Tumor endothelial marker 1–specific DNA vaccination targets tumor vasculature

Tumor endothelial marker 1 (TEM1; also known as endosialin or CD248) is a protein found on tumor vasculature and in tumor stroma. Here, we tested whether TEM1 has potential as a therapeutic target for cancer immunotherapy by immunizing immunocompetent mice with Tem1 cDNA fused to the minimal domain of the C fragment of tetanus toxoid (referred to herein as Tem1-TT vaccine). Tem1-TT vaccination elicited CD8⁺ and/or CD4⁺ T cell responses against immunodominant TEM1 protein sequences. Prophylactic immunization of animals with Tem1-TT prevented or delayed tumor formation in several murine tumor models. Therapeutic vaccination of tumor-bearing mice reduced tumor vascularity, increased infiltration of CD3⁺ T cells into the tumor, and controlled progression of established tumors. Tem1-TT vaccination also elicited CD8⁺ cytotoxic T cell responses against murine tumor-specific antigens. Effective Tem1-TT vaccination did not affect angiogenesis-dependent physiological processes, including wound healing and reproduction. Based on these data and the widespread expression of TEM1 on the vasculature of different tumor types, we conclude that targeting TEM1 has therapeutic potential in cancer immunotherapy.

Angiogenesis, the growth of new blood vessels from preexisting vessels, is essential for many physiological processes, such as pregnancy, embryonic development, and wound healing (1). However, uncontrolled or defective angiogenesis also contributes to many pathological processes, including tumor growth and metastasis (2).

Targeting angiogenesis is a promising strategy for the treatment of cancer. Tumor-associated blood vessels differ from vessels of normal tissue both structurally and functionally and can offer unique molecular targets for therapeutic interventions (3–5). There are several advantages to targeting the vasculature rather than the tumor cells proper. Tumor-associated vascular antigens are broadly expressed on cancers of different histological types, whereas they are generally absent or poorly expressed on most normal tissues (6–8). Immunotherapy may also elicit a significant bystander effect, because each capillary is thought to support hundreds of tumor cells and adaptive or antibody-mediated immune responses can easily access the tumor vasculature, whereas tumor islets may be compartmentalized and less accessible. Also, adaptive immunotherapy can persist, leaving a long-lasting memory response that protects the host against tumor recurrence. Finally, tumor cells are intrinsically more genetically unstable than vascular endothelial cells, and targeting them directly may promote tumor escape mechanisms (9).

Plasmid DNA offers a safe and promising method for vaccination and is approved for cancer immunotherapy for veterinary use (10). However, the immune responses induced to date by DNA vaccines in humans have been relatively weak compared with conventional vaccines, an issue that is particularly relevant for cancer vaccines because of the limited immunogenicity of tumor antigens. A combination of approaches are being studied to boost immune responses. In vivo electroporation of plasmid DNA or electrogene transfer increases DNA uptake, leading to enhanced expression in treated muscle and a concomitant increase in immune responses to the target antigen (11). The minimized domain of the C fragment of tetanus toxoid (TT) has been used to elicit antigen-specific immune responses (12). For example, DNA vectors encoding the tumor-associated antigen carcinoembryonic antigen (CEA) fused to TT can break tolerance to CEA and elicit significant antigen-specific CD8⁺ T cell–mediated immune responses (13).

Human tumor endothelial marker 1 (TEM1; also known as endosialin or CD248) is overexpressed in the vasculature of carcinomas and brain tumors as well as in the vasculature and stroma of most sarcomas (14–17). Expression of TEM1 is variable among tumor types; it may be expressed on endothelial cells, pericytes, and fibroblasts in both mouse tumors (18) and human tumors (19, 20). Human TEM1 gene expression is also induced by hypoxia under the regulation of HIF-2α (21). In the mouse, TEM1 is overexpressed during embryonic development, but is largely absent in adult tissue (18, 22). TEM1 is one of the most abundantly expressed tumor endothelial or stromal antigens in human tumors, making it a prime candidate for immunological targeting.

We hypothesized that effective DNA vaccination using TT as an adjuvant would be able to break tolerance to TEM1 and generate adaptive immunity targeted to the tumor vasculature. We propose that such vaccination can elicit potent antitumor activity in TEM1-expressing tumors by ablation of the tumor vasculature without affecting normal physiological functions that are dependent on angiogenesis. We tested these hypotheses by genetically fusing mouse Tem1 cDNA with the first domain of fragment C of TT, creating a DNA vaccine (referred to herein as Tem1-TT) that we administered i.m. via electrogene transfer.

DNA vaccination with Tem1-TT breaks tolerance to TEM1. Consistent with previous work in humans and mice, we found that Tem1 was overexpressed in actively growing tumors of different background or histology (TC1, CT26, and LLC models) and was expressed at very low to undetectable levels in mouse normal tissues, as quantified by quantitative RT-PCR (qRT-PCR; Figure 1A and data not shown). In the hypervascularized CT26 mouse colon carcinoma model, Tem1 mRNA was found to localize with, or to be in close juxtaposition to, CD31 mRNA (Figure 1B and Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI67382DS1), which suggests that TEM1 is expressed by ECs and/or pericytes. These findings support the notion that TEM1 is an overexpressed vasculature-associated antigen in CT26 colon tumors, which can serve as a target for antitumor vascular therapy.

Figure 1

Physiological and plasmid DNA-induced TEM1 expression. (A) Tem1 was expressed in tumors in vivo. qRT-PCR was performed with a Tem1 probe. An 18S probe was used as an endogenous control, and samples were normalized with mouse liver. Post–qRT-PCR calculations to analyze relative gene expression were performed by the 2^–ΔΔCt method. Error bars denote SD (n = 5). (B) Tem1 RNA colocalized with CD31⁺ cells. CT26 tumor sections were subjected to immunofluorescence with a CD31 antibody (red) and single-molecule RNA FISH for Tem1 (yellow) or CD31 (cyan). Bright spots in the RNA FISH channels correspond to individual transcript molecules. DAPI staining (purple) shows cell nuclei. Original magnification, ×100. Images shown are 85 μm wide. (C) Expression of the Tem1-TT DNA plasmid. A fusion Tem1-TT construct was generated by fusing codon-optimized Tem1 cDNA (nt 1–2,297; aa 1–765) with the cDNA of the amino terminal domain of the fragment C of TT (819 bp, 273 aa). Expression of Tem1 or TT after transfection of the Tem1-TT construct was tested with CHO cells and assessed by qRT-PCR. Error bars denote SD (n = 3).

Using pcDNA3.1 as a DNA plasmid vector, the Tem1 gene sequence was fused to the first domain of the C fragment of the TT sequence (TT 865–1,120) at its carboxyl terminal (see Methods). Expression levels of both sequences were measured by qRT-PCR after transient transfection of CHO cells (Figure 1C). This fusion construct was used to vaccinate healthy mice at 1-week intervals for a total of 3 immunizations. Splenocytes harvested from vaccinated mice were stimulated with a TEM1 peptide library consisting of 15 mers that overlap by 10 aa. The TEM1 library encompassing the entire protein sequence was divided into 4 pools: pool A (sequences spanning aa 1–197), pool B (aa 187–377), pool C (aa 367–557), and pool D (aa 547–765). This peptide-based approach facilitates the detection of CD8⁺ and CD4⁺ T cells that are specific for TEM1 sequences in the context of MHC class I and MHC class II, respectively (23). IFN-γ enzyme-linked immunosorbent spot assay (ELISpot) and intracellular staining (ICS) indicated that the majority of CD8⁺ and CD4⁺ T cell epitopes in BALB/c mice were contained within pool C (Figure 2A). Spleen-derived CD8⁺ T cells from vaccinated C57BL/6 mice exhibited reactivity against pool D, whereas CD4⁺ T cell responses were not detected (Figure 2B). As hypothesized, only the Tem1-TT DNA fusion vaccine was able to break tolerance to TEM1; specific responses were not detected in mice of either strain that were vaccinated with DNA encoding Tem1 or TT adjuvant alone.

Figure 2

Characterization of TEM1 immunodominant epitopes. (A and B) Specific peptide pools restricted in H-2^d (A) and H-2^b (B) haplotypes. 1 × 10⁶ splenocytes from BALB/c (A) or C57BL/6 (B) mice vaccinated with Tem1-TT were cultured with the 4 TEM1 peptide pools and tested by ELISpot and ICS for IFN-γ secretion. (A) Splenocytes from Tem1-TT–vaccinated BALB/c mice recognized pool C (left), which induced CD3⁺CD4⁺ (middle) and CD3⁺CD8⁺ (right) T cell responses. (B) Splenocytes from Tem1-TT–vaccinated C57BL/6 mice recognized pool D (left). By ICS, there was a specific CD3⁺CD8⁺ response (right). Data are mean ± SD (n = 5 per group) from 1 of 3 experiments. (C and D) Characterization of TEM1-derived peptides specific for H-2^d (C) and H-2^b (D) haplotypes. Peptides from pool C (C) or pool D (D) were divided into 7 minipools of 15 mers, so each individual peptide was present in 2 minipools and could be identified through an experimental matrix design. (C) Reactivity to pool C was present in minipools f, g, and 5. (D) Reactivity to pool D was present in minipools a and 6. Data are from 1 of 3 independent experiments. (E) TEM1_696–710 induced IFN-γ secretion and cytotoxic effector activity. Splenocytes from TT-, Tem1-, or Tem1-TT–vaccinated mice was used in peptide-stimulated cultures and tested in an IFN-γ secretion ELISA (left) and CTL assay (right). Tem1-TT exclusively primed and expanded antigen-specific CD8⁺ T cells that killed ECs in the context of MHC class I/TEM1_696–710 peptide, but not control peptides.

To characterize the TEM1 sequences recognized by CD4⁺ and CD8⁺ T cells generated by the Tem1-TT vaccine, pools C and D were deconvoluted using a matrix scheme. For the BALB/c strain, pool C was further divided into minipools. TEM1-specific reactivity was found against minipools f, g, and 5 (Figure 2C). IFN-γ ICS analysis revealed that minipools g and 5, corresponding to TEM1_516–530 (ITSATHPARSPPYQP), induced a stronger CD8⁺ T cell response compared with minipools f and 5, corresponding to TEM1_511–525 (GHKPGITSATHPARS). Both TEM1_516–530 and TEM1_511–525 peptides stimulated a CD4⁺ T cell response (data not shown). We therefore decided to use the TEM1_516–530 peptide for subsequent monitoring of the immune response in BALB/c mice. Using the same approach to further divide pool D into minipools for testing the C57BL/6 background, a specific TEM1 response was detected for minipools a and 6, corresponding to the TEM1_696–710 peptide (GQSQRDDRWLLVALL) (Figure 2D). Using IFN-γ ICS, this sequence was found to specifically activate CD8⁺ T cells (data not shown), which indicates that peptide TEM1_696–710 contains the immunodominant CD8⁺ T cell epitope for C57BL/6 mice.

To confirm that the TEM1_696–710 peptide mediates target cell recognition and killing by CTL, splenocytes from vaccinated C57BL/6 mice were harvested and tested in mixed leukocyte peptide–stimulated culture (MLPC). MBL2 leukemia cells were used as the stimulatory cells. MLPCs were pulsed with TEM1_696–710 or control peptides for 5 days and tested for IFN-γ secretion by ELISA and in a ⁵¹Cr cytotoxicity CTL assay. IFN-γ ELISA revealed that Tem1-TT vaccination selectively generated antigen-specific T cells that recognize the TEM1_696–710 peptide, but not the control peptides TEM1_691–705 or β-gal_96–103 (Figure 2E). Splenocytes from vaccinated C57BL/6 mice were also able to recognize unpulsed or TEM1_696–710–pulsed H5V ECs, which express endogenous TEM1 protein, whereas MS1 ECs (which do not express TEM1; ref. 24) required TEM1 peptide pulsing to stimulate splenocytes from Tem1-TT–vaccinated mice (Figure 2E). In CTL assays, splenocytes from Tem1-TT–vaccinated mice efficiently lysed all 3 target cell types when pulsed with TEM1 peptide (Figure 2E). Together, these data suggest that Tem1-TT can break tolerance and elicit functional CD8⁺ T cells that are specific for TEM1 epitopes. Furthermore, ECs can naturally process the endogenous TEM1 antigen and are able to present the cognate TEM1 peptide in an MHC class I–restricted fashion.

Tem1-TT DNA vaccine suppresses tumor growth and induces CD3⁺ T cell tumor infiltration. To evaluate the ability of Tem1-TT vaccination to control tumor growth in vivo, we tested both prophylactic and therapeutic vaccine approaches using CT26, TC1, and LLC tumor models. Mice were vaccinated 3 times i.m. at weekly intervals, followed by tumor challenge (prophylactic), or tumor cells were inoculated first and then 3–5 days later were given 3 weekly vaccinations (therapeutic). Prophylactic treatment with Tem1-TT induced significant tumor protection compared with single Tem1 and TT constructs in all 3 tumor models (Figure 3A). These results reinforced the notion that the fusion construct Tem1-TT vaccine is essential to break TEM1 tolerance and generate protective immunity.

Figure 3

Tem1-TT vaccination controls tumor progression. (A) Tumor control in a prophylactic setting. BALB/c and C57BL/6 mice were immunized 3 times with Tem1, TT, or Tem1-TT vaccines, then injected s.c. with 1 × 10⁵ CT26, TC1, or LLC cells. Data are mean ± SD (n = 10 per group) from 1 of 5 experiments. P < 0.05, Tem1-TT vs. Tem1 and Tem1-TT vs. TT (CT26, TC1, and LLC), pairwise multiple-comparison Tukey test. (B and C) Inhibition of CT26 and TC1 tumor progression and induction of CD3⁺ T cell infiltration in a therapeutic setting. BALB/c mice were injected s.c. with 1 × 10⁵ CT26 cells (B), and C57BL/6 mice were injected with 1 × 10⁵ TC1 cells (C). Mice were immunized 1 day, 1 week, and 2 weeks later with Tem1, TT, or Tem1-TT vaccine and euthanized 33 (B) or 29 (C) days after tumor challenge. Left: Data points are mean ± SD (n = 10 mice/group) from 1 of 3 experiments. P < 0.05, Tem1-TT vs. Tem1 and Tem1-TT vs. TT (CT26 and TC1), Tukey test. CD3⁺ T cells in corresponding tumor sections and quantification are also shown. Original magnification, ×20. Data are mean ± SD of a representative experiment (n = 5 per group).

Therapeutic Tem1-TT vaccination conferred significant tumor protection compared with single construct controls in CT26 and TC1 tumor-bearing mice (Figure 3, B and C). Analysis of CD3⁺ T cell tumor infiltration by immunohistochemistry revealed consistent, heavy infiltration of CD3⁺ T cells after therapeutic vaccination with Tem1-TT within CT26 (42 ± 5 CD3⁺ cells/high-powered field [hpf]; P < 0.05 vs. TT) and TC1 tumors (54 ± 11 CD3⁺ cells/hpf; P < 0.05 vs. TT) compared with the single constructs and saline control (Figure 3, B and C). Cumulatively, these data indicate that the Tem1-TT vaccine induces significant T cell infiltration within tumors and exerts potent antitumor activity.

To demonstrate that the antitumor response by Tem1-TT vaccine is indeed immune mediated, and to determine whether the immune response is cellular or humoral in nature, we performed adoptive transfer of CD3⁺ T cells or sera from Tem1-TT–immunized mice into TC1 tumor-bearing mice (Supplemental Figure 2). Splenocytes from Tem1-TT–immunized mice were enriched for CD3⁺ cells and inoculated into naive mice. In simultaneous experiments, the serum from immunized mice was also transferred to naive mice. Mice were then sublethally irradiated and challenged with TC1 tumor. The antitumor response mediated by Tem1-TT vaccine was based on the induction of a CD3⁺ T cell response, whereas serum had no therapeutic efficacy (Supplemental Figure 2). Therefore, although antibodies may be generated by the Tem1-TT vaccine, the results of our adoptive transfer experiments ruled out any humoral contribution to antitumor efficacy.

Tem1-TT vaccination inhibits tumor vascularization. To determine whether Tem1-TT vaccination controls tumor progression by targeting the tumor vasculature, we used common readouts for measuring the effects on the vasculature, but also measured the functional dynamics of the vasculature in the hypervascularized CT26 model (25). To ensure that tumors of the same size were studied, mice that were immunized with Tem1-TT were sacrificed 10 days later than mice immunized with TT vaccine, and available tumors were harvested. Tumors of similar volume (∼200 mm³) from both groups were assessed for tumor hemoglobin levels by ELISA, as a readout for blood perfusion. Tumors from mice vaccinated with Tem1-TT had significantly decreased hemoglobin levels compared with those of TT-vaccinated mice (Figure 4A), suggestive of reduced red blood cell perfusion in these tumors.

Figure 4

Tem1-TT vaccination inhibits CT26 tumor vascularization. (A) Tem1-TT vaccination reduced tumor hemoglobin content. Tumors at approximately 200 mm³ were excised from TT- or Tem1-TT–vaccinated mice and inspected grossly. Tumors from Tem1-TT–vaccinated mice appeared pale relative to control tumors. Reduced hemoglobin levels in tumors from Tem1-TT–vaccinated mice were observed by ELISA. (B) Tem1-TT vaccine reduces tumor vascularity. Tumors from TT- or Tem1-TT–vaccinated mice were analyzed by Doppler ultrasound. Perfused tumor area and real blood flux are shown, measured and calculated by Doppler image analysis. (C) CT26 tumors from Tem1-TT–immunized mice had significantly decreased CD31 expression compared with TT vaccination. Also note the abnormal blood vessel shape. Original magnification, ×20. (D) CAIX expression was increased in tumors from Tem1-TT–immunized animals. CAIX expression was visualized by immunohistochemistry, and Caix was independently quantified by qRT-PCR in tumors from mice vaccinated with either TT or Tem1-TT. Original magnification, ×20. Data in A–D are mean ± SD of a representative experiment (n = 5 per group). Statistical analyses were performed with Student’s t test.

We then assessed the effects of Tem1-TT vaccination on the functional vasculature by measuring tumor vessel blood perfusion using ultrasound for each treatment group when tumors reached ∼600 mm³ in volume. Gas-filled microbubbles were injected i.v. and acted as a contrast agent as they flowed through the tumor blood vessels. Regions of perfusion in the tumors were visualized by ultrasound pulsing and contrast-enhanced Doppler imaging, which enabled us to determine the area of the tumor that was perfused (as a percentage of total tumor area) as well as the red blood cell flux (rate of blood flow) per unit area of the tissue (color-weighted fractional area) (26, 27). Contrast-enhanced images (after perfusion) showed a marked reduction in tumor perfusion (colored regions) in the Tem1-TT–treated group compared with TT-treated controls (Figure 4B). The reductions in both area of perfusion and blood flux in Tem1-TT–vaccinated tumors were statistically significant (P = 0.008 and P = 0.010, respectively; Figure 4B), which suggests that Tem1-TT vaccination disrupts the functional vasculature within the CT26 tumor.

We next assessed the apoptotic index of CT26 tumors by TUNEL assay. Compared with CT26 tumors from TT-immunized mice, cellular apoptosis increased in tumors from Tem1-TT–immunized mice (P = 0.014; Supplemental Figure 3A). We also observed colocalization of these apoptotic cells with the CD31 antigen (Supplemental Figure 3B), indicative of cell death of ECs of the tumor vasculature. Lastly, we also observed a reduction of cells positive for Ki-67, a marker for proliferating cells, within tumors from Tem1-TT– versus TT-immunized mice, which indicates that Tem1-TT vaccination lowers the proliferative capacity of the tumor, a likely consequence of disruption of the tumor vasculature (data not shown).

Next, the microvasculature density (MVD) of the tumors was measured by immunohistochemical staining of CD31. CT26 tumors from Tem1-TT–treated mice displayed reduced MVD compared with TT-treated tumors (Figure 4C). In addition to fewer CD31⁺ ECs per hpf, structural disorganization of the vasculature was noted: Tem1-TT–treated tumor vessels appear flattened with less luminal space (Figure 4C), suggestive of vessel collapse as well as difficulty assembling new vasculature. Lastly, we tested whether reduced tumor perfusion in Tem1-TT–vaccinated mice was associated with an increase in tumor-associated hypoxia. Using immunohistochemistry, an increase in carbonic anhydrase IX (CAIX), a cellular biomarker of hypoxia, was observed in tumors of Tem1-TT– versus TT-vaccinated mice (Figure 4D). Analysis by qRT-PCR also showed an increase in Caix message levels in tumors from Tem1-TT–treated mice relative to controls (Figure 4D). These data suggest an association between reduced tumor blood perfusion and hypoxia in CT26 tumors from Tem1-TT–vaccinated mice.

Tem1-TT vaccination induces T cell responses against tumor-associated antigens as well as TEM1 antigen. Next, we tested whether immunization against TEM1 by Tem1-TT vaccine results in epitope spreading, an immunological phenomenon in which immunotherapy or chemotherapy kills tumor cells and induces cross-presentation of tumor-associated antigens followed by cross-priming (28, 29). Tumor antigen–specific vaccines can also induce epitope spreading, inducing specificity against antigens unrelated to the original vaccine target (30, 31).

We hypothesized that the antivascular effects described above would provide a rich source of dead or dying tumor/stromal cells capable of supporting a corollary cross-priming event that would generate immune responses to antigens other than TEM1. We used the immunodominant AH1 (gp70_423–431) peptide in the immunological assays. AH1 is the MHC class I–associated epitope for CT26 tumor, and as such, AH1-specific CD8⁺ T cells can cure mice of established CT26 tumor (32, 33). AH1 peptide is derived from an endogenous retroviral gene product, gp70, expressed by CT26 cells (32). Splenocytes from CT26 tumor-bearing BALB/c mice vaccinated with Tem1-TT exhibited a specific response against TEM1_516–530 but not against the TEM1_506–520 control peptide, as expected, but they also exhibited a vigorous response against the AH1 peptide, as measured by IFN-γ ELISpot (Figure 5A). Tumor-bearing mice immunized with control TT vaccine did not respond to any of these peptides (data not shown). As measured by IFN-γ ICS, Tem1-TT vaccine induced both CD4⁺ and CD8⁺ T cell responses against TEM1_516–530, while the response to AH1 was restricted to CD8⁺ cells, as expected (Figure 5A). The frequency of the TEM1 CD8⁺ T cell response from Tem1-TT–vaccinated mice was inversely correlated with tumor volume (Figure 5A), as was the CD4⁺ T cell response (data not shown). A similar inverse correlation was found between the AH1-specific CD8⁺ T cell response from Tem1-TT–vaccinated mice and tumor volume (Figure 5A).

Figure 5

Tem1-TT vaccination induces secondary tumor cell–specific cytotoxic T cell responses in tumor-bearing mice. BALB/c (A) and C57BL/6 (B) mice were challenged, vaccinated, and euthanized as in Figure 3, B and C, respectively. 1 × 10⁶ splenocytes from TT-, Tem1-, or Tem1-TT–vaccinated mice were tested for their capacity to respond to the specific TEM1 peptide (TEM1_516–530), and to AH1 antigen (A) or the TC1 tumor-associated E7 antigen (B). (A) Tem1-TT–vaccinated splenocytes mounted specific responses against the immunodominant peptide of TEM1 (TEM1_516–530) and the AH1 antigen, but not control peptide (TEM1_506–520), as measured by ELISpot. Tem1-TT induced CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell responses against TEM1 antigen. The CD3⁺CD8⁺ T cell response from Tem1-TT–vaccinated mice correlated with suppression of tumor growth. ICS confirmed that Tem1-TT immunization induced an AH1-specific CD3⁺CD8⁺ T cell response that also correlated with tumor volume. (B) Splenocytes from mice vaccinated with Tem1-TT recognized TEM1_696–710 peptide and the E7 antigen, but not control peptide (TEM1_691–705). Only Tem1-TT vaccination was able to induce both a TEM1 and an E7 CD3⁺CD8⁺ T cell response, both of which correlated with tumor volume. Correlation analysis was performed by Spearman’s rank correlation.

To further understand the potency of the antivascular TEM1 response and the subsequent antitumor AH1 response, we performed adoptive T cell transfer from Tem1-TT–immunized naive or CT26 tumor–bearing mice to recipient mice that were challenged with CT26 tumor. CD3⁺ T cells from Tem1-TT–immunized naive and tumor-bearing donor mice both significantly increased the survival of recipient mice injected with CT26 (Supplemental Figure 4, A and B). This result showed that the anti-TEM1 immune response generated by Tem1-TT in the non–tumor-bearing donor mice (without concomitant cross-priming and epitope spreading, as in the tumor-bearing mice) was sufficiently potent to control tumor growth, comparable to CD3⁺ cells adoptively transferred from Tem1-TT–immunized tumor-bearing mice (Supplemental Figure 4, A and B). As expected, adoptively transferred CD3⁺ cells from Tem1-TT–immunized naive mice exhibited a response against TEM1_516–530, but not against AH1 as in the tumor-bearing mice.

To confirm the epitope spreading phenomenon, we analyzed a second tumor model, TC1, a C57BL/6 epithelial lung tumor cell transformed by HPV E6 and E7 viral oncogenes. E7-derived peptide (E7_49–57) is the immunodominant epitope that can cure mice of established tumors (34). Mice immunized with Tem1-TT or control vaccine were challenged with TC1, as described previously. Splenocytes from Tem1-TT–vaccinated mice displayed CD8⁺-specific immune responses against TEM1_696–710 peptide (but not the control peptide TEM1_691–705), as well as against the E7_49–57 epitope (Figure 5B). As we observed in the CT26 model, the frequency of both TEM1- and E7-specific CD8⁺ immune responses correlated with TC1 tumor control (Figure 5B). Thus, effective Tem1-TT vaccination elicited a T cell–mediated response against both the tumor vasculature antigen (TEM1) and also against antigens expressed by the tumor cells themselves (gp70 and E7).

Tem1-TT vaccination does not affect wound healing or reproduction. Physiologic angiogenesis is critical for wound healing, and antiangiogenic agents such as bevacizumab have been associated with important safety concerns, including wound healing (35–37). We assessed whether wound repair was impaired by Tem1-TT immunization in C57BL/6 mice by wounding animals on their backs after immunization. There was no significant difference in time to wound closure between Tem1-TT– and control-vaccinated groups, despite generation of robust anti-TEM1 responses as measured by IFN-γ ELISpot and ICS (Figure 6, A–C).

Figure 6

Wound healing is not delayed by Tem1-TT immunization. (A) Time course of wound healing. C57BL/6 mice were vaccinated 5 times with Tem1-TT or TT vaccine. 10 days after the final vaccination, 2 circular wounds were inflicted on the upper back of the mice. The wound area was measured every 2 days. Data are mean ± SD of 2 experiments (n = 10 per group for each experiment). P = 0.726, Tem1-TT vs. TT, pairwise multiple-comparison Tukey test. (B and C) TEM1-specific response. 20 days after the punch biopsies, mice were sacrificed to check the immune response against TEM1 antigen by IFN-γ ELISpot (B) and ICS (C) of bulk splenocytes (1 × 10⁶). Statistical analyses were performed with Student’s t test.

Reproductive organs are sites of de novo angiogenesis, with both the corpus luteum and placenta being heavily dependent on formation of new blood vessels (38–40). Antiangiogenic compounds have been associated with defective luteogenesis, reproductive dysfunction, pregnancy loss, and teratogenesis (41–43). Previous studies have reported TEM1 expression in several tissues of the mouse embryo (22, 44). Postnatally, although TEM1 expression is downregulated in most organs, some expression persists in the renal glomerulus and in the adult uterus, where the expression pattern varies with the stage of the estrous cycle (45). Immunological targeting of the vascular TEM1 antigen could therefore have significant side effects on the reproductive system.

To test whether Tem1-TT immunization affects various aspects of mouse reproduction, female mice were immunized with Tem1-TT or TT vaccine and then mated (see Methods). Tem1-TT immunization had no effect on pregnancy success rate (P = 0.572), time to gestation (P = 0.698), total litter size (P = 0.552), and pup weight at birth (P = 0.409) compared with control immunization (Figure 7A). Furthermore, there were no anatomical or histological abnormalities in embryos from Tem1-TT– or control-immunized groups during early (E10) or late (E19) gestation (Figure 7B). We also assessed the potential effects of Tem1-TT immunization on various organs of vaccinated pregnant mice postpartum and found no abnormality in heart, lungs, liver, uterus, ovary, or placenta (Figure 7C). Similarly, organs from nonpregnant mice, including the uterus and the renal glomeruli, had no abnormalities after Tem1-TT immunization. To ensure that pregnancy did not dampen the effectiveness of Tem1-TT vaccination, we measured TEM1-specific T cell responses (TEM1_696–710) by IFN-γ ELISpot and ICS in the same female mice as above that were impregnated and gave birth, as well as in Tem1-TT–immunized mice that did not become pregnant after vaccination (Figure 7E and Supplemental Figure 5). TEM1 responses were roughly equal between pregnant and nonpregnant mice that were Tem1-TT immunized (Figure 7E). The ICS assay similarly showed that the TEM1 response was roughly of equal frequency between pregnant and nonpregnant mice and CD8⁺ specific (Supplemental Figure 5), as expected with this TEM1 peptide.

Figure 7

Tem1-TT vaccine does not affect reproduction. C57BL/6 female mice were vaccinated 5 times with Tem1-TT or TT vaccine followed by mating 1 week later. (A) Tem1-TT did not alter reproduction. Vaccination had no effect on fertility, gestation time, number of pups, or weight of pups (n = 15 per group). Data are representative of 2 independent experiments. Statistical analyses were performed with Student’s t test. (B) Tem1-TT vaccine did not affect embryonic development. H&E staining of embryos of female mice vaccinated with TT and Tem1-TT showed no abnormalities detected for either group. (C) Tem1-TT vaccination did not induce tissue pathology. Histological analysis of lung, heart, liver, spleen, kidney, colon, ovary, uterus, and placenta from pregnant (postpartum) and nonpregnant mice (no placenta) revealed no pathological effects by either vaccine. Original magnification, ×10, except spleen and ovary (×4). (D) Tem1-TT vaccination did not affect the estrus cycle. In a separate independent experiment, sera from immunized mice had synchronized estrus cycles and were evaluated for reproductive hormones. No fluctuations were noted. (E) TEM1-specific T cell responses were unaltered by pregnancy. 1 × 10⁶ splenocytes of vaccinated pregnant and nonpregnant mice were tested against TEM1_696–710 peptide or control peptide (TEM1_691–705) using IFN-γ ELISpot. Mice were boosted with a single injection of Tem1-TT or TT 45 days after the last immunization, then sacrificed 2 weeks later. There was significant recognition of TEM1_696–710 peptide by Tem1-TT– versus TT-vaccinated splenocytes from both pregnant and nonpregnant mice splenocytes (n = 5).

Lastly, because antiangiogenic agents may disrupt luteogenesis (38, 46, 47), we also tested reproductive endocrine function after Tem1-TT vaccination in a separate independent experiment. Mice were vaccinated only, then had their estrous cycle synchronized by medroxyprogesterone, and serum levels of 17β-estradiol (E2), progesterone (P4), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured longitudinally. Patterns of E2, P4, FSH, and LH expression were similar between TT- and Tem1-TT–vaccinated groups (Figure 7D); therefore, Tem1-TT did not modulate hormone levels during estrus. Collectively, Tem1-TT vaccination appears to have no untoward effects on physiological processes in mice that depend on de novo angiogenesis.

Tumor vasculature is critical for tumor growth and presents unique immunological epitopes, becoming an attractive target for therapy. Although many antiangiogenic and vascular-disrupting therapeutic approaches have been developed, immunotherapy offers the unique advantage of protective memory. Active immunization against whole tumor–derived ECs has produced encouraging preclinical results, but is not practical (48–50). Development of active vaccination targeting vascular epitopes has been previously reported, but safety has not been adequately addressed (31, 51). In some experiments using adoptive therapy of T cells engineered to recognize the vascular growth factor 2 receptor, there was severe dose-dependent toxicity (52). Thus, identification of unique epitopes on the tumor vasculature that are suitable for immunotherapy is of high significance for the tumor immunology field. TEM1 is a surface protein expressed by tumor endothelial and endothelial progenitor cells (53) as well as pericytes and tumor-associated fibroblasts (19, 54, 55) and is found in the vasculature of tumors (4, 14, 56–61), but not in normal vessels (57, 58). TEM1 is implicated in vascular cell adhesion, migration, development (62, 63), neoangiogenesis (57, 58), and tumor progression (20), and its overexpression is associated with poor survival (61). Importantly, Tem1^–/– mice were previously shown to resist tumor growth, invasiveness, and metastasis, but were otherwise healthy and exhibited normal wound healing (64). Thus, knockout of TEM1 greatly attenuated tumor establishment and progression, without affecting physiologic angiogenesis. However, whether this null phenotype can be reproduced through therapeutic means is not known. We showed here for the first time that effective immunization against TEM1 closely phenocopied the Tem1^–/– mouse, severely prohibiting the establishment of tumors and inhibiting the progression of tumor growth without affecting reproduction and/or would healing.

We developed a DNA vaccine that expresses the full-length mouse Tem1 cDNA fused to TT adjuvant. Immunization with the Tem1-TT vaccine reduced tumor vasculature compared with mice given control vaccine, as measured by MVD, functional imaging (ultrasound imaging of blood perfusion and blood flux), and hematocrit levels and indirectly supported by increased levels of CAIX in the tumor, suggestive of localized hypoxia therein. Splenocytes from Tem1-TT–vaccinated mice actively secreted IFN-γ and lysed TEM1-expressing ECs in vitro and specifically induced a cellular immune response that controlled tumor progression. Cumulatively, these findings indicate that vaccination with Tem1-TT specifically targets the TEM1-expressing cells of the vascular system and can functionally disrupt the tumor vasculature.

Breaking tolerance to TEM1 with DNA vaccine required the fusion of Tem1 and TT genes. TT may enhance immunogenicity through several mechanisms; the whole C fragment of TT activates DCs to secrete cytokines involved in CD4⁺ T cell activation (11, 65). In addition, fragment C contains universal T helper epitopes (p30, p2), which are effective across different MHC class II haplotypes in mice and humans and elicit strong CD4⁺ responses (66). Peptides within domain 2 of fragment C are strong CD8⁺ CTL epitopes, which may compete with the fusion peptides. Elimination of domain 2 enhances CTL responses to fused peptides (67), and thus we eliminated it from our TT construct accordingly. In addition, we generated localized inflammation by in vivo electrogene therapy during both prime and boost injections. Electrogene therapy enhances responsiveness (68, 69) and has facilitated translation of DNA vaccines into clinical trials (12, 70, 71). All of these attributes may be contributing to the generation of potent anti-TEM1 immune responses we observed with Tem1-TT immunization.

Our approach used full-length antigen rather than peptide, thus bypassing a major limitation of peptide-based cancer vaccines, namely MHC restriction. Genetic fusion of TT to the 3′ end of full-length antigens has been tested previously by us and others (13, 72). Our DNA vaccine elicited CD4⁺ and CD8⁺ anti-TEM1 responses in the BALB/c mouse and a CD8⁺ response in the C57BL/6 mouse. Given the prime/boost approach we used, it is likely that the responses observed are directed against the immunodominant TEM1 epitopes in each mouse strain (11, 73). Other adjuvant formulations may identify alternative dominant and/or subdominant epitopes.

In previous mouse studies, DNA vaccination with the tumor-associated antigens AH1 and E7 induced CD8⁺ cell–dependent regression of established tumors that expressed those antigens (33, 74). In the studies presented here, fusion of the TEM1 antigen to the TT adjuvant increased the immunogenicity of the target antigen and the efficacy of tumor control. It also generated robust CTL activity against tumor cell proper immunodominant epitopes, such as AH1 and E7. This was not achieved by direct targeting of these antigens, but rather indirectly by targeting the vasculature antigen TEM1. Taken together, our data suggest a model whereby Tem1-TT vaccination induces expansion of TEM1-specific T lymphocytes (Figure 8). These effector T cells target TEM1-expressing cells in the tumor vasculature, including tumor-associated ECs and/or pericytes. This immune attack impairs the tumor vasculature, with increases in local tumor-associated hypoxia and likely inflammation-related mediators, resulting in apoptosis of local tumor cells. These apoptotic bodies are scavenged by macrophages and DCs that process and present additional tumor-associated antigens. This secondary cross-priming event then expands tumor-specific cytotoxic CD8⁺ T cells, which work hand in hand with the anti-TEM1 immune response to eradicate larger numbers of tumor cells, resulting in improved control of tumor growth. The anti-TEM1 response and the antitumor immune responses against AH1 and E7 significantly correlated with control of tumor growth. Further studies will address the kinetics of the TEM1 and AH1 immune response. Our findings are consistent with the general paradigm of epitope spreading as a mechanism underlying superior immunotherapeutic outcomes and extend this concept to include T cell specificities against tumor-associated vasculature antigens (75).

Figure 8

Proposed mechanism of action of Tem1-TT vaccination. (i) Tem1-TT vaccine induces expansion of TEM1-specific CD8⁺ T cells that target the CT26 tumor vasculature. These cytolytic effectors target and kill TEM1-expressing cells, most likely tumor-associated endothelial cells and/or pericytes. (ii) This event results in reduction of the tumor vasculature, inducing inflammation and tumor hypoxia, which results in localized tumor cell death. (iii) Dead tumor cells are scavenged by APCs, which process relevant tumor-associated antigens (TAA; e.g., AH1 antigen in CT26 tumor model). (iv) This cross-presentation event results in a secondary cross-priming event that expands tumor-specific cytotoxic CD8⁺ T cells to tumor cell–derived epitopes. (v) Tumor-specific cytotoxic CD8⁺ T cells lyse and eliminate tumor cells, resulting in control of tumor growth.

The results of our adoptive transfer experiments (Supplemental Figure 2) suggest that tumor control by Tem1-TT vaccination is mediated primarily by CD3⁺ cells, while serum (containing antibodies) does not play any major therapeutic role. We did not directly measure anti-TEM1 antibodies potentially generated by Tem1-TT, and therefore cannot entirely rule out the possibility that anti-TEM1 antibodies contribute to the observed efficacy of Tem1-TT vaccine in some capacity. Adoptive cell transfer also illustrated the effective generation of memory responses: mice challenged with tumor 6 months after adoptive transfer of CD8⁺ T cells remained tumor free (data not shown), which illustrated that Tem1-TT generated robust, long-lasting adaptive immune responses. The adoptive transfer experiments with the CT26 model showed that Tem1-TT vaccination generated TEM1-specific CD3⁺ T cells that, on their own, could directly delay tumor growth and confer survival advantage to mice. These data also showed that Tem1-TT vaccination generated a dually specific T cell response, with a primary TEM1-specific response targeting the tumor vasculature generated by Tem1-TT vaccine, and a secondary response targeting the tumor cell–specific antigen AH1 generated by epitope spreading.

An ideal tumor vasculature antigen is one that is overexpressed in tumors and poorly expressed in peripheral tissues. Expression of TEM1 in the mouse is spatiotemporally regulated. By using a lacZ knockin model, Tem1 gene expression was initially detectable in the kidney of prenatal embryos (45). Postnatally, Tem1 expression decreases in most organs, but persists in the renal glomerulus and in the adult mouse uterus. In mice immunized with Tem1-TT, we did not observe toxicity in association with angiogenic processes, such as reproduction or wound healing. It is perhaps surprising that the Tem1-TT vaccine could mediate tumor vessel destruction without causing significant toxicity during pregnancy and wound healing. However, angiogenesis during tumor development differs significantly from the uterine NK cell–driven angiogenesis seen during implantation and placenta development (76, 77) and from the highly organized, slow growth of wound healing. In addition, tissue specificity and cells involved in tumor vessel recruitment differ from superficial wounds, although many similarities exist (78).

Cumulatively, our data indicated that immunization with a DNA vaccine encoding Tem1-TT induces TEM1-specific cell-mediated immune responses, disrupts the tumor vasculature, and controls tumor growth without affecting important normal physiological functions. Our preclinical data set can serve as a foundation for clinical studies targeting human TEM1 tumor vasculature antigen.

View Supplemental data

This project was supported by the Basser Research Center for BRCA, the Alliance for Cancer Gene Therapy, the NIH Director’s New Innovator Award (1DP2OD008514), and the Pennsylvania Department of Health (4100051725).

Address correspondence to: Andrea Facciabene, Ovarian Cancer Research Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. Phone: 215.746.7071; Fax: 215.573.5408; E-mail: facciabe@mail.med.upenn.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2014;124(4):1497–1511. doi:10.1172/JCI67382.

1. Chung AS, Lee J, Ferrara N. Targeting the tumour vasculature: insights from physiological angiogenesis. Nat Rev Cancer. 2010;10(7):505–514.
   View this article via: PubMed CrossRef Google Scholar
2. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285(21):1182–1186.
   View this article via: PubMed CrossRef Google Scholar
3. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407(6801):249–257.
   View this article via: PubMed CrossRef Google Scholar
4. St Croix B, et al. Genes expressed in human tumor endothelium. Science. 2000;289(5482):1197–1202.
   View this article via: PubMed CrossRef Google Scholar
5. Jain RK. Molecular regulation of vessel maturation. Nat Med. 2003;9(6):685–693.
   View this article via: PubMed CrossRef Google Scholar
6. Teicher BA. Newer vascular targets: endosialin (review). Int J Oncol. 2007;30(2):305–312.
   View this article via: PubMed Google Scholar
7. Buckanovich RJ, et al. Tumor vascular proteins as biomarkers in ovarian cancer. J Clin Oncol. 2007;25(7):852–861.
   View this article via: PubMed CrossRef Google Scholar
8. Sasaroli D, et al. Novel surface targets and serum biomarkers from the ovarian cancer vasculature. Cancer Biol Ther. 2011;12(3):169–180.
   View this article via: PubMed CrossRef Google Scholar
9. Khong HT, Restifo NP. Natural selection of tumor variants in the generation of “tumor escape” phenotypes. Nat Immunol. 2002;3(11):999–1005.
   View this article via: PubMed CrossRef Google Scholar
10. Liu MA. DNA vaccines: an historical perspective and view to the future. Immunol Rev. 2010;239(1):62–84.
    View this article via: PubMed CrossRef Google Scholar
11. Rice J, Ottensmeier CH, Stevenson FK. DNA vaccines: precision tools for activating effective immunity against cancer. Nat Rev Cancer. 2008;8(2):108–120.
    View this article via: PubMed CrossRef Google Scholar
12. Stevenson FK, Mander A, Chudley L, Ottensmeier CH. DNA fusion vaccines enter the clinic. Cancer Immunol Immunother. 2011;60(8):1147–1151.
    View this article via: PubMed CrossRef Google Scholar
13. Facciabene A, et al. DNA and adenoviral vectors encoding carcinoembryonic antigen fused to immunoenhancing sequences augment antigen-specific immune response and confer tumor protection. Hum Gene Ther. 2006;17(1):81–92.
    View this article via: PubMed CrossRef Google Scholar
14. Brady J, Neal J, Sadakar N, Gasque P. Human endosialin (tumor endothelial marker 1) is abundantly expressed in highly malignant and invasive brain tumors. J Neuropathol Exp Neurol. 2004;63(12):1274–1283.
    View this article via: PubMed Google Scholar
15. Rouleau C, et al. Endosialin protein expression and therapeutic target potential in human solid tumors: sarcoma versus carcinoma. Clin Cancer Res. 2008;14(22):7223–7236.
    View this article via: PubMed CrossRef Google Scholar
16. Carson-Walter EB, et al. Characterization of TEM1/endosialin in human and murine brain tumors. BMC Cancer. 2009;9:417.
    View this article via: PubMed CrossRef Google Scholar
17. Bagley RG. Endosialin: from vascular target to biomarker for human sarcomas. Biomark Med. 2009;3(5):589–604.
    View this article via: PubMed CrossRef Google Scholar
18. MacFadyen J, Savage K, Wienke D, Isacke CM. Endosialin is expressed on stromal fibroblasts and CNS pericytes in mouse embryos and is downregulated during development. Gene Expr Patterns. 2007;7(3):363–369.
    View this article via: PubMed CrossRef Google Scholar
19. MacFadyen JR, et al. Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium. FEBS Lett. 2005;579(12):2569–2575.
    View this article via: PubMed CrossRef Google Scholar
20. Christian S, et al. Endosialin (Tem1) is a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells. Am J Pathol. 2008;172(2):486–494.
    View this article via: PubMed CrossRef Google Scholar
21. Ohradanova A, et al. Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2. Br J Cancer. 2008;99(8):1348–1356.
    View this article via: PubMed CrossRef Google Scholar
22. Rupp C, et al. Mouse endosialin, a C-type lectin-like cell surface receptor: expression during embryonic development and induction in experimental cancer neoangiogenesis. Cancer Immun. 2006;6:10.
    View this article via: PubMed Google Scholar
23. Draenert R, et al. Comparison of overlapping peptide sets for detection of antiviral CD8 and CD4 T cell responses. J Immunol Methods. 2003;275(1–2):19–29.
    View this article via: PubMed CrossRef Google Scholar
24. Zhao A, Nunez-Cruz S, Li C, Coukos G, Siegel DL, Scholler N. Rapid isolation of high-affinity human antibodies against the tumor vascular marker Endosialin/TEM1, using a paired yeast-display/secretory scFv library platform. J Immunol Methods. 2011;363(2):221–232.
    View this article via: PubMed CrossRef Google Scholar
25. Kano MR, et al. Comparison of the effects of the kinase inhibitors imatinib, sorafenib, and transforming growth factor-beta receptor inhibitor on extravasation of nanoparticles from neovasculature. Cancer Sci. 2009;100(1):173–180.
    View this article via: PubMed CrossRef Google Scholar
26. Wood AK, Bunte RM, Cohen JD, Tsai JH, Lee WM, Sehgal CM. The antivascular action of physiotherapy ultrasound on a murine tumor: role of a microbubble contrast agent. Ultrasound Med Biol. 2007;33(12):1901–1910.
    View this article via: PubMed CrossRef Google Scholar
27. Seiler GS, Ziemer LS, Schultz S, Lee WM, Sehgal CM. Dose-response relationship of ultrasound contrast agent in an in vivo murine melanoma model. Cancer Imaging. 2007;7:216–223.
    View this article via: PubMed CrossRef Google Scholar
28. van der Most RG, Currie A, Robinson BW, Lake RA. Cranking the immunologic engine with chemotherapy: using context to drive tumor antigen cross-presentation towards useful antitumor immunity. Cancer Res. 2006;66(2):601–604.
    View this article via: PubMed CrossRef Google Scholar
29. Ribas A, Timmerman JM, Butterfield LH, Economou JS. Determinant spreading and tumor responses after peptide-based cancer immunotherapy. Trends Immunol. 2003;24(2):58–61.
    View this article via: PubMed CrossRef Google Scholar
30. Seavey MM, Paterson Y. Anti-angiogenesis immunotherapy induces epitope spreading to Her-2/neu resulting in breast tumor immunoediting. Breast Cancer (London). 2009;1:19–30.
    View this article via: PubMed Google Scholar
31. Zhao X, et al. Vaccines targeting tumor blood vessel antigens promote CD8(+) T cell-dependent tumor eradication or dormancy in HLA-A2 transgenic mice. J Immunol. 2012;188(4):1782–1788.
    View this article via: PubMed CrossRef Google Scholar
32. Huang AY, et al. The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product. Proc Natl Acad Sci U S A. 1996;93(18):9730–9735.
    View this article via: PubMed CrossRef Google Scholar
33. Rice J, Buchan S, Stevenson FK. Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen. J Immunol. 2002;169(7):3908–3913.
    View this article via: PubMed Google Scholar
34. Feltkamp MC, et al. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol. 1993;23(9):2242–2249.
    View this article via: PubMed CrossRef Google Scholar
35. Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med. 1999;341(10):738–746.
    View this article via: PubMed CrossRef Google Scholar
36. Scappaticci FA, et al. Surgical wound healing complications in metastatic colorectal cancer patients treated with bevacizumab. J Surg Oncol. 2005;91(3):173–180.
    View this article via: PubMed CrossRef Google Scholar
37. Sharma K, Marcus JR. Bevacizumab and wound-healing complications: mechanisms of action, clinical evidence, and management recommendations for the plastic surgeon. Ann Plast Surg. 2013;71(4):434–440.
    View this article via: PubMed CrossRef Google Scholar
38. Ferrara N, et al. Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nat Med. 1998;4(3):336–340.
    View this article via: PubMed Google Scholar
39. Fraser HM, Wulff C. Angiogenesis in the corpus luteum. Reprod Biol Endocrinol. 2003;1:88.
    View this article via: PubMed CrossRef Google Scholar
40. Demir R, Kayisli UA, Cayli S, Huppertz B. Sequential steps during vasculogenesis and angiogenesis in the very early human placenta. Placenta. 2006;27(6–7):535–539.
    View this article via: PubMed CrossRef Google Scholar
41. Klauber N, Rohan RM, Flynn E, D’Amato RJ. Critical components of the female reproductive pathway are suppressed by the angiogenesis inhibitor AGM-1470. Nat Med. 1997;3(4):443–446.
    View this article via: PubMed CrossRef Google Scholar
42. Rutland CS, Mukhopadhyay M, Underwood S, Clyde N, Mayhew TM, Mitchell CA. Induction of intrauterine growth restriction by reducing placental vascular growth with the angioinhibin TNP-470. Biol Reprod. 2005;73(6):1164–1173.
    View this article via: PubMed CrossRef Google Scholar
43. Ito T, Ando H, Handa H. Teratogenic effects of thalidomide: molecular mechanisms. Cell Mol Life Sci. 2011;68(9):1569–1579.
    View this article via: PubMed CrossRef Google Scholar
44. Lax S, et al. CD248/Endosialin is dynamically expressed on a subset of stromal cells during lymphoid tissue development, splenic remodeling and repair. FEBS Lett. 2007;581(18):3550–3556.
    View this article via: PubMed CrossRef Google Scholar
45. Huang HP, et al. Gene targeting and expression analysis of mouse Tem1/endosialin using a lacZ reporter. Gene Expr Patterns. 2011;11(5–6):316–326.
    View this article via: PubMed CrossRef Google Scholar
46. Kamat BR, Brown LF, Manseau EJ, Senger DR, Dvorak HF. Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Role in corpus luteum development. Am J Pathol. 1995;146(1):157–165.
    View this article via: PubMed Google Scholar
47. Delli Carpini J, Karam AK, Montgomery L. Vascular endothelial growth factor and its relationship to the prognosis and treatment of breast, ovarian, and cervical cancer. Angiogenesis. 2010;13(1):43–58.
    View this article via: PubMed CrossRef Google Scholar
48. Rafii S. Vaccination against tumor neovascularization: Promise and reality. Cancer Cell. 2002;2(6):429–431.
    View this article via: PubMed CrossRef Google Scholar
49. Wei YQ, et al. Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine. Nat Med. 2000;6(10):1160–1166.
    View this article via: PubMed CrossRef Google Scholar
50. Okaji Y, et al. Vaccination with autologous endothelium inhibits angiogenesis and metastasis of colon cancer through autoimmunity. Cancer Sci. 2004;95(1):85–90.
    View this article via: PubMed CrossRef Google Scholar
51. Zhao X, et al. Intratumoral IL-12 gene therapy results in the crosspriming of Tc1 cells reactive against tumor-associated stromal antigens. Mol Ther. 2011;19(4):805–814.
    View this article via: PubMed CrossRef Google Scholar
52. Chinnasamy D, et al. Gene therapy using genetically modified lymphocytes targeting VEGFR-2 inhibits the growth of vascularized syngenic tumors in mice. J Clin Invest. 2010;120(11):3953–3968.
    View this article via: JCI PubMed CrossRef Google Scholar
53. Bagley RG, et al. Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248. Mol Cancer Ther. 2008;7(8):2536–2546.
    View this article via: PubMed CrossRef Google Scholar
54. Simonavicius N, Robertson D, Bax DA, Jones C, Huijbers IJ, Isacke CM. Endosialin (CD248) is a marker of tumor-associated pericytes in high-grade glioma. Mod Pathol. 2008;21(3):308–315.
    View this article via: PubMed CrossRef Google Scholar
55. Wesseling P, Schlingemann RO, Rietveld FJ, Link M, Burger PC, Ruiter DJ. Early and extensive contribution of pericytes/vascular smooth muscle cells to microvascular proliferation in glioblastoma multiforme: an immuno-light and immuno-electron microscopic study. J Neuropathol Exp Neurol. 1995;54(3):304–310.
    View this article via: PubMed CrossRef Google Scholar
56. Carson-Walter EB, Watkins DN, Nanda A, Vogelstein B, Kinzler KW, St Croix B. Cell surface tumor endothelial markers are conserved in mice and humans. Cancer Res. 2001;61(18):6649–6655.
    View this article via: PubMed Google Scholar
57. Christian S, et al. Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium. J Biol Chem. 2001;276(10):7408–7414.
    View this article via: PubMed CrossRef Google Scholar
58. Rettig WJ, Garin-Chesa P, Healey JH, Su SL, Jaffe EA, Old LJ. Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer. Proc Natl Acad Sci U S A. 1992;89(22):10832–10836.
    View this article via: PubMed CrossRef Google Scholar
59. Buckanovich RJ, et al. Use of immuno-LCM to identify the in situ expression profile of cellular constituents of the tumor microenvironment. Cancer Biol Ther. 2006;5(6):635–642.
    View this article via: PubMed CrossRef Google Scholar
60. Walter-Yohrling J, et al. Murine endothelial cell lines as models of tumor endothelial cells. Clin Cancer Res. 2004;10(6):2179–2189.
    View this article via: PubMed CrossRef Google Scholar
61. Davies G, Cunnick GH, Mansel RE, Mason MD, Jiang WG. Levels of expression of endothelial markers specific to tumour-associated endothelial cells and their correlation with prognosis in patients with breast cancer. Clin Exp Metastasis. 2004;21(1):31–37.
    View this article via: PubMed CrossRef Google Scholar
62. Becker R, et al. Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis-related protein Mac-2 BP/90K. FASEB J. 2008;22(8):3059–3067.
    View this article via: PubMed CrossRef Google Scholar
63. Tomkowicz B, et al. Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proc Natl Acad Sci U S A. 2007;104(46):17965–17970.
    View this article via: PubMed CrossRef Google Scholar
64. Nanda A, et al. Tumor endothelial marker 1 (Tem1) functions in the growth and progression of abdominal tumors. Proc Natl Acad Sci U S A. 2006;103(9):3351–3356.
    View this article via: PubMed CrossRef Google Scholar
65. Sparwasser T, et al. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol. 1998;28(6):2045–2054.
    View this article via: PubMed CrossRef Google Scholar
66. Panina-Bordignon P, Tan A, Termijtelen A, Demotz S, Corradin G, Lanzavecchia A. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol. 1989;19(12):2237–2242.
    View this article via: PubMed CrossRef Google Scholar
67. Rice J, Elliott T, Buchan S, Stevenson FK. DNA fusion vaccine designed to induce cytotoxic T cell responses against defined peptide motifs: implications for cancer vaccines. J Immunol. 2001;167(3):1558–1565.
    View this article via: PubMed Google Scholar
68. Buchan S, Gronevik E, Mathiesen I, King CA, Stevenson FK, Rice J. Electroporation as a “prime/boost” strategy for naked DNA vaccination against a tumor antigen. J Immunol. 2005;174(10):6292–6298.
    View this article via: PubMed Google Scholar
69. Ahlen G, et al. In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells. J Immunol. 2007;179(7):4741–4753.
    View this article via: PubMed Google Scholar
70. Stevenson FK, Rice J, Ottensmeier CH, Thirdborough SM, Zhu D. DNA fusion gene vaccines against cancer: from the laboratory to the clinic. Immunol Rev. 2004;199:156–180.
    View this article via: PubMed Google Scholar
71. Stevenson FK, Ottensmeier CH, Rice J. DNA vaccines against cancer come of age. Curr Opin Immunol. 2010;22(2):264–270.
    View this article via: PubMed CrossRef Google Scholar
72. Chen CH, et al. Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene. Cancer Res. 2000;60(4):1035–1042.
    View this article via: PubMed Google Scholar
73. Yewdell JW, Bennink JR. Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol. 1999;17:51–88.
    View this article via: PubMed CrossRef Google Scholar
74. Oosterhuis K, et al. Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7. Int J Cancer. 2011;129(2):397–406.
    View this article via: PubMed CrossRef Google Scholar
75. Seavey MM, Maciag PC, Al-Rawi N, Sewell D, Paterson Y. An anti-vascular endothelial growth factor receptor 2/fetal liver kinase-1 Listeria monocytogenes anti-angiogenesis cancer vaccine for the treatment of primary and metastatic Her-2/neu+ breast tumors in a mouse model. J Immunol. 2009;182(9):5537–5546.
    View this article via: PubMed CrossRef Google Scholar
76. van den Heuvel MJ, et al. A review of trafficking and activation of uterine natural killer cells. Am J Reprod Immunol. 2005;54(6):322–331.
    View this article via: PubMed CrossRef Google Scholar
77. Bilinski MJ, et al. Uterine NK cells in murine pregnancy. Reprod Biomed Online. 2008;16(2):218–226.
    View this article via: PubMed CrossRef Google Scholar
78. Schafer M, Werner S. Cancer as an overhealing wound: an old hypothesis revisited. Nat Rev Mol Cell Biol. 2008;9(8):628–638.
    View this article via: PubMed Google Scholar
79. Facciabene A, et al. Vectors encoding carcinoembryonic antigen fused to the B subunit of heat-labile enterotoxin elicit antigen-specific immune responses and antitumor effects. Vaccine. 2007;26(1):47–58.
    View this article via: PubMed CrossRef Google Scholar
80. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods. 2008;5(10):877–879.
    View this article via: PubMed CrossRef Google Scholar
81. Mennuni C, et al. Efficient induction of T-cell responses to carcinoembryonic antigen by a heterologous prime-boost regimen using DNA and adenovirus vectors carrying a codon usage optimized cDNA. Int J Cancer. 2005;117(3):444–455.
    View this article via: PubMed CrossRef Google Scholar
82. Sehgal CM, et al. Renal blood flow changes induced with endothelin-1 and fenoldopam mesylate at quantitative Doppler US: initial results in a canine study. Radiology. 2001;219(2):419–426.
    View this article via: PubMed CrossRef Google Scholar
83. Sehgal CM, Arger PH, Rowling SE, Conant EF, Reynolds C, Patton JA. Quantitative vascularity of breast masses by Doppler imaging: regional variations and diagnostic implications. J Ultrasound Med. 2000;19(7):427–440.
    View this article via: PubMed CrossRef Google Scholar
84. Cha HR, et al. Mucosa-associated epithelial chemokine/CCL28 expression in the uterus attracts CCR10+ IgA plasma cells following mucosal vaccination via estrogen control. J Immunol. 2011;187(6):3044–3052.
    View this article via: PubMed CrossRef Google Scholar
85. Li Y, et al. Active immunization against the vascular endothelial growth factor receptor flk1 inhibits tumor angiogenesis and metastasis. J Exp Med. 2002;195(12):1575–1584.
    View this article via: PubMed CrossRef Google Scholar