A DNA Vaccine Targeting Angiomotin Inhibits Angiogenesis and Suppresses Tumor Growth

A DNA vaccine targeting angiomotin inhibits angiogenesis and suppresses tumor growth

Lars Holmgren*, Elena Ambrosino†, Olivier Birot*, Carl Tullus*, Niina Veitonma¨ ki*, Tetyana Levchenko*, Lena-Maria Carlson*, Piero Musiani‡, Manuela Iezzi‡, Claudia Curcio†, Guido Forni†§, Federica Cavallo†¶, and Rolf Kiessling*

*Department of Oncology and Pathology, Cancer Centre Karolinska, Karolinska Institutet, SE171 76 Stockholm, Sweden; †Department of Clinical and Biological Sciences, University of Turin, I-10043 Orbassano, Italy; ‡Aging Research Center, ‘‘Gabriele d’Annunzio’’ University Foundation, I-66013 Chieti, Italy; and §Molecular Biotechnology Center, University of Turin, I-10123 Turino, Italy

Communicated by Judah Folkman, Harvard Medical School, Boston, MA, April 20, 2006 (received for review September 28, 2005) Endogenous angiogenesis inhibitors have shown promise in pre- terization of the angiostatin receptor angiomotin (Amot) that is clinical trials, but clinical use has been hindered by low half-life in expressed in tumor and placental endothelium (15). Other recep- circulation and high production costs. Here, we describe a strategy tors for angiostatin have also been identified: for example, ATP that targets the angiostatin receptor angiomotin (Amot) by DNA synthase, the integrin ␣v␤3, and c-Met (16). Amot is a membrane- vaccination. The vaccination procedure generated antibodies that associated protein that mediates angiostatin inhibition of endothe- detected Amot on the endothelial cell surface. Puriﬁed Ig bound to lial migration and tube formation in vitro (17–20). A role of Amot the endothelial cell membrane and inhibited endothelial cell mi- in cell motility is also indicated by the finding that Amot-deficient gration. In vivo, DNA vaccination blocked angiogenesis in the mouse embryos exhibit a migratory defect in the anterior visceral matrigel plug assay and prevented growth of transplanted tumors endoderm at embryonic day 7.5 (21). High Amot mRNA levels also for up to 150 days. We further demonstrate that a combination of have been correlated to poor survival in breast cancer patients (22). DNA vaccines encoding Amot and the extracellular and transmem- Therefore, the functional role of Amot as an angiostatin receptor brane domains of the human EGF receptor 2 (Her-2)͞neu oncogene and its expression in angiogenic vessels makes it a possible target for inhibited breast cancer progression and impaired tumor vascular- antiangiogenic therapy. ization in Her-2͞neu transgenic mice. No toxicity or impairment of The development of active immunotherapy of cancer has been normal blood vessels could be detected. This work shows that DNA hampered by limited success in the clinic, related to difficulties of vaccination targeting Amot may be used to mimic the effect of breaking tolerance against weak self-antigens on tumor cells and angiostatin. the genetic variability of tumor cells resulting in immunologic escape variants. This limited clinical efficacy has spurred the cancer vaccines ͉ neoplasia ͉ neovascularization ͉ breast cancer ͉ development of combination approaches in which tumor vaccines angiostatin are combined with cancer therapies that target the tumor stroma, which is more genetically stable. Recent evidence has shown the he expansion of the circulatory system by the mechanism of feasibility of targeting molecules that are expressed by angiogenic Tangiogenesis is a driving force behind diseases such as cancer, endothelium [for example, VEGF-R2 (23, 24)] and that synergy macular degeneration, and atherosclerosis (1). Inhibition of the between tumor immunotherapy and antiangiogenic therapy can be signaling pathways underlying pathological angiogenesis therefore achieved (25). Here, we have used DNA vaccination to break offers a possible way of intervening with the progress of the disease. tolerance and invoke an immune response against Amot. Our Indeed, recent evidence has shown that treatment with antibodies approach generates Amot-specific Ig, resulting in inhibition of targeting VEGF in combination with chemotherapy prolongs life in angiogenesis and tumor growth without detectable toxicity and thus patients with metastatic colon, breast, and lung cancer (2, 3). At circumventing the problems experienced with endogenous angio- least in the case of tumor growth, inhibition of one signaling genesis inhibitors. pathway may not be sufficient, because compensatory pathways Results may be activated when VEGF signaling is inhibited (4, 5). Tumor angiogenesis may therefore involve a coordinate expression of a Amot Is Expressed in Endothelial Cells During Angiogenesis. For DNA variety of angiogenic factors, such as IL-8, basic fibroblast growth vaccination, we used cDNA encoding the human p80 isoform of factor (bFGF), and others (6). The implication of these findings is Amot inserted into the pcDNA3 vector (Fig. 1A). Transient trans- that therapies that target more than one pathway or target endo- fection of this vector into HeLa cells yielded an expected 80-kDa thelial functions downstream of these pathways could improve the band similar to that of endogenous p80 Amot in bovine capillary efficacy of antiangiogenic therapies. endothelial cells (Fig. 1B). To verify that the target protein was The induction of tumor angiogenesis is likely regulated by a present in endothelial cells during angiogenesis, we analyzed Amot balance between endogenous proangiogenic and antiangiogenic levels in lobular mammary carcinomas that spontaneously arise in BALB͞c mice that are transgenic for the human EGF receptor 2 factors (7). At least 16 endogenous angiogenesis inhibitors have ͞ been isolated that exhibit antiangiogenic and tumor suppressive (Her-2) neu oncogene (BALB-neuT mice) (26). p80 Amot was activity (8, 9). One of the earliest inhibitors to be reported, clearly detectable by Western blot analysis of two individual car- angiostatin, was shown to be specific for endothelial cells and could cinomas from 22- and 24-week-old BALB-neuT mice. However, maintain dormancy of established metastases in vivo (10, 11). Amot was not detectable in normal breast tissue from a virgin However, the serum half-life of angiostatin in patients is Ϸ3h,

making it necessary to administer high amounts of angiostatin at Conﬂict of interest statement: L.H. and R.K. are supported by grants from BioInvent frequent intervals (12). Thus, the pharmacodynamics of angiostatin International (Lund, Sweden). and other inhibitors such as endostatin constitute a major obstacle Abbreviations: Amot, angiomotin; bFGF, basic ﬁbroblast growth factor; Her-2, human EGF for the use of these agents in cancer patients (13, 14). To design receptor 2; KO, knockout; MAE, mouse aortic endothelial cell; PECAM, platelet endothelial alternative molecules more suited for antiangiogenic therapy, it is cell adhesion molecule. of importance to identify the receptors that mediate the antiangio- ¶To whom correspondence should be addressed. E-mail: [email protected]. genic effect. We have previously reported the cloning and charac- © 2006 by The National Academy of Sciences of the USA

9208–9213 ͉ PNAS ͉ June 13, 2006 ͉ vol. 103 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603110103 Downloaded by guest on October 1, 2021 Fig. 1. Expression of Amot in angiogenic vessels. (A) The human p80 isoform of Amot was cloned into the pCDNA3 vector, which was then used for DNA vaccination in mice. (B) Western blot analysis of Amot protein levels. Bovine capillary endothelial cells (BCE) expressed both p80 and p130 isoforms of Amot. Transient transfection with the pcDNA3-Amot construct into HeLa cells yielded a band corresponding to 80 kDa (Amot), whereas vector control was negative. The TUBO mouse breast cancer cell line is derived from BALB-neuT transgenic mice and did not express detectable levels of Amot. In contrast, Fig. 2. Amot DNA vaccination inhibits tumor growth. (A) WT BALB͞c mice were total lysates from tumors harvested at 22 and 24 weeks after birth from vaccinated twice with either Amot or control vector pcDNA3 21 and 7 days before BALB-neuT transgenic mice showed detectable expression of p80 Amot (BALB- s.c. challenge with the TUBO mouse breast cancer cell line. In three independent neuT). Breast tissue harvested from a normal virgin female mouse was below experiments, Amot vaccination suppressed tumor growth for Ͼ150 days in 12 of detection (Br). (C) The top row shows a vessel positive for Amot and for the 18 treated mice; in two mice, tumors recurred 150 days after challenge. (B)To endothelial marker PECAM in a tumor derived from a BALB-neuT mouse. The determine whether the induction of the antitumor activities depended on CD4ϩ two middle rows show angiogenic vessels activated by bFGF in the in vivo T cells, we conducted an in vivo T cell depletion of this subset during DNA matrigel plug assay that were strongly positive for Amot as shown by immu- vaccination. Mice were treated with either anti-CD4 or control IgG before and nofluorescence analysis using polyclonal anti-Amot antibodies. Functional during vaccinations with Amot or empty vector. The antitumor effect of vacci- vessels were visualized by i.v. FITC-dextran injection before euthanization of nation against Amot was abrogated in the CD4-depleted mice. The size of tumors the animal. The bottom row shows vessels of the normal stroma that did not in Amot-vaccinated mice receiving control IgG or anti-CD4 Ig was significantly stain positively for Amot. (Scale bars, 100 ␮m.) different between 7 and 35 days after challenge (n ϭ 5; P Ͻ 0.0001, unpaired Student t test). (C) Amot plasmid vaccination was also performed in ␮-chain- deficient BALB͞c mice lacking B cells (B cell KO mice). No significant difference in female of the same age (Fig. 1B). Immunofluorescence analysis of the growth of TUBO tumor cells could be detected between Amot- and control the cellular localization of Amot expression in these tumors showed pcDNA3 plasmid-vaccinated mice. that positive staining overlapped with that of the endothelial marker PECAM (platelet endothelial cell adhesion molecule) (Fig. 1C). Furthermore, bFGF-induced vessels in the in vivo angiogenesis Amot Vaccination Inhibits Tumor Growth. To break tolerance against matrigel plug assay were positive for Amot, whereas surrounding Amot and consequently activate an Amot-specific immune re- stromal tissue was negative (Fig. 1C). This finding is in accordance sponse, we vaccinated BALB͞c mice by intramuscular injection with our previously published data that showed that Amot is followed by electroporation with pcDNA3-Amot or empty vector as primarily expressed in cytotrophoblasts of the placenta as well as in a negative control as described in ref. 27. The use of a human Amot endothelial cells of angiogenic tissues (15). The spatiotemporal DNA construct was motivated by the ability of xenogeneic proteins restricted expression of Amot by angiogenic vessels is therefore to break immunological tolerance against self-antigens (28). The consistent with a candidate target for antiangiogenic vaccination. mice were vaccinated twice, with a 2-week interval. To assess the MEDICAL SCIENCES

Holmgren et al. PNAS ͉ June 13, 2006 ͉ vol. 103 ͉ no. 24 ͉ 9209 Downloaded by guest on October 1, 2021 Fig. 3. Combined vaccination with Amot and EC-TMneu plasmids prevents tumor onset in BALB-neuT transgenic mice. (A) BALB-neuT transgenic mice were vaccinated twice at 6 and 8 weeks (before the angiogenic switch). Anti-Amot vaccination as a single therapy did not significantly delay the onset of tumor formation. (B) A similar result was observed when mice were vaccinated at 10 and 12 weeks (after the angiogenic switch). The EC-TMneu DNA vaccine targeting the Her-2͞neu oncogene in the tumor cells delayed tumor progression. However, the combination of the Amot EC-TMneu vaccinations prevented tumor formation in 80% of transgenic mice for Ͼ70 weeks. (pcDNA3 vs. pcDNA3-Amot, P Ͻ 0.01; EC-TMneu vs. Amot, P Ͻ0.0001; EC-TMneu vs. Amot plus EC-TMneu, P Ͻ 0.0001; n ϭ 5 animals). (C) Whole-mount analysis of mammary glands from vaccinated mice at 14 and 18 weeks of age. The mice were vaccinated at the time points indicated in A. Mammary glands were stained with ferric hematoxylin to visualize normal mammary structures and neoplastic formation in the whole gland (arrowheads indicate neoplastic foci). No significant reduction in mammary neoplastic lesions, detected as dark masses (the large, oval-shaped dark area is the lymph node, L), was observed in pcDNA3-vaccinated mice. In EC-TMneu-vaccinated mice, neoplastic lesions are reduced, and they are absent in mice vaccinated with both Amot and EC-TMneu plasmids. (Scale bar, 2 mm.) (D) Image analyses of the tumor burden of mammary glands from animals vaccinated with vector control, Amot, EC-TMneu, or a combination as indicated in the graph. Sixteen tumors from four animals in each group were analyzed at 14 weeks, and 20 tumors from five animals were analyzed at 18 weeks (***, P Ͻ 0.0001 compared with control).

protective response elicited by vaccination, mice were challenged protection elicited by Amot plasmid vaccination, ␮-chain-deficient with a lethal dose of TUBO carcinoma cells. TUBO is a cloned cell BALB͞c mice lacking B cells [B cell knockout (KO) mice] were line established from a mammary carcinoma of BALB-neuT mice vaccinated with pCDNA3-Amot or empty vector. In these mice, (29). TUBO cells display a high cytoplasmic and membrane ex- Amot vaccination did not inhibit tumor growth (Fig. 2C). There- pression of Her-2͞neu but no detectable levels of Amot (Fig. 1B). fore, we conclude that B cells, and probably antibodies, are required Injection of TUBO cells in control-vaccinated animals resulted in for the observed antitumor effect. rapidly growing s.c. tumors in all (n ϭ 13) mice, which were Transgenic tumor models differ from tumor transplantation euthanized 40–60 days after injection. Suppression of tumor models in that the tumor cells are derived from normal cells that growth was detected in 12 of 18 mice electroporated with the progress through multiple stages in the progression to neoplasia. To pcDNA3-Amot construct in three independent experiments. In study the effect of Amot, we used the BALB-neuT transgenic breast Amot-vaccinated animals, only a minority (6 of 18) of the mice cancer model system. These mice are transgenic for the rat trans- developed tumors that grew progressively, albeit at a slower rate forming Her-2͞neu oncogene driven by the mouse mammary than in the control-vaccinated mice. Moreover, in one of three tumor virus promoter (which confers expression to the mouse performed experiments, mice were monitored for tumor growth for mammary epithelium). Tumors progress through the following 200 days after TUBO challenge (Fig. 2A). In this experiment, Amot stages: at 6–7 weeks, female BALB-neuT mice develop atypical vaccination induced a state of no growth for Ͼ150 days, after which ductal hyperplasia; the angiogenic switch occurs at weeks 8–10, and tumors recurred in two of five animals. To determine whether CD4 mammary lesions progress to invasive cancers at weeks 23–30. The T cells were responsible for the antitumor activities, we conducted mice have to be killed between weeks 27 and 33, when the tumors an in vivo T cell depletion assay in DNA-vaccinated mice. Mice were exceed the size of 1.5 cm in mean diameter (30, 31). treated with anti-CD4 or control Ab before and during vaccinations To assess the effect of pcDNA3-Amot vaccination on the angio- with Amot or empty vector. The antitumor effect of vaccination genic switch and tumor progression, BALB-neuT mice were vac- against Amot was abrogated in CD4-depleted mice (Fig. 2B), cinated either before (6–8 weeks) or after (10–12 weeks) the onset demonstrating that CD4 T cell help was necessary for the induction of tumor angiogenesis. Although Amot plasmid vaccination had no of antitumor activity. To examine the role of antibodies in the significant effect on tumor progression (Fig. 3), significant alter-

9210 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603110103 Holmgren et al. Downloaded by guest on October 1, 2021 Fig. 4. Amot vaccination inhibits angiogenesis in vivo. Tumor-induced angiogenesis was analyzed by the matrigel plug assay. Matrigel is a solubilized basement membrane preparation from Engelbreth–Holm–Swarm mouse sarcoma that polymerizes at 37°C. (A) Angiogenesis was induced by suspending 75,000 TUBO cells into 500 ␮l of matrigel and injecting s.c. in vaccinated mice as indicated. Seven days later, matrigel plugs were extracted, sectioned, and stained with PECAM antibodies to visualize vessels, and cell nuclei were visualized by DAPI staining. (B) Bar diagram shows vascular density of matrigel plugs stained for the endothelial marker PECAM (**, P Ͻ 0.001 compared with control). (C) bFGF (200 ng͞ml) was added to the matrigel before injection into nonvaccinated or mice vaccinated with Amot or EC-TMneu plasmids as indicated. (D) Bar diagram shows vascular density of matrigel plugs stained for the endothelial marker PECAM (**, P Ͻ 0.001 compared with control). (Scale bars, 100 ␮m.)

ations were evident in the vasculature of vaccinated lesions. The and D). No negative effect on the vasculature of the stromal vessels percentage of the area occupied by blood vessels was lower in adjacent to the matrigel plug or on the vessels of retinas isolated Amot-vaccinated tumors (4.38 Ϯ 1.31%) as compared with control from vaccinated mice could be detected (data not shown; see Fig. tumors (7.12 Ϯ 0.95%; P Ͻ 0.0005). 6, which is published as supporting information on the PNAS web To test the potential synergistic effect of targeting both the site). endothelial and tumor compartments, we combined vaccination against Amot with a plasmid encoding the extracellular and trans- Vaccination Generates Amot-Reactive Antibodies That Inhibit Endo- membrane domains of Her-2 (EC-TMneu). Single vaccinations with thelial Cell Migration. Because the antitumor effect was lost in B cell the EC-TMneu plasmid significantly delayed tumor growth, but, KO animals, we investigated whether our DNA vaccination strategy eventually, tumors progressed in 80% of the mice, which is in generated antibodies that reacted with Amot. Sera were harvested accordance with our previously published results (Fig. 3B) (27). from BALB͞c mice vaccinated two times (with a 2-week interval) However, when EC-TMneu was combined with Amot vaccination, with the Amot construct. Binding to recombinant mouse Amot was 80% of the treated mice were tumor-free for Ͼ70 weeks (Fig. 3B). analyzed by ELISA (Fig. 5A). The results showed that vaccination DNA-vaccinated BALB-neuT mice were killed at the age of 14 and with human Amot cDNA resulted in the generation of Ig that 18 weeks. Whole-mount image analysis of mammary glands from cross-reacted with the mouse orthologue. Sera from all control mice pcDNA3-Amot-vaccinated mice revealed no significant inhibition were negative. Cell surface binding of purified Ig fractions was of tumor growth as compared with control animals (Fig. 3 C and analyzed by incubation with live Amot-transfected mouse aortic D). In the EC-TMneu-vaccinated mice, the number of neoplastic endothelial (MAE) cells. A distinct binding could be detected at lesions was similar to that in the control mice, but the lesions were intercellular junctions with Ig from Amot vaccinated animals, markedly reduced in size. Interestingly, the combination of the whereas no reactivity was detected with control Ig (Fig. 5B). This Amot and EC-TMneu plasmids resulted in mammary glands with an reactivity was specific for Amot; MAE vector-transfected cells apparent normal phenotype with few neoplastic lesions (Fig. 3 C lacking Amot expression did not exhibit any positive staining with and D). Amot or control Ig. Angiostatin specifically inhibits migration of Amot-transfected Amot Vaccination Inhibits Angiogenesis in Vivo. To investigate the cells in the modified Boyden chamber assay (15, 20). We therefore antiangiogenic effect of Amot DNA vaccination, we injected Amot- tested whether the Amot-reactive Ig generated by DNA vaccination or vector-vaccinated animals with 500 ␮l of matrigel containing could mimic the effect of angiostatin. We first tested whole sera and either 75,000 TUBO breast cancer cells or 200 ng͞ml bFGF s.c. into could show that dilution up to 1,000 times could completely block the lower abdomen as described in ref. 32. The mice were eutha- basal migration of Amot-expressing MAE cells (Fig. 5C). We then nized 7 days later, and the plugs were harvested and analyzed for used purified Ig to test the inhibition of bFGF- and VEGF- neovessel formation. Vessel in-growth was analyzed by isolectin B4 stimulated endothelial migration (Fig. 5 D and E). Significant and PECAM staining. Tumor-induced angiogenesis was signifi- inhibition of migration could be achieved with as little as 0.2 ␮g͞ml cantly inhibited in Amot-vaccinated mice (Fig. 4A). Furthermore, total Ig. These results thus show that DNA vaccination generates the matrigel plugs containing bFGF harvested from Amot- antibodies that mimic the effect of angiostatin in vitro. vaccinated animals did not differ significantly from the controls lacking bFGF (Fig. 4 C and D). Animals were also vaccinated with Discussion the EC-TMneu construct, which did not affect the bFGF-induced In this report, we describe a strategy that circumvents the problems in-growth of vessels into the matrigel (Fig. 4C). The microvascular of low half-life in circulation and expensive production of the density of all plugs was also assessed by scoring PECAM-positive endogenous angiogenesis inhibitor angiostatin. We report that cells under high-power magnification with similar results (Fig. 4 B DNA vaccination targeting the angiostatin receptor Amot mimics MEDICAL SCIENCES

Holmgren et al. PNAS ͉ June 13, 2006 ͉ vol. 103 ͉ no. 24 ͉ 9211 Downloaded by guest on October 1, 2021 Fig. 5. DNA vaccination generates Amot-specific antibodies that inhibit endothelial migration. (A) Sera from vaccinated mice were analyzed for the presence of antibodies that are reactive to mouse Amot as analyzed by ELISA. (B) Purified Ig from sera of Amot-vaccinated animals bound to the surface of live MAE cells transfected with p80 Amot. (Scale bar, 10 ␮m.) (C) Sera from Amot-vaccinated animals inhibited specifically the migration of MAE cells transfected with Amot. (D and E) Purified Ig abolishes migration stimulated by bFGF (D) or VEGF (E) in the Boyden chamber assay (***, P Ͻ 0.001 compared with control).

the effect of angiostatin and inhibits angiogenesis and tumor growth with the TUBO cell line and showed a dramatic synergistic effect in mice. when used together with a vaccine targeting the Her-2 oncogene in Recent evidence has shown that breaking immune tolerance BALB-neuT transgenic mice. This two-compartment therapy could against angiogenic-associated molecules through active immuno- prevent tumor formation for Ͼ70 weeks in 80% of the mice, which therapy that targets endothelial cell-specific proteins provides an is in line with the findings of Nair et al. (25), who reported an innovative strategy to block tumor angiogenesis (33). One major improved antitumor effect by combining endothelial and tumor advantage of this strategy, compared with targeting tumor antigens antigens. Furthermore, an early phase clinical study recently directly, is that the target cells are readily accessible to the blood- showed the added effect of combining herceptin (which targets stream. Therefore, vaccination approaches that break immune Her-2) with antibodies targeting VEGF in Her-2͞neu-positive ʈ tolerance may deliver cytotoxic T lymphocytes and antibodies breast cancer. Amot vaccination used as a single therapy affected directly to the activated endothelial cells in the tumor. We hypoth- the vascular density of the mammary lesions but did not show any esized that a DNA vaccination approach targeting Amot could inhibitory effect on tumor growth in the BALB-neuT mice. The mimic the effect of angiostatin. First, we show that Amot expression explanation for this finding is not yet clear. It is possible that these is up-regulated in angiogenic vessels in the tumors of BALB-neuT tumors also depend on other ways to expand their microcirculatory mice. Second, Amot is highly expressed during bFGF-driven an- network (e.g., intussusception, vessel cooption, or vascular mim- giogenesis in the matrigel assay. Importantly, the surrounding icry). Another explanation is that we could not generate high nonangiogenic stroma did not express detectable levels of Amot. enough antibody titers in these mice to efficiently suppress tumor These data argue that Amot is up-regulated during angiogenesis growth. and therefore is a target for antiangiogenic vaccination. We argue that the observed antiangiogenic and antitumor effect Several approaches have been used to vaccinate against targets is antibody-dependent because (i) B cell KO mice were not pro- on tumor endothelial cells. One study has used cross-immunization tected against tumor challenge and (ii) antibodies generated by with xenogeneic endothelial cells as a vaccine to induce endothelial- DNA vaccination bound Amot and could inhibit endothelial cell specific immune responses against tumor vasculature (34). Others migration in vitro. In addition, the importance of CD4 T cell help have immunized against VEGF-R2 with in vitro-generated den- for the induction of tumor protection is compatible with the antibody dependency. These findings provide the rationale for dritic cells pulsed with soluble VEGF-R2 or with a vaccine con- generating antibodies that bind to Amot and thereby could inhibit sisting of attenuated Salmonella typhimurium expressing VEGFR-2 angiogenesis. The use of therapeutic antibodies would potentially cDNA (23, 24). These immunization strategies generated VEGF- ϩ be a marked advantage, because generation of an efficient antibody R2-specific neutralizing antibody and͞or CD8 cytotoxic T cell titer would not depend on breaking tolerance against Amot, and the responses, thereby demonstrating that tolerance to self-VEGF-R2 dosage of antibody could therefore be more easily controlled. It antigen was broken. We used the method of DNA vaccination where intramuscular injections of pDNA were followed by electroporation. This ap- ʈPegram, M. D., Yeon, C., Ku, N. C. Gaudreault, J., Durna, L. & D. J., Slamon (2004) Breast proach protected a majority of the vaccinated mice from challenge Cancer Res. Treat. 88, Suppl. 1, S124 (abstr.).

9212 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0603110103 Holmgren et al. Downloaded by guest on October 1, 2021 should be noted, however, that although antibodies appear as the Supporting Text, which is published as supporting information on major component of the observed antiangiogenic effect, it does not the PNAS web site. exclude an involvement of CD4 or CD8 T cells. A major concern when activating the immune system to recog- Plasmids and in Vivo Electroporation. pcDNA3 and Amot plasmid nize self-antigens is possible toxicity. We have analyzed the normal and EC-TMneu plasmids were produced as previously described. vasculature of the retinas of animals vaccinated against Amot, and Twenty-five micrograms of plasmid in 20 ␮l of 0.9% NaCl with 6 we could find no significant changes in animals at 16 weeks or 70 mg͞ml polyglutamate was injected bilaterally into the tibial muscle weeks after vaccination. In addition, adjacent normal stroma in the of the hind legs of anesthetized mice. Two 25-ms transcutaneous matrigel plug assay did not express Amot and remained functional electric pulses were generated by a T820 electroporator (BTX, San as analyzed by FITC-dextran injection. Others have also reported Diego) with a field strength of 375 V͞cm, as described in detail in that antiangiogenic vaccination targeting VEGF-R2 inhibits tumor refs. 27 and 36. In BALB͞candBcellKOBALB͞c mice, plasmids angiogenesis without affecting the wound-healing process (23). were electroporated 21 and 7 days before TUBO cell challenge, Angiostatin was originally identified by its ability to maintain whereas transgenic BALB-neuT mice were electroporated with dormancy of lung metastasis in the Lewis lung carcinoma mouse plasmids at 10 and 12 weeks of age. model system (10, 11). Our observations that some tumors recur after 150 days of latency show that, similar to angiostatin, Amot Whole-Mount Image Analyses. A detailed description of the staining DNA vaccination can maintain dormancy of established tumors. procedure of tumor imaging and vessel quantification can be found The escape from dormancy may be explained by the fact that either in Supporting Text. the tumor has generated resistance to Amot therapy or that a continuous vaccination regimen is required for maintaining inhibition of tumor angiogenesis. In conclusion, our report provides the In Vivo Matrigel Plug Angiogenesis Assay. Five hundred microliters of matrigel (BioSite, Ta¨by,Sweden) mixed with either 75,000 rationale for targeting Amot to inhibit tumor angiogenesis, which ͞ may be achieved through either DNA vaccination or the use of TUBO cells or 200 ng ml human bFGF (PeproTech, Rocky Hill, therapeutic mAbs (35). NJ) were injected s.c. in the midventral abdominal region of BALB͞c mice as described by Passaniti et al. (32). Migration assays Materials and Methods were performed in a modified Boyden chamber as described in ref. Cell Lines. TUBO is a cloned cell line established in vitro from 15. A more detailed description of this assay can be found in lobular carcinomas that arose in a BALB-neuT mouse. TUBO and Supporting Text. HeLa cells were cultured in DMEM (BioWhittaker) with 20% FBS (Life Technologies, Paisley, Scotland). MAE cells stably expressing The authors dedicate this paper to the memory of Carl Tullus (1979– mouse p80 Amot (15) were cultured in DMEM (Sigma) and 10% 2006). We thank Kristina Berggren (BioInvent International, Lund, FCS (GIBCO). Sweden) for expert help with the Amot ELISA. This work was supported by grants from the Italian Association for Cancer Research, the Italian Ministries for the Universities and Health, and the Center of Excellence Tumor Experiments in Mice. WT BALB͞cnAnCr (BALB͞c) on Aging (University of Chieti, Chieti, Italy); grants from the Swedish (Charles River Breeding Laboratories) and BALB͞cmiceKOfor ␮ Cancer Society, the Cancer Society of Stockholm, the Swedish Research the Ig -chain (B cell KO mice) were kindly provided by T. Council, BioInvent International, and the Karolinska Institutet (to L.H.); Blankenstein (Institute of Immunology, Charite Campus Benjamin a postdoctoral stipend from the Wenner–Gren Foundation (to O.B.); Franklin, Berlin). BALB-neuT mice transgenic for transforming rat and grants from the Swedish Cancer Society, the Cancer Society of Her-2͞neu (Charles River Breeding Laboratories) were generated Stockholm͞King Gustaf V Jubilee Fund, the European Community, the as described in ref. 30. Mice were treated according to European Swedish Medical Research Council, BioInvent International, and the Community guidelines. Additional information can be found in National Institutes of Health (CA102280) (to R.K.).

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