tumor.

Previously we have shown that neu mice were tolerant to neu antigens. However, these animals have a residual low-avidity repertoire for neu antigens with antitumor activity.4 We demonstrated that to induce an antitumor immune response in neu mice it was necessary to apply multiple vaccinations with DCs pulsed with neu antigens in the presence of IL-2 and anti-OX40 mAb. At best this therapeutic intervention retarded tumor growth. Considering that immunotherapy or antiangiogenic therapy alone was not sufficient to completely eliminate the tumor in neu mice, we tested the combination of immunotherapy and antiangiogenic therapy. Our results showed that there was a cooperative effect when these monotherapies were combined. In the dorsal skin chamber model, tumors were completely eliminated. It is important to note that at the end of the experiment no neovascularization was observed and all of the tumor cells were eliminated. In the s.c. tumor model, with a larger tumor burden, it was possible to control the tumor growth. It is worth noting the difference in tumor size at different times among the animals. For example, animals that received the combination therapy had very slow tumor growth, with a tumor size of ~250 mm3 on day 60, whereas control animals with implanted N202.1A parental cells on day 35 had a tumor size of ~1800–2000 mm3, at which point they needed to be sacrificed. These studies demonstrated that although neu mice were immunotolerant to neu antigens, it was possible to manipulate the antitumor immune responses and enhance the effect of immunotherapy by combining it with another form of therapy, such as antiangiogenic therapy. With the neu mice at different stages of the disease, we are presently testing the effect of combining immunotherapy and antiangiogenic therapy with the recombinant sFlt-1 protein for preventing and eliminating spontaneous tumors.

Her-2/neu is considered a self-protein with a normal physiological effect, particularly during development (31) . Her-2/neu is expressed at low levels in a few normal tissues, such as the kidney and intestines in mice (32) . Immunohistological analysis of neu mice after immunotherapeutic intervention revealed no damage in normal tissues (e.g., kidneys or intestines) or accumulation of T-cell infiltrates in these tissues after immunization as well as no signs of an autoimmune pathology.4 Thus, the observation that the low-affinity T-cells that persist in a tolerant host do not induce an overt autoimmune response and that they recognize and kill tumor cells offers a window of opportunity to exploit these cells for the induction of antitumor responses as described in these studies.

It is well established that tumor cells secrete VEGF and other angiogenic factors (13) . Another advantage of using this combination therapy could be that the elimination of tumors with the use of immunotherapy would reduce the angiogenic activity of the tumor, thereby making the antiangiogenic therapy more effective. Taken together, our results show that there is a cooperative effect once immunotherapy and antiangiogenic therapy are combined and that this combination is superior to each monotherapy alone and can serve as a novel strategy for the treatment of solid tumors.


   ACKNOWLEDGMENTS
 
We thank Lynn Zacker for secretarial assistance.


   FOOTNOTES
 
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 by NIH Grant CA 78579 (to J. L.). Back

2 To whom requests for reprints should be addressed, at Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121. Phone: (858) 410-4190; Fax: (858) 450-3251; E-mail: jlustgarten{at}skcc.org Back

3 The abbreviations used are: TAA, tumor-associated antigen; DC, dendritic cell; IL, interleukin; mAb, monoclonal antibody; VEGF, vascular endothelial growth factor; Flt-1, VEGF receptor 1; Flk-1, VEGF receptor 2; GM-CSF, granulocyte macrophage colony-stimulating factor; FACS, fluorescence-activated cell sorting; rIL, recombinant IL. Back

4 J. Lustgarten, A. L. Dominguez, and C. Cuadros. The CD8 T cell repertoire against neu antigens in Her-2/neu transgenic mice is of low affinity with antitumor activity, submitted for publication. Back

Received 12/18/02. Revised 6/19/03. Accepted 7/21/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. van der Bruggen P., Traversari C., Chomez P., Lurquin C., De Plaen E., Van den Eynde B., Knuth A., Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science (Wash. DC), 254: 1643-1647, 1991.[Abstract/Free Full Text]
  2. Van den Eynde B. J., Boon T. Tumor antigens recognized by T lymphocytes. Int. J. Clin. Lab. Res., 27: 81-86, 1997.[Medline]
  3. Sotomayor E. M., Borrello I., Levitsky H. I. Tolerance and cancer: a critical issue in tumor immunology. Crit. Rev. Oncog., 7: 433-456, 1996.[Medline]
  4. Morgan D. J., Kreuwel H. T., Fleck S., Levitsky H. I., Pardoll D. M., Sherman L. A. Activation of low avidity CTL specific for a self epitope results in tumor rejection but not autoimmunity. J. Immunol., 160: 643-651, 1998.[Abstract/Free Full Text]
  5. Muller W. J., Sinn E., Pattengale P. K., Wallace R., Leder P. Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene. Cell, 54: 105-115, 1988.[Medline]
  6. Guy C. T., Webster M. A., Schaller M., Parsons T. J., Cardiff R. D., Muller W. J. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc. Natl. Acad. Sci. USA, 89: 10578-10582, 1992.[Abstract/Free Full Text]
  7. Hanahan D., Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 86: 353-364, 1996.[Medline]
  8. Carmeliet P., Jain R. K. Angiogenesis in cancer and other diseases. Nature (Lond.), 407: 249-257, 2000.[Medline]
  9. Feldman A. L., Libutti S. K. Progress in antiangiogenic gene therapy of cancer. Cancer (Phila.), 89: 1181-1194, 2000.
  10. Nicosia R. F. What is the role of vascular endothelial growth factor-related molecules in tumor angiogenesis?. Am. J. Pathol., 153: 11-16, 1998.[Free Full Text]
  11. Gale N. W., Yancopoulos G. D. Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. Genes Dev., 13: 1055-1066, 1999.[Free Full Text]
  12. Puri M. C., Rossant J., Alitalo K., Bernstein A., Partanen J. The receptor tyrosine kinase TIE is required for integrity and survival of vascular endothelial cells. EMBO J., 14: 5884-5891, 1995.[Medline]
  13. Yancopoulos G. D., Davis S., Gale N. W., Rudge J. S., Wiegand S. J., Holash J. Vascular-specific growth factors and blood vessel formation. Nature (Lond.), 407: 242-248, 2000.[Medline]
  14. Ferrara N. Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int., 56: 794-814, 1999.[Medline]
  15. Borgstrom P., Hillan K. J., Sriramarao P., Ferrara N. Complete inhibition of angiogenesis and growth of microtumors by anti-vascular endothelial growth factor neutralizing antibody: novel concepts of angiostatic therapy from intravital videomicroscopy. Cancer Res., 56: 4032-4039, 1996.[Abstract/Free Full Text]
  16. Prewett M., Huber J., Li Y., Santiago A., O’Connor W., King K., Overholser J., Hooper A., Pytowski B., Witte L., Bohlen P., Hicklin D. J. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res., 59: 5209-5218, 1999.[Abstract/Free Full Text]
  17. Li C. Y., Shan S., Huang Q., Braun R. D., Lanzen J., Hu K., Lin P., Dewhirst M. W. Initial stages of tumor cell-induced angiogenesis: evaluation via skin window chambers in rodent models. J. Natl. Cancer Inst. (Bethesda), 92: 143-147, 2000.[Abstract/Free Full Text]
  18. Goldman C. K., Kendall R. L., Cabrera G., Soroceanu L., Heike Y., Gillespie G. Y., Siegal G. P., Mao X., Bett A. J., Huckle W. R., Thomas K. A., Curiel D. T. Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate. Proc. Natl. Acad. Sci. USA, 95: 8795-8800, 1998.[Abstract/Free Full Text]
  19. Lollini P. L., Nicoletti G., Landuzzi L., De Giovanni C., Rossi I., Di Carlo E., Musiani P., Muller W. J., Nanni P. Down regulation of major histocompatibility complex class I expression in mammary carcinoma of HER-2/neu transgenic mice. Int. J. Cancer, 77: 937-941, 1998.[Medline]
  20. Hasumi Y., Mizukami H., Urabe M., Kohno T., Takeuchi K., Kume A., Momoeda M., Yoshikawa H., Tsuruo T., Shibuya M., Taketani Y., Ozawa K. Soluble FLT-1 expression suppresses carcinomatous ascites in nude mice bearing ovarian cancer. Cancer Res., 62: 2019-2023, 2002.[Abstract/Free Full Text]
  21. Hoshida T., Sunamura M., Duda D. G., Egawa S., Miyazaki S., Shineha R., Hamada H., Ohtani H., Satomi S., Matsuno S. Gene therapy for pancreatic cancer using an adenovirus vector encoding soluble flt-1 vascular endothelial growth factor receptor. Pancreas, 25: 111-121, 2002.[Medline]
  22. Sun X., Kanwar J. R., Leung E., Lehnert K., Wang D., Krissansen G. W. Angiostatin enhances B7.1-mediated cancer immunotherapy independently of effects on vascular endothelial growth factor expression. Cancer Gene Ther., 8: 719-727, 2001.[Medline]
  23. Kuo C. J., Farnebo F., Evan Y. Y., Christofferson R., Swearingen R. A., Carter R., von Recum H. A., Yuan J., Kamihara J., Flynn E., et al Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer. Proc. Natl. Acad. Sci. USA, 98: 4605-4610, 2001.[Abstract/Free Full Text]
  24. Torres Filho I. P., Hartley-Asp B., Borgstrom P. Quantitative angiogenesis in a syngeneic tumor spheroid model. Microvasc. Res., 49: 212-226, 1995.[Medline]
  25. Todaro G. J., Habel K., Green H. Antigenic and cultural properties of cells doubly transformed by polyoma virus and SV40. Virology, 27: 179-185, 1965.[Medline]
  26. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65: 55-63, 1983.[Medline]
  27. Inaba K., Inaba M., Romani N., Aya H., Deguchi M., Ikehara S., Muramatsu S., Steinman R. M. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med., 176: 1693-1702, 1992.[Abstract/Free Full Text]
  28. Taraboletti G., Margosio B. Antiangiogenic and antivascular therapy for cancer. Curr. Opin. Pharmacol., 1: 378-384, 2001.[Medline]
  29. Pedley R. B., El-Emir E., Flynn A. A., Boxer G. M., Dearling J., Raleigh J. A., Hill S. A., Stuart S., Motha R., Begent R. H. Synergy between vascular targeting agents and antibody-directed therapy. Int. J. Radiat. Oncol. Biol. Phys., 54: 1524-1531, 2002.[Medline]
  30. Sacco M. G., Soldati S., Mira Cato E., Cattaneo L., Pratesi G., Scanziani E., Vezzoni P. Combined effects on tumor growth and metastasis by anti-estrogenic and antiangiogenic therapies in MMTV-neu mice. Gene Ther., 9: 1338-1341, 2002.[Medline]
  31. Lee K. F., Simon H., Chen H., Bates B., Hung M. C., Hauser C. Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature (Lond.), 378: 394-398, 1995.[Medline]
  32. Nagata Y., Furugen R., Hiasa A., Ikeda H., Ohta N., Furukawa K., Nakamura H., Kanematsu T., Shiku H. Peptides derived from a wild-type murine proto-oncogene c-erbB-2/HER2/neu can induce CTL and tumor suppression in syngeneic hosts. J. Immunol., 159: 1336-1343, 1997.[Abstract]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
S. Sharma, A. L. Dominguez, S. Z. Manrique, F. Cavallo, S. Sakaguchi, and J. Lustgarten
Systemic Targeting of CpG-ODN to the Tumor Microenvironment with Anti-neu-CpG Hybrid Molecule and T Regulatory Cell Depletion Induces Memory Responses in BALB-neuT Tolerant Mice
Cancer Res., September 15, 2008; 68(18): 7530 - 7540.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. L. Dominguez and J. Lustgarten
Implications of Aging and Self-Tolerance on the Generation of Immune and Antitumor Immune Responses
Cancer Res., July 1, 2008; 68(13): 5423 - 5431.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Lustgarten, A. L. Dominguez, and C. Pinilla
Identification of Cross-Reactive Peptides Using Combinatorial Libraries Circumvents Tolerance against Her-2/neu-Immunodominant Epitope
J. Immunol., February 1, 2006; 176(3): 1796 - 1805.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. W. B. Jeffes, J. G. Zhang, N. Hoa, A. Petkar, C. Delgado, S. Chong, A. Obenaus, R. Sanchez, S. Khalaghizadeh, T. Khomenko, et al.
Antiangiogenic Drugs Synergize with a Membrane Macrophage Colony-Stimulating Factor-Based Tumor Vaccine to Therapeutically Treat Rats with an Established Malignant Intracranial Glioma
J. Immunol., March 1, 2005; 174(5): 2533 - 2543.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
A. L. Rakhmilevich, A. T. Hooper, D. J. Hicklin, and P. M. Sondel
Treatment of experimental breast cancer using interleukin-12 gene therapy combined with anti-vascular endothelial growth factor receptor-2 antibody
Mol. Cancer Ther., August 1, 2004; 3(8): 969 - 976.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cuadros, C.
Right arrow Articles by Lustgarten, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cuadros, C.
Right arrow Articles by Lustgarten, J.

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