Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer

Abstract

Endoglin (CD105), a co-receptor in the TGF-β receptor complex, is over-expressed on proliferating endothelial cells in the breast tumor neovasculature and thus offers an attractive target for anti-angiogenic therapy. Here we report the anti-angiogenic/anti-tumor effects achieved in a prophylactic setting with an oral DNA vaccine encoding murine endoglin, carried by double attenuated Salmonella typhimurium (dam ⁻, AroA ⁻) to a secondary lymphoid organ, i.e., Peyer’s patches . We demonstrate that an endoglin vaccine elicited activation of antigen-presenting dendritic cells, coupled with immune responses mediated by CD8⁺ T cells against endoglin-positive target cells. Moreover, we observed suppression of angiogenesis only in mice administered with the endoglin vaccine as compared to controls. These data suggest that a CD8⁺ T cell-mediated immune response induced by this vaccine effectively suppressed dissemination of pulmonary metastases of D2F2 breast carcinoma cells presumably by eliminating proliferating endothelial cells in the tumor vasculature. It is anticipated that vaccine strategies such as this may contribute to future therapies for breast cancer.

Introduction

Angiogenesis, the growth of new capillary blood vessels from preexisting vasculature, is an essential feature of tumor growth and metastasis. In fact, anti-angiogenenic therapy, originally proposed by Folkman more than 30 years ago [10] has become a most attractive concept, receiving ever increasing attention during the last decade [12, 14, 34, 41]. The goal of this approach has been to deliver anti-angiogenic agents to appropriate targets in the tumor vasculature to eliminate or suppress blood supply to tumors, resulting in their ablation or growth suppression without seriously disturbing blood flow to normal tissues [3]. Several approaches have been reported to suppress murine tumor growth and metastasis through anti-angiogenesis by targeting specific molecules such as vascular endothelial growth factor receptor 2 (VEGF-R2) [31]. Our laboratory also demonstrated that a survivin-based oral DNA vaccine, coexpressing the chemokine CCL21, induced effective suppression of angiogenesis by triggering potent CTLs against tumor cells and proliferating endothelial cells expressing survivin, resulting in the suppression or eradication of metastases in a murine tumor model [47].

Endoglin (CD105) is a 180-kDa homodimeric transmembrane glycoprotein, primarily expressed on endothelial cells. It acts as an auxiliary protein that interacts with the ligand-binding receptors of multiple members of the transforming growth factor beta (TGF-β) superfamily [4]. Studies have suggested that endoglin offers an excellent target for anti-angiogenic therapy since it is over-expressed on proliferating endothelial cells in blood vessels of tumor tissue. In fact, endoglin and its ligand, TGF-β, are significant modulators of angiogenesis [16, 19]. Moreover, endoglin expression on endothelial cells is up-regulated by TGF-β and hypoxic conditions [40]. In solid tumors such as breast carcinoma, endoglin is almost exclusively expressed on endothelial cells of both peri- and intratumoral blood vessels and on tumor stromal components [5]. Furthermore, a monoclonal endoglin Ab was reported to react with small and immature tumor blood vessels in prostate and breast cancer [46], and to strongly stain endothelial cells, but not smooth muscle cells associated with blood vessels within all tumor lesions investigated [7]. In addition, quantifying tumor microvessel density with this same Ab also proved to be an independent prognostic parameter for survival of colorectal cancer patients [45]. Taken together, these data suggest the involvement of endoglin in tumor angiogenesis and point it as a candidate for vascular targeting in tumor therapy, especially since endoglin is not detectable in blood vessels within normal tissues [18, 20, 38]. In fact, Seon et al. [42] successfully applied an anti-human endoglin immunotoxin to inhibit growth of subcutaneous MCF7 human breast carcinoma in SCID mice. Recently, synergy was demonstrated between endoglin mAbs and TGFβ in growth suppression of human endothelial cells in vitro, suggesting that TGF-β plays a key role by synergistically enhancing the anti-angiogenic activity of such antibodies [43]. In addition, endoglin-based xenogeneic vaccination was shown to effectively elicit both protective and therapeutic anti-tumor immunity in several mouse tumor models [44]. Preliminary data obtained in our laboratory suggested that a DNA vaccine encoding the entire murine endoglin gene suppressed angiogenesis and pulmonary metastases of murine breast carcinoma [29].

Here, we demonstrate that a DNA vaccine encoding murine endoglin was delivered orally by attenuated Salmonella typhimurium to secondary lymphoid organs such as Peyer’s patches (PPs). This vaccine overcame peripheral T cell tolerance and induced a robust CD8⁺ T cell mediated immune response that inhibited angiogenesis, resulting in suppression of pulmonary breast tumor metastases and increased life-span of tumor bearing, syngeneic BALB/c mice in a prophylactic setting.

Materials and methods

Animals, bacterial strains, and cell lines

Female BALB/c mice, 6–8 weeks of age, were purchased from the Scripps Research Institutes (La Jolla, CA, USA) Rodent Breeding Facility. All animal experiments were performed according to the NIH Guides for the Care and Use of Laboratory Animals.

The double-attenuated S. typhimurium strain RE88 (dam ⁻; AroA ⁻) was obtained from Remedyne Inc. (Santa Babara, CA, USA).

The murine D2F2 breast cancer cell line was kindly provided by Dr. W-Z. Wei (Karmanos Cancer Institute, Detroit, MI, USA) and cultured as previously described [49]. The murine high endothelial venule cell line (HEVc) was a gift from Dr. J.M. Cook-Mills (University of Cincinnati, OH, USA). The mEndo⁺–D2F2 cell, were obtained by transfecting vector encoding full-length endoglin into D2F2 cells with DMRIE-C Reagent (Invitrogen, Carlsbad, CA, USA). Endoglin positive cells were purified by FACSsort and maintained in selection medium containing Neomycin (1 mg/ml).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted with the RNeasy mini kit or RNeasy tissue kit (Qiagen, Valencia, CA, USA) from D2F2, HEVc cells or normal mouse spleen or liver. Reverse transcription was performed with 1 μg of total RNA followed by PCR with specific endoglin primers: TCG ATA GCA GCA CTG GAT (forward), and ATC TAG CTG GAC TGT GAC (reverse). Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as control.

Immunohistochemistry

Frozen sections were fixed and stained with anti-endoglin Ab (BD PharMingen, San Diego, CA, USA), followed by treatment with biotinylated anti-rat IgG Ab and HRP-conjugated streptavidin (Vector Laboratories, Inc, Burlingame, CA, USA). The DAB substrate (Sigma, St. Louis, MO, USA) was added and slides were examined microscopically.

Vector construction and Western blotting

Full-length murine endoglin was cloned from tumor tissue. The vector was constructed based on the pCMV vector (Invitrogen). The empty vector served as a control. Western blot analysis was performed with transiently transfected COS-7 cell lysates using monoclonal rat anti-mouse endoglin Ab (Cymbus Biotechnology, UK).

Transformation of attenuated S. typhimurium and expression of endoglin in vivo

Attenuated S. typhimurium (dam ⁻; AroA ⁻) were transformed with DNA vaccine plasmids by electroporation [26]. Freshly prepared bacteria (1×10⁸) were mixed with plasmid DNA (2 μg) on ice in a 0.2-cm cuvette and electroporated at 2.5 kV, 25 μF, and 200 Ω. Resistant colonies harboring the vaccine vectors were cultured and stored at −70°C after confirmation of their coding sequence.

Peyer’s Patches were dissected from the mouse small intestines [22] 24 h after vaccination. Frozen sections were fixed, blocked and stained with unlabeled anti-endoglin Ab and Alexa 568-conjugated goat-anti-rat Ab (Molecular Probes), followed by biotin labeled anti-CD11c Ab and streptavidin-Alexa 488 (Molecular Probe). Slides were air dried and mounted with Vectashield (Vector Laboratories) and analyzed by confocal microscopy with a Zeiss Axioplan/BioRad MRC 1024 confocal microscope.

Oral immunization and tumor challenge

BALB/c mice were divided into three experimental groups (n=8) and immunized three times at 1 week intervals by gavage with 100 μl 5% sodium bicarbonate containing approximately 1×10⁸ double attenuated S. typhimurium harboring either empty vector or murine endoglin vector (mEndoglin). All mice were challenged by i.v. injection of 1.5×10⁵ D2F2 murine breast carcinoma cells 1 week after the last immunization. Mice were monitored and sacrificed as indicated.

In vivo depletion of CD4⁺ or CD8⁺ T cell populations

mAbs against CD8 (2.43: rat mAb, IgG_2b) or CD4 (GK1.5: rat mAb, IgG_2b) were purchased from The National Cell Culture Center (NCCC, Minneapolis, MN, USA). Immunized mice were injected i.v. with anti-CD4 or anti-CD8 mAb (0.5 mg/mouse) 1 day before D2F2 tumor cell challenge, followed by weekly i.p. injection of mAbs until sacrifice.

In vitro cytotoxicity assay

Cytotoxicity was performed by a standard [³⁵S] release assay [24, 25]. Splenocytes were prepared from immunized mice 14 days after tumor cell challenge, and re-stimulated in vitro for 4 days on a monolayer of irradiated (1,000 Gy) and mitomycin C-treated (80 μg/10⁷ cells, 45 min at 37°C) mEndo⁺-D2F2 cells. Viable lymphocytes were separated by Lympholyte-M (Cenderlane, ON, Canada) gradient centrifugation and mixed at different ratios with [³⁵S] methionine-labeled target cells for 5 h. Supernatants (100 μl) were harvested and measured in a mixture with scintillation fluid. Percent specific lysis was calculated by the formula; [(E−S)/(T−S)]×100, where E is the average experimental release, S the average spontaneous release, and T the average total release.

Flow cytometric analysis

T cell activation was assessed by staining freshly isolated splenocytes from vaccinated mice with FITC-labeled anti-CD8 Ab in combination with PE-conjugated anti-CD28 Ab. DCs were analyzed by PE-conjugated anti-CD80/CD86 mAbs in combination with FITC-labeled anti-CD11c mAbs. All reagents were obtained from BD Pharmingen . D2F2 or HEVc cells were stained with PE-labeled rat anti-mouse endoglin mAb or isotype control Ab (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Flow cytometry were performed with a FACScan (Becton Dickinson, San Jose, CA, USA) and the data analyzed with FlowJo software (Tree Star, Inc, Stanford, CA, USA).

ELISPOT assay

The number of IFN-γ secreting cells was determined with an ELISPOT kit (BD Pharmingen) according to the manufacturer’s instructions. Briefly, splenocytes were collected 10 days after the last immunization from all experimental groups. T cells were isolated from splenocytes on a Nylon Wool Column (Polysciences, Inc., Warrington, PA, USA). Purified T cells (2×10⁵/well) were cultured for 24 h with 2×10⁴/well of irradiated (1,000 Gy) D2F2 cells, mEndo⁺-D2F2 cells or HEVc cells.

Evaluation of anti-angiogenic activity

One week after the last vaccination, mice were injected s.c. near the abdominal midline with 500 μl of growth factor reduced Matrigel (BD Pharmingen) containing 400 ng/ml bFGF (PeproTech, Princeton, NJ, USA). Mice were injected 6 days later with 200 μl (0.1 mg/ml) isolectin B4 conjugated with fluorescein (Vector Laboratories) to stain the endothelium. Mice were sacrificed 15 min thereafter and Matrigel plugs were homogenized with RIPA lysis buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). After centrifugation, the fluorescein content in the supernatant was quantified by fluorimetry at 490 nm. Background fluorescence found in the non-injected control was subtracted in each case [1, 27].

Statistical analysis

The statistical significance of different finding between experimental groups and controls was determined by Student’s t test and considered significance it two-tailed P values was <0.05.

Results

Determination of endoglin expression in vitro and in vivo

Endoglin expression by the murine breast tumor cell line D2F2, the murine endothelial cell line HEVc and normal mouse spleen and liver was assessed by RT-PCR. Results indicated that HEVc strongly express endoglin (Fig. 1a). However, endoglin is absent in D2F2 cells or normal liver tissue under these experimental conditions. Low levels of endoglin expression in spleen were also observed (Fig. 1a). We further confirmed this finding by FACS analysis: HEVc cells express endoglin on the surface, but endoglin is not detectable on D2F2 tumor cells (Fig. 1b). However, endothelial cells in metastatic D2F2 lung tumor tissue highly express endoglin, while endoglin expression is barely detectable in normal lungs (Fig. 1c). Thus, these results confirm that the expression level of endoglin is significantly up-regulated on proliferating endothelial cells, despite the fact that D2F2 breast tumor cells themselves do not express detectable levels of endoglin [17].

Fig. 1.

Expression of murine endoglin by tumor cells lines and normal mouse tissues. a RT-PCR analysis of endoglin expression by wild-type D2F2 breast carcinoma cells, HEVc endothelial cells, as well as normal mouse spleen and liver. Total RNA extraction and RT-PCR were performed according to “Material and methods” and GAPDH was used as control for total RNA loading. b FACS analyses of endoglin expression on the cell surface of D2F2 cells and HEVc cells, both stained with PE-conjugated anti-endoglin Ab. PE-labeled Rat IgG Ab served as isotype control (black line). c Comparison of endoglin expression levels on normal lungs and lung metastases of D2F2 tumor induced by i.v. injection of breast tumor cells (1×10⁶ cells/mouse). Mice were sacrificed when inoculated animals were moribund and frozen lung sections analyzed by immunostaining with anti-endoglin Ab (×20 magnification)

The mEndoglin vaccine is delivered to PP

To test our hypothesis that an oral DNA vaccine encoding endoglin induces a T cell-mediated immune response, we first inserted the entire gene encoding murine endoglin into the pCMV/myc/cyto expression vector (Fig. 2a). Protein expression of endoglin was demonstrated by a single band of expected molecular weight (90 kDa) detected by Western blots of lysates of COS-7 cells transiently transfected with mEndoglin (Fig. 2a).

Fig. 2.

Schematic of a vector encoding murine endoglin and expression in vitro and in vivo. a Protein expression of endoglin was detected by Western blotting of endoglin-transfected COS-7 cell lysates with anti-endoglin Ab. b Endoglin expression of CD11c⁺ DCs in Peyer’s patches isolated 24 h after vaccination. Sections were stained with Ab against CD11c (green) and Ab against endoglin (red), and examined for expression by confocal microscopy. Samples derived from mice administered with empty vector served as a negative control

Our oral DNA vaccination strategy using double attenuated S. typhimurium (dam ⁻; AroA ⁻) is designed to achieve successful in vivo delivery of plasmids to secondary lymphoid organs, i.e. PPs, to facilitate subsequent priming of specific T cells. To confirm endoglin expression after vaccination, mice were sacrificed 24 h after oral vaccine administration and PP collected from the thoroughly washed small intestine. Confocal microscopy demonstrated that CD11c⁺ DC sub-populations expressed endoglin intracellularly in PP of mEndoglin-vaccinated mice (Fig. 2b). However, endoglin was not detected in CD11c⁺ DC cells from PPs of control mice.

The mEndoglin vaccine induces suppression of D2F2 breast tumor metastases

We tested the efficacy of mEndoglin vaccine in a prophylactic setting, in which disseminated pulmonary metastases were induced in mice challenged by i.v. injection of 1.5×10⁵ D2F2 breast carcinoma cells 1 week after the last vaccination. Whenever control mice showed signs of morbidity, all animals were sacrificed and evaluated for lung metastases and lung weights. Results (Fig. 3a) indicated that all mice receiving either PBS or empty vector presented with extensive disseminated pulmonary metastases. In contrast, all mEndoglin-vaccinated mice exhibited significant suppression of pulmonary metastases when compared to control mice (P<0.05). In addition, in survival studies, all control mice (PBS or CMV groups) died within 4 week after tumor cell challenge due to extensive metastases; however, mice immunized with the mEndoglin vaccine had a 60% prolongation in life span (Fig. 3b).

Fig. 3.

Suppression of pulmonary metastases of D2F2 breast carcinoma by the mEndoglin vaccine in prophylactic tumor models. Lung metastases were induced by i.v. injection of 1.5×10⁵ D2F2 cells, 1 week after the last immunization. Experiments were terminated 27 days after tumor cell inoculation and the extent of pulmonary tumor metastases evaluated based on lung weights. a Lung weights are depicted following immunization with PBS, empty vector or mEndoglin vaccine. Symbols within bar graphs of experimental groups each represent a single mouse (n=8). Normal lung weight is approximately 200 mg. *P<0.05 when compared with either PBS or empty vector. b Kaplan–Meier survival plots of different groups of mice (n=8). Differences in survival times between the mEndoglin group and control groups were statistically significant according to Kaplan–Meier analyses (P<0.05). Experiments were repeated three times with similar results

The anti-tumor effects induced by the mEndoglin vaccine are mediated by CD8⁺ T cells

To determine the roles of cell subpopulations played in mEndoglin vaccine-induced suppression of pulmonary metastases, in vivo depletions of CD4⁺ or CD8⁺ were performed (Fig. 4). We observed that non-depleted, vaccinated mice effectively suppressed D2F2 pulmonary metastases when compared to the empty control vector mice (P<0.05); however, this suppression of pulmonary metastases was abrogated in mice depleted of CD8⁺ T cells (Fig. 4), indicating that CD8⁺ T cells play a major role in suppressing D2F2 pulmonary metastases. In contrast, in vivo depletion of CD4⁺ T cells did not significantly affect suppression of D2F2 pulmonary metastases, suggesting that CD4⁺ T cells do not play a major role in anti-tumor effects induced by the mEndoglin vaccine.

Fig. 4.

Increased incidence of pulmonary D2F2 metastases of vaccinated mice that were depleted of CD8⁺ T cell populations. Mice were immunized orally three times at weekly intervals with either DNA encoding endoglin or the empty vector. Anti-CD8 mAb (2.43: mouse mAb, IgG2b) or anti-CD4 mAb (GK1.2: rat mAb, rat IgG2b), were each injected i.v. 7 days after the last immunization. The following day, experimental lung metastases were induced in a prophylactic setting by i.v. injection of 1.5×10⁵ D2F2 cells followed by additional weekly i.p. injection of anti-CD8 or anti-CD4 mAb to maintain the depleted state of subset T cells until the termination of the experiments. Mice were sacrificed 27 days after D2F2 tumor cell challenge and lung weights established to determine the extent of pulmonary metastases. The data shown in bar graphs reveal the average ± SD of lung weights in each experimental group (n=8). Statistically significant differences in lung weights between groups of mice treated with either the empty vector and non-depleted as well as CD4⁺ and CD8⁺ depleted T cells are shown. The data are representative of two independent experiments

The mEndoglin vaccine induces T cell and DC activation

We then investigated whether the anti-tumor activity of the mEndoglin vaccine correlated with T cell activation. This was evident from the increased expression of CD28, an important marker of activated T cells (Fig. 5), especially since optimal T cell activation is critically dependent on the ligation of CD28 with co-stimulatory molecules CD86 and CD80 on DCs. In this regard, FACS analyses of splenocytes obtained from vaccinated mice clearly demonstrated that the expression of both CD80 and CD86 on CD11c⁺ DCs was up-regulated when compared with control animals (Fig. 5).

Fig. 5.

The mEndoglin vaccine induced activation of T cell and DCs. Two-color flow cytometric analyses were performed with splenocytes from vaccinated mice 1 week after tumor cell challenge. For T cell activation, PE-conjugated anti-CD28 mAbs were used in combination with FITC-conjugated anti-mouse CD8 mAb (upper panel). For DC analyses, splenocytes were stained with FITC-labeled anti-CD11c mAb in combination with PE-conjugated anti-CD80 (middle panel) or anti-CD86 mAbs (lower panel). Y-axis represents % of double positive cells compared to the total CD8⁺ or CD11c⁺ cells (mean±SD, n=4). Differences between the control groups and the treatment group were statistically significant (P<0.05)

Immunization with the mEndoglin vaccine evokes endoglin-specific CTLs

In order to assess whether CD8⁺ T cells are able to specifically lyse endoglin-positive target cells, we generated endoglin-expressing D2F2 cells (mEndo⁺-D2F2) by transfection of D2F2 cells with the endoglin plasmid. These cells expressed endoglin on the surface (Fig. 6a), in comparison to wild-type D2F2 cells that did not express endoglin (Fig. 1b).

Fig. 6.

Vaccination against endoglin induces specific T-cell responses. a FACS analyses of endoglin expression on endoglin-transfected D2F2 tumor cells (mEndo⁺–D2F2). Cells were stained with PE conjugated anti-endoglin Ab. Rat IgG Ab was used as an isotype control (black line). b ELISPOT analyses of IFNγ producing cells using different stimulator cells. Splenocytes, enriched for CD8⁺ cells, were isolated from vaccinated mice and incubated for 24 h with either irradiated wild-type D2F2 cells, mEndo⁺-D2F2, or HEVc endothelial cells. The mean spot number of each group is shown (n=3, mean±SD). c T cell-specific cytotoxicity against endoglin-positive HEVc endothelial target cells. Splenocytes were isolated from vaccinated mice 10 days after tumor cell challenge. A [³⁵S]-release assay was performed at different effector-to-target cell ratios with splenocytes being re-stimulated with irradiated mEndo⁺–D2F2 cells for 5 days and [³⁵S] methionine labeled HEVc used as target cells. The data depict average ± SD of triplicate wells. Similar results were obtained in three independent experiments. d Sensitivity of mEndo⁺–D2F2 and wild-type D2F2 cells (mEndo⁻–D2F2) to CTL killing. [³⁵S] methionine labeled wild-type D2F2 or mEndo⁺ D2F2 target cells were co-incubated with effectors at E/T =1:12.5. Similar results were obtained in three independent experiments. *P<0.05 compared with control wild-type D2F2 target cells

ELISPOT analysis for IFNγ secretion was performed to determine the frequency of endoglin-specific T cells in mEndoglin-vaccinated mice. The number of spots markedly increased when such cells were co-incubated with irradiated mEndo⁺-D2F2 cells as stimulators when compared to stimulation with wild-type D2F2 cells (Fig. 6b). These data indicate the success in expanding endoglin-specific cells in mEndoglin-vaccinated mice.

Furthermore, we determined whether such activated T cells could eliminate endoglin-expressing endothelial target cells. The results (Fig. 6c) indicate that endothelial HEVc target cells, which naturally express endoglin, are susceptible to lysis by effector cells obtained from mEndoglin-vaccinated mice. In contrast, T cells from control mice showed low level of killing (Fig. 6c).

We next examined the specificity of the vaccine-induced cytotoxicity. In fact, mEndo⁺–D2F2 target cells were two times more sensitive to CTL killing than wild-type D2F2 cells (P<0.05, Fig. 6d). Moreover, mEndo⁺–D2F2 cells were more susceptible to lysis by effector cells obtained from mEndoglin-vaccinated mice than by those from control mice (Fig. 6d). These data indicate that the mEndoglin vaccine effectively induced the specific elimination of endoglin-positive target cells.

The mEndoglin vaccine elicits suppression of angiogenesis

We assessed whether the mEndoglin vaccine could suppress angiogenesis. In this regard, a Matrigel assay revealed a significant decrease in neovascularization only in mice immunized with mEndoglin vaccine (Fig. 7). In fact, quantification of relative fluorescence intensity, measured after in vivo staining of mouse endothelium with FITC-conjugated lectin, clearly indicated that the angiogenic process in such experimental animals decreased significantly in comparison to control mice (P<0.05).

Fig. 7.

Suppression of angiogenesis by the mEndoglin vaccine. One week after the last vaccination, Matrigel was implanted s.c. into the midline of the abdomen of either control mice (n=7) or vaccine-treated mice (n=8), and vessel growth quantified by staining of endothelium with FITC-labeled Isolectin B4 as described in Material and methods. The average fluorescence of extracts is measured by fluorimetry at 490 nm and depicted by bar graphs (mean±SD; P<0.05)

Discussion

An oral DNA vaccine encoding murine endoglin, which is primarily over-expressed by proliferating endothelial cells in the angiogenic tumor vasculature, effectively induced an endoglin-specific CD8⁺ T cell-mediated immune response. This immune response broke peripheral tolerance against the endoglin self-antigen and presumably suppressed tumor angiogenesis, resulting in the suppression of pulmonary D2F2 breast carcinoma metastases in a prophylactic setting.

The rationale for using double attenuated S. typhimurium as a vaccine carrier is based on our prior data [30, 32, 33, 48], including the finding that transformation of such bacteria with a DNA plasmid encoding a tumor antigen and their subsequent oral administration by gavage leads to delivery of the vaccine via the small intestine and M cells into PPs. There, the attenuated bacteria are phagocytosed, primarily by DCs in the subepithelial dome of this secondary lymphoid organ [23], and then die due to their mutations and liberate the DNA. This is followed by transcription and translation of the DNA and processing of proteins or peptides in the proteosomes of these APCs, ultimately leading to the formation of antigen peptide/MHC class I Ag complexes in the cytosol, which are delivered to the cell surface and presented to T cell receptors. In this regard, intralymphatic immunization with naked DNA was reported to be most effective since it is 100–1,000- fold more efficient inducing strong and biologically relevant CD8+ CTL responses over traditional i.m., s.c., or i.v. routes of immunization [28]. Consequently, vaccination with naked DNA appears to be optimal when targeted to secondary lymphoid organs such as PP. In addition, in this draining lymph node, effective cross-priming of CD8+ cells may also possibly be achieved without CD4+ T cell help [50].

Antiangiogenic therapies generally take two approaches: (1) targeting preexisting blood vessels or (2) preventing the development of the tumor neovasculature. Since our vaccination was performed in a prophylactic setting, where vaccination preceded tumor cell challenge, CD8⁺ T cell responses induced by our mEndoglin vaccine likely interfered with the development of the tumor angiogenic blood vessels which, in turn, prevented the establishment of D2F2 pulmonary metastases.

The well-established, high-level expression of endoglin by proliferating endothelial cells of both peri- and intratumoral blood vessels [6, 15, 39] makes endoglin an excellent target for antiangiogenic therapy, particularly in attempts to prevent the development of tumor blood vessels. Since in our experimental system, endoglin is only over-expressed by proliferating endothelial cells in angiogenic blood vessels (Fig. 1), targeting proliferating endothelial cells has several additional advantages over targeting tumor cells. These include the following: first, the avoidance of tumor antigen heterogeneity and the down-regulation of MHC class I antigens, both of which seriously limit effective T cell-mediated immune responses against tumor cells; second, the specific targeting of the antiangiogenic intervention to proliferating endothelial cells in the tumor neovasculature limits its toxicity; third, the direct contact of the vasculature with the circulation makes for efficient access of therapeutic agents since the target tissue can be reached unimpaired by anatomical barriers such as the blood-brain barrier or encapsulated tumor tissue [2, 9, 13, 37]; fourth, since the therapeutic target is tumor-independent, killing of proliferating endothelial cells in the tumor microenvironment could be effective against a variety of solid tumors [8, 11, 21, 35, 36].

Taken together, our data indicate that the endoglin-based DNA vaccine, delivered to PP in the small intestine by double attenuated S. typhimurium, evoked an effective CD8⁺ T cell-mediated anti-tumor immune response. Importantly, this response was shown to be specifically directed against endoglin expressed by proliferating endothelial cells, and presumably resulted in the suppression of angiogenesis in the breast tumor neovasculature. This included the ability of T cells from mEndoglin-vaccinated mice to specifically lyse both mEndo⁺–D2F2 and endothelial target cells, the latter which naturally express endoglin. The up-regulation of the T cell activation marker, CD28, and of co-stimulatory molecules CD80/CD86 on DCs provided further evidence for the activation of these cells. This type of activation is presumably of key importance to achieve a T cell-mediated immune response leading to the limitation of angiogenesis in the tumor vasculature, as well as to the suppression of breast tumor growth and pulmonary metastases in a prophylactic tumor model.

In conclusion, we anticipate that this novel, oral DNA vaccine targeting endoglin might ultimately lead to a successful clinical application aiding in the prevention and therapy of human breast cancer.