Targeting tumor vasculature: expanding the potential of DNA cancer vaccines

Targeting the tumor vasculature with anti-angiogenesis modalities is a bona fide validated approach that has complemented cancer treatment paradigms. Tumor vasculature antigens (TVA) can be immunologically targeted and offers multiple theoretical advantages that may enhance existing strategies against cancer. We focused on tumor endothelial marker 1 (TEM1/CD248) as a model TVA since it is broadly expressed on many different cancers. Our DNA-based vaccine approach demonstrated that CD248 can be effectively targeted immunologically; anti-tumor responses were generated in several mouse models; and CD8+/CD4+ T cell responses were elicited against peptides derived from CD248 protein. Our work supports our contention that CD248 is a novel immunotherapeutic target for cancer treatment and highlights the efficient, safe and translatable use of DNA-based immunotherapy. We next briefly highlight ongoing investigations targeting CD248 with antibodies as a diagnostic imaging agent and as a therapeutic antibody in an early clinical trial. The optimal approach for generating effective DNA-based cancer vaccines for several tumor types may be a combinatorial approach that enhances immunogenicity such as combination with chemotherapy. Additional combination approaches are discussed and include those that alleviate the immunosuppressive tumor microenvironment induced by myeloid-derived suppressor cells and T regulatory cells. Targeting the tumor vasculature by CD248-based immunological modalities expands the armamentarium against cancer.

Tumor vasculature, Tumor endothelial marker (TEM), DNA cancer vaccine, PIVAC-2014

Vasculature formation depends on the process of angiogenesis, which is responsible for the remodeling and expansion of the existing vasculature network and vasculogenesis, a process whereby endothelial precursor cells (EPC) migrate and differentiate in response to local signals (such as growth factors from stroma and extracellular matrix) to form new blood vessels. Vasculogenesis is a fundamental process during embryogenesis and in adults takes place during tumor growth, following trauma and in endometriosis [1]. In normal tissues, angiogenesis is a tightly regulated physiological process that comprises two different mechanisms: endothelial cells (EC) sprouting and intussusceptive microvascular growth (IMG), which are regulated by several endogenous activator (pro-angiogenic factors) and inactivator (anti-angiogenic factors) molecules. Pro-angiogenic factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMP), transforming growth factor (TGF)-β and angiopoietins among others. Anti-angiogenic factors include interferon (IFN)-γ, CXCL9, CXCL10, thrombospondin 1, endostatin and angiostatin [2]. For cancer progression, angiogenesis is a necessary step in the uncontrolled proliferation, invasion, and metastatic dissemination of tumor cells, involving the formation and remodeling of new vessels to “feed” the tumor and promote its progression. Cancer angiogenesis begins when tumor nodules reach dimensions over a few millimeters, and nutrient/oxygen diffusion is no longer sufficient to sustain both the physiological cell cycle and basic cell metabolism. In response to these unfavorable conditions, cancer cells produce pro-angiogenic signals that are able to activate resting EC and stromal cells. The activated EC acquire the ability to proliferate, migrate, differentiate and remodel adjacent extracellular matrix in association with pericytes to stabilize the new blood vessels [3]. Anti-vascular strategies are mainly classified into two groups: anti-angiogenic and vascular-disrupting therapies. The VEGF–VEGF receptor (VEGFR) signaling axis has emerged as one of the most promising angiogenic targets because of its central role in tumor angiogenesis and growth. VEGFA (commonly referred to as “VEGF”) has been identified as the predominant pro-angiogenic growth factor expressed by tumor cells and binds both VEGFR1 and VEGFR2 [4, 5]. Angiogenesis has become an accepted target for anticancer therapy since 2004 when bevacizumab, a VEGF-binding antibody, became the first angiogenesis inhibitor to be approved in the USA for the treatment of several metastatic cancer types (as a single or in combination with different cytotoxic chemotherapy) including colorectal cancer, non-squamous non-small cell lung cancer, renal cell carcinoma, cervical cancer, ovarian cancer and as a monotherapy for glioblastoma (Avastin Prescribing Information, 2015). Major side effects of bevacizumab result from the undesirable targeting of normal vasculature including gastrointestinal perforation, wound healing complications, hemorrhaging and effects on reproduction (Avastin Prescribing Information, 2015). The VEGFR signaling pathway as well as other angiogenic receptors (EGFR, PDGFR, FGFR) has also been targeted with the development of small molecule tyrosine kinase inhibitors such as vandetanib, cabozantinib, sorafenib, ponatinib and lenvatinib [6]. Vascular-disrupting agents (VDA), a relatively new class of drugs in clinical development, target and destroy preexisting tumor vessels, resulting in tumor cell death from ischemia and necrosis [7]. Preclinical data using VDA have confirmed the extraordinary potential of this approach to control tumor growth by inducing massive necrosis of cancer cells. VDA small molecules, such as tubulin-depolymerizing agents and flavonoids, have already produced promising preclinical results, although they lack robust specificity and induce neurological and cardiovascular side effects as observed in a phase I clinical trial [8]. The development of novel, effective and safe VDA for cancer therapy has been currently hindered by both the paucity of selective targets for different tumors and the lack of reagents with optimal affinity. In the last several decades, anticancer vaccination strategies have been developed by different approaches preferentially based on targeting tumor cells expressing tumor-associated antigens (TAA) [9]; however, tumor cells are genetically unstable and when immune pressure is applied, can rapidly give rise to antigen escape variants, resulting in treatment failures. Development and delivery of immunotherapy directed against tumor vasculature antigens (TVA) offer multiple theoretical advantages: (1) TVA are overexpressed on cancers of different histological types, whereas they are generally absent or poorly expressed on most normal tissue; (2) the tumor vasculature and stroma are genetically stable as compared to tumor cells, which can minimize escape variants [10, 11]; (3) TVA-targeting therapies can be broadly applicable to different cancers [12]; (4) TVA targeting is endowed with powerful effector lymphocytes that can persist in tumor-bearing hosts, leaving a long-lasting memory response that protects the host against tumor recurrence; (5) TVA-targeting efficacy can be enhanced in combination with different pharmacological/immunological strategies as we describe in the last section. In this review, we describe recent findings on the application of targeting the tumor vasculature with a CD248-specific DNA vaccine, highlighting its rationale, its therapeutic effectiveness and latest developments as well as potential future areas of clinical improvement. CD248 background As reported by Brad St. Croix et al. [13], colorectal tumor endothelium was found to up-regulate several genes compared to the normal intestinal endothelium; nine genes were overexpressed up to tenfold in tumor-derived versus normal-derived endothelium and were dubbed tumor endothelial marker (TEM). TEM overexpression in tumor vessels was confirmed by reverse transcriptase polymerase chain reaction and in situ hybridization [14]. These TEMs are found on the cell surface and although are not structurally related (TEM1, 3, 5, 7, 8), they are functionally conserved between mouse and human tumor endothelium. These two attributes make TEMs attractive candidates for immunotherapeutic interventions because these targets are directly accessible to the immune cells via the tumor vasculature. The most differentially expressed TEM, TEM1 (CD248) was found to be the same antigen, previously dubbed endosialin, a highly sialylated glycoprotein found on many different tumor types described first in the Rettig seminal paper [15]. Human CD248 mRNA and/or protein has been characterized in various human tumor settings including colorectal cancer, brain tumors and sarcomas [16–18]. CD248 has also been reported to be expressed by tumor vessel-associated pericytes [19] and stromal fibroblasts [20] on a broad variety of human tumors with different histology but not in normal vessels [21]. Moreover, CD248 has also been classified as a marker of tumor vessel-associated pericyte cells and tumor-associated myofibroblasts [22] as well as a selective EPC marker [23]. Pericytes provide structural support for EC and therefore stabilize the vasculature. CD248 plays a role in physiological processes such as cell adhesion, neo-angiogenesis, cell migration and tissue development [24] and has an essential role in promoting tumor growth; its overexpression negatively correlated with the clinical outcome of cancer patients [25]. Using a knockout mouse model for CD248 expression (Tem1−/− mice), researchers were able to identify the physiological and biological role of CD248 in promoting tumor progression. Tumor growth was inhibited in Tem1−/− mice, and metastatic dissemination was also reduced. Normal wound healing was observed in Tem1−/− mice compared to wild-type mice [24]. These data suggest that targeting CD248 may generate efficient therapeutic effects with minimal toxicity. CD248 targeting by DNA vaccine as a useful approach to strike tumor vasculature To test the hypothesis that CD248 is a suitable candidate for cancer immunotherapy, we designed an active immunotherapeutic approach based on a DNA-based vaccine in conjunction with in vivo gene transfer [26]. To increase the immunogenicity of CD248 and break murine tolerance, we developed a DNA vaccine based on fusing mouse CD248 cDNA with the cDNA of the amino terminal domain of fragment C of tetanus toxoid (TT). The TT fragment DNA was introduced at the 3′ end of the CD248 coding sequence, generating the plasmid called pcDNA3.1/TEM1-TT. The TEM1-TT DNA sequence was codon-optimized to increase expression yields up to 100-fold as well as inclusion of CpG motifs which activate TLR9 to increase immunogenicity of the DNA vaccine. As an adjuvant, TT may enhance immunogenicity through several mechanisms; the whole C fragment of TT activates dendritic cells (DC) to secrete cytokines involved in CD4+ T cell activation and Th1 polarization, such as interleukin (IL)-12, IL-23 and tumor necrosis factor (TNF)-α [27]. In addition, fragment C contains universal T helper epitopes (p30, p2), which are effective across different major histocompatibility complex (MHC) class II haplotypes in mice and humans and elicits strong CD4+ T responses [28]. Lastly, electrogene therapy induces local inflammation and promotes a favorable microenvironment that is able to stimulate T cell fitness and functionality [29]. We have reported that mice vaccinated with TEM1-TT plasmid vaccine were able to develop T lymphocytes with strong reactivity against a CD248-specific peptide; vaccinated C57BL/6 mice generated a specific CD8+ T cell response toward the TEM1696–710 peptide (GQSQRDDRWLLVALL). TEM1-TT-vaccinated BALB/c mice responded with elicitation of a CD8+ and a CD4+ T cell response against two CD248 peptides: TEM1516–530 (ITSATHPARSPPYQP) and TEM1511–525 (GHKPGITSATHPARS). Moreover, the identified CD248-derived peptides were able to expand CD248-specific cytotoxic T lymphocytes (CTL) in vitro that selectively recognized CD248-expressing endothelium immortalized cells such as H5V cells. Our data are the first demonstration of CD248 immunogenicity and establishes a foundation upon which to develop selective immunotherapy against this potential TVA. TEM1-TT vaccine was able to control in vivo tumor progression of different transplantable tumor models by both prophylactic and therapeutic immunization [26]. The therapeutic effectiveness of our vaccine can be summarized in three parts as schematically shown in Fig. 1. Firstly, TEM1-TT vaccine elicits anti-CD248 CTLs that specifically attack the tumor vasculature, resulting in significant inhibition and ablation of the tumor vasculature [26]. The TEM1-TT vaccine modified the tumor vessel architecture as demonstrated by reduction of CD31+ EC and/or pericytes in the CT26 tumor model. By RNA FISH, CD248 mRNA was found to localize with, or to be in close juxtaposition to, CD31 mRNA, suggesting that EC and/or pericytes are expressing CD248 [26]. We also show that in TEM1-TT-vaccinated mice, CT26 tumor contained CD31+ cells that were also TUNEL positive, suggesting that CD31+ EC/pericytes were in an apoptotic state [26]. Pericyte coverage on EC provides the structural support for the vasculature. Since CD248 is expressed by tumor vessel-associated pericyte cells [22], TEM1-TT vaccine may also target pericytes directly, with subsequent destabilization and inhibition of the tumor endothelial cell network. This selective inhibition of tumor vascularity was also verified by in vivo ultrasound imaging which allowed us to define physical vascular parameters of the tumor mass such as the perfused tumor area and the red blood cell flux per unit area of tissue. Collectively, our data demonstrate that TEM1-TT vaccine functionally destabilizes the tumor vasculature, induces vessel collapse, as well as possibly impedes the assembly of new vasculature [26]. The second part of our proposed mechanism includes the consequences of tumor vessel destruction mediated by the TEM1-TT vaccine. We hypothesize that the lack of a constant stream of metabolites and oxygen leads to an increased hypoxic and inflammatory tumor microenvironment that promotes tumor cell death. Tumor cells from TEM1-TT-vaccinated mice showed an increase in tumor cell apoptosis. We verified that tumor masses isolated from TEM1-TT-vaccinated mice showed higher levels of carbonic anhydrase IX (CAIX), a cellular biomarker of hypoxia, which correlated with an elevated presence of apoptotic tumor areas. Furthermore, we evaluated tumor cells of TEM1-TT-vaccinated mice for Ki67 expression and found Ki67 down-regulation, indicating a less proliferative state compared to tumor cells from control-treated mice (Andrea Facciabene, unpublished results). The ability of our vaccine to provide a secondary tumor-specific immune response represents the third and last step of the therapeutic effectiveness of TEM1-TT vaccine. All of these above events (hypoxia, tumor EC/pericyte apoptosis, tumor cells’ proliferative status/apoptosis) can generate a source of antigenic determinants that can be engulfed by antigen-presenting cells (APC) such as DC and macrophages. These APC are able to process specific tumor cell antigens by cross-priming, promoting a secondary immune response [30] against the tumor-specific antigens. In our tumor models, CT26- and TC1-associated tumor apoptotic/necrotic bodies were processed by APC inducing a secondary immune response against specific TAA; the immunodominant antigen of CT26, gp70423–431 (AH1 peptide) is derived from viral antigen Gp70 encoded by the genome of BALB/c mice [31]. Gp70 is poorly expressed in normal tissues [32]; AH1-specific T cells escape negative selection that can therefore be activated and expanded by inflammation associated during tumor progression [33]. In the TC-1 tumor model, a C57BL/6 epithelial lung tumor, tumorigenesis is maintained by the expression of the viral proteins E6 and E7 of HPV origin. E7 is recognized by the immune system and elicits a selective CTL response against the E7 immunodominant peptide E749–57 [34]. Our data clearly demonstrate that splenocytes from TEM1-TT-vaccinated mice contained CD8+ T cell specific immune responses against CD248 immunodominant peptide (but not against the control CD248 peptide), as well as against the AH1 epitope in CT26-tumor-bearing mice or E749–57 epitope in TC1-tumor-bearing mice [26]. Moreover, the frequency of the specific TAA-related immune response correlated with the tumor volume, suggesting that the magnitude of the secondary immune response was important to control tumor progression. The induction of this secondary TAA-specific anti-tumor response amplifies the magnitude of the therapeutic effectiveness of the CD248-targeting strategy. To define the immune system component (adaptive and/or humoral) that was responsible for the anti-tumor effects, we performed CD3+ T cell adoptive transfer or adoptive serum transfer from TEM1-TT-immunized naïve donor mice into naïve mice that were irradiated and challenged with CT26 tumor and found that CD3+ T cells exclusively mediated the anti-tumor effects [26]. To characterize the potency of the TEM1-TT vasculature-targeting component with the secondary TAA immune response component, we performed CD3+ T cell adoptive transfer from either TEM1-TT-immunized mice (vaccinated only) donor mice or TEM1-TT-immunized tumor-bearing (vaccinated followed by tumor challenged) donor mice into recipient mice that were challenged with CT26 tumor. Both types of adoptively transferred donor cells significantly increased the survival of recipient mice once challenged with CT26. These data demonstrate the anti-CD248 immune response in the recipient mice adoptively transferred with CD3+ T cells from vaccinated only donor mice (without concomitant cross-priming and epitope spreading) was sufficiently potent to control tumor growth [26]. This observation suggests that the therapeutic ability of TEM1-TT vaccine is not secondary to development of an anti-TAA response but is solely capable, on its own, to limit tumor progression. We hypothesize that the anti-CD248 cellular immune response is the initial triggering event that generates and sustains a secondary TAA cross-priming event, expanding TAA tumor-specific cytotoxic CD8+ T cells that control tumor growth. Since neo-angiogenesis is also a physiological process that occurs during wound healing and reproduction, we investigated whether these two physiological processes were negatively affected by the TEM1-TT vaccine. Our results demonstrate that CD248 targeting did not mediate normal vessel damage in the skin by inducing toxicity during wound healing. Moreover, we showed that TEM1-TT immunization did not alter the pregnancy success rate, the time to gestation, the total litter size and the pup weight at birth compared with control immunization; there were also no anatomical or histological abnormalities in embryos from TEM1-TT- or control-immunized groups during early or late gestation. Lastly, our vaccine did not affect normal luteogenesis; in fact, TEM1-TT-immunized mice presented the same serum levels of 17β-estradiol, progesterone, luteinizing hormone and follicle-stimulating hormone during their estrus cycle compared to control-vaccinated mice [26]. These results are critical to demonstrate that TEM1-TT vaccine is a safe immunization strategy because it does not induce an immune response against physiological neo-angiogenesis compared with other anti-vascular therapy such as bevacizumab, which is associated with important safety concerns, including wound healing [35] and pregnancy [36]. Overall, our data demonstrate that targeting the vasculature with a TEM1-TT DNA-based vaccine is a safe immunotherapeutic approach and further investigations are warranted. Future perspective and potential applications Identification and characterization of TVA such as CD248 and other TEM family members have created strong rationale and motivation to develop cancer immunotherapy against the tumor vasculature. The feasibility of this approach has been verified by experiments based on the use of Tem1−/− mice, where it was demonstrated that the genetic ablation of CD248 was not lethal and was associated with both control of tumor progression, local invasion and a reduction of metastases [24]. Our strategy to break CD248 self-tolerance using the sequence of modified TT adjuvant lays the foundation for developing other potential fusion protein constructs using cDNA of CD248 or other TEMs in association with the cDNA of either other microbial-derived proteins, such as the heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli [37, 38], or other immunogenic molecules such as pathogen-associated molecular patterns (PAMP) [39]. The therapeutic effectiveness of electrogene transfer with the fusion vaccine encoding human CD248 (hCD248) cDNA will be tested in guiding clinical trials since the safety of DNA-based modalities has already been demonstrated [40]. Identification of the immunodominant peptide(s) of hCD248 should be a source of innovative clinical trials in which patients will be treated with new formulations of either emulsionated peptide immunization in which CD248 peptide will be injected with effective adjuvants such as montanide [41] or CD248 peptide-loaded DC vaccination protocols (Fig. 2). The most effective immunotherapy approach today is based on adoptive cell therapy (ACT) [42]. To test the potential of CD248-specific T cells, we adoptively transferred T cells from CD248-immunized mice into tumor-bearing mice and clearly demonstrate CD248-specific T cells are capable to reject established tumors. Engineered high-affinity anti-CD248 T cell ACT is a potential innovative approach in which the gene sequences of TCR α and β chains can be isolated and inserted into a lentiviral/retroviral vector carrying a bidirectional promoter capable of robust and coordinated expression of the two transgenes. Transduction of donor peripheral blood mononucleated cells (PBMC) with the lentiviral vector leads to generation of specific anti-hCD248-positive T cells. This strategy can overcome the obstacles of ACT in the clinic including technical factors limiting the availability of sufficient numbers of tumor-specific T cells as well as the availability of an antigen shared by tumors of different histologic patterns and conserved during tumor progression [43]. The potential of engineered human anti-CD248 T cells for adoptive transfer could be useful to treat a broad spectrum of histologically different cancers since many tumor types express CD248 to varying degrees. In addition to targeting CD248 with T cell-based immunotherapy protocols, other investigators have been developing both diagnostic and therapeutic antibodies against CD248 antigen. Radiolabeled 125I-Fc78 antibody was designed and based on a CD248-specific fully human single-chain antibody (scFv) 78, shown to bind with high affinity to both human and mouse CD248 [44, 45]. An ideal antibody-based therapy platform should have an antibody that can be used for diagnostic and therapeutic use (theranostic). The diagnostic antibody should be amenable to complexing to an imaging agent as well as complexing the antibody to a cytotoxic agent. The theranostic potential of MORAb-004 as a PET imaging tracer and naked antibody therapy for CD248+ tumor has recently been demonstrated [46]. (124)I-MORAb-004, a humanized monoclonal antibody targeting an extracellular epitope of hCD248, was evaluated for its ability to specifically and sensitively detect vascular cells expressing hCD248 in vivo [47]. Lastly, since CD248 is a cell surface marker, MORAb-004 antibody is currently undergoing phase I clinical trials for the treatment of patients with various cancers [15, 48]. With great enthusiasm, it is possible to use CD248-scFV to create chimeric antigen receptor (CAR)-bearing T cells to recognize hCD248 on endothelial targets in vivo. For example, CAR T cells directed against VEGFR have been shown to destroy the tumor vasculature and impair tumor growth [49]. Strikingly, up to 90 % of acute lymphocytic leukemia (ALL) patients and up to 50 % of chronic lymphocytic leukemia (CLL) patients are in durable remission with T cells engineered with CARs [50]. All of the aforementioned potential immunotherapeutic strategies based on CD248 targeting are summarized in Fig. 2. CD248-based immunotherapy in combination with chemotherapy can be a suitable approach for translation into the clinic. The majority of chemotherapeutic compounds, when used at clinically useful doses, are endowed with intrinsic immunosuppressive properties owing to the fact that they preferentially kill rapidly proliferating cells. However, most of these drugs are able to stimulate the immune system against cancer by various different mechanisms: directly activating CD4+, CD8+ or γδ T cells, promoting the production of IL-2, IFN-γ and IL-17, facilitating the maturation or activation of DC, inhibiting or depleting immunosuppressive cell subsets, triggering immunogenic cell death of tumor cells, up-regulating MHC-I molecules on cancer cells, increasing the permeability of tumor cells to granzyme B and inhibiting immunosuppressive cytokine release such as IL-10 or up-regulating the expression of type I IFN [51]. These chemotherapy immune-modulating properties can be used to improve the therapeutic efficacy of CD248-based immunization both in preclinical and in clinical settings as previously described for different tumor antigens [52]. CD248-based immunotherapy might be combined with other anti-vasculature agents such as small molecule inhibitors (sunitinib, axitinib, pazopanib, vandetanib, cabozantinib, sorafenib) that target VEGFR2 and other receptor tyrosine kinases [53]. Finally, there is emerging evidence that tumor resistance occurs in cancer patients treated with conventional anti-vascular therapy. Several hypotheses to explain this resistance have been already proposed, and they include an increased redundancy of other angiogenic factors that compensate for the specific pathway blockade [54], the presence of a hypoxic microenvironment that modifies signaling pathways in both tumor and vasculature cells [55] and epigenetic mechanisms [56]. Moreover, bone marrow myeloid cells recruited by tumor-derived factors (TDF) play an important role in promoting and sustaining tumor angiogenesis/vasculogenesis and include Tie2-expressing monocytes, neutrophils and myeloid-derived suppressor cells (MDSC), generally defined as CD11b+Gr-1+ cells [57]. A role for CD11b+Gr1+ cells in tumor resistance to anti-VEGF antibody treatment was recently proposed by Ferrara et al. [58]. This effect depends on the up-regulated production and secretion of protein Bv8 (also known as prokineticin-2) by CD11b+Gr-1+ cells specifically induced by granulocyte colony-stimulating factor (G-CSF). Thus, G-CSF overproduction by tumors has a deleterious effect promoting vasculature alteration through a Bv8-dependent pathway that bypasses VEGF and renders tumors refractory to anti-VEGF therapy. Pharmacological/immunological MDSC inhibitors are able to either inhibit MDSC differentiation/maturation from precursors or inhibit MDSC accumulation in lymphoid organs [59] and can be used to synergize with CD248-targeting modalities. Moreover, as recently described in the literature, the accumulation of T regulatory lymphocytes (Treg) at tumor sites has been correlated with biomarkers of tumor-associated angiogenesis such as VEGF overexpression and increased microvessel density in endometrial [60] and breast cancers [61] providing clinical indication for an association between Treg and angiogenesis. Treg can contribute to tumor angiogenesis through both indirect and direct mechanisms. Treg sustain angiogenesis indirectly by eliminating effectors T lymphocytes that secrete angiostatic soluble molecules such as TNF-α, IFN-γ, CXCL9, CXCL10 and CXCL11. Moreover, Treg are able to directly promote angiogenesis in ovarian cancer by production of CCL28 that increases VEGF levels in the tumor microenvironment [62]. Depletion of Treg significantly down-regulates VEGF expression in the tumor, and subsequently angiogenesis within tumor sites, and therefore favors an anti-tumor immune response. Immunological/pharmacological anti-Treg treatments include cyclophosphamide, gemcitabine, mitoxantrone, fludarabine, thalidomide analogues, cyclooxygenase 2 inhibitors, as well as Denileukin diftitox (Ontak), a fusion protein of human IL-2 and diphtheria toxin, able to reduce the frequency of Treg in peripheral blood of chronic T cell lymphoma patients [63]. Combination treatment with any of the above anti-Treg treatments with a CD248-targeting strategy may form a complementary approach against the tumor vasculature network. Moreover, the CD248-targeting strategy can be used to treat non-cancer-related inflammatory diseases such as arthritis; in fact, it has recently been reported that CD248 genetic deletion using Tem1−/− mice promotes osteoblast-mediated bone formation and reduces local inflammation [64]. In conclusion, our DNA-based immunotherapy approach targeting the tumor vasculature with a novel TVA demonstrates that CD248 can be targeted immunologically and generates potent anti-CD248 T cell responses as well as anti-tumor responses. Alternate therapeutic approaches targeting CD248 and other modalities in combination with CD248 targeting are warranted. Acknowledgments Financial support for A.F. is from the Basser Research Center for BRCA (Breast Cancer early onset) at Abramson Cancer Center, University of Pennsylvania; the Alliance for Cancer Gene Therapy, the National Institutes of Health (NIH) Director’s New Innovator Award (1DP2OD008514), and the Pennsylvania Department of Health (4100051725). The Italian Association for Cancer Research (AIRC) supported Stefano Ugel with Fellowship for studies abroad (2010). Abbreviations - ACT Adoptive cell therapy - ALL Acute lymphocytic leukemia - APC Antigen-presenting cells - bFGF Basic fibroblast growth factor - CAIX Carbonic anhydrase IX - CAR Chimeric antigen receptor - CLL Chronic lymphocytic leukemia - CTL Cytotoxic T lymphocytes - CXCL Chemokine (C-X-C motif) ligand - DC Dendritic cells - E2 17β-estradiol - EC Endothelial cells - EPC Endothelial progenitor cells - G-CSF Granulocyte colony-stimulating factor - HGF Hepatocyte growth factor - IFN Interferon - IL Interleukin - IMG Intussusceptive microvascular growth - MDSC Myeloid-derived suppressor cells - MHC Major histocompatibility complex - MMP Matrix metalloproteinases - PDGF Platelet-derived growth factor - scFv Single-chain antibody - TAA Tumor-associated antigen - TCR T cell receptor - TEM Tumor endothelial marker - TGF Transforming growth factor - TNF Tumor necrosis factor - Treg T regulatory lymphocytes - TT Tetanus toxoid - TVA Tumor vasculature antigen - VDA Vascular-disrupting agents - VEGF Vascular endothelial growth factor - VEGFR Vascular endothelial growth factor receptor Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Footnotes This paper is a Focussed Research Review based on a presentation given at the Fourteenth International Conference on Progress in Vaccination against Cancer (PIVAC 14), held in Rome, Italy, 24th–26th September, 2014. It is part of a Cancer Immunology, Immunotherapy series of Focussed Research Reviews and meeting report. Stefano Ugel and John G. Facciponte have contributed equally to this paper.

- 1.Chung AS, Lee J, Ferrara N. Targeting the tumour vasculature: insights from physiological angiogenesis. Nat Rev Cancer. 2010;10(7):505–514. doi: 10.1038/nrc2868. [DOI] [PubMed] [Google Scholar] - 2.Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature. 2005;438(7070):967–974. doi: 10.1038/nature04483. [DOI] [PubMed] [Google Scholar] - 3.Walsh DA. Pathophysiological mechanisms of angiogenesis. Adv Clin Chem. 2007;44:187–221. doi: 10.1016/S0065-2423(07)44006-9. [DOI] [PubMed] [Google Scholar] - 4.Dvorak HF. Vascular permeability factor/vascular endothelial growth factor: a critical cytokine in tumor angiogenesis and a potential target for diagnosis and therapy. J Clin Oncol. 2002;20(21):4368–4380. doi: 10.1200/JCO.2002.10.088. [DOI] [PubMed] [Google Scholar] - 5.Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004;25(4):581–611. doi: 10.1210/er.2003-0027. [DOI] [PubMed] [Google Scholar] - 6.Spannuth WA, Sood AK, Coleman RL. Angiogenesis as a strategic target for ovarian cancer therapy. Nat Clin Pract Oncol. 2008;5(4):194–204. doi: 10.1038/ncponc1051. [DOI] [PubMed] [Google Scholar] - 7.Thorpe PE. Vascular targeting agents as cancer therapeutics. Clin Cancer Res. 2004;10(2):415–427. doi: 10.1158/1078-0432.CCR-0642-03. [DOI] [PubMed] [Google Scholar] - 8.Subbiah IM, Lenihan DJ, Tsimberidou AM. Cardiovascular toxicity profiles of vascular-disrupting agents. Oncologist. 2011;16(8):1120–1130. doi: 10.1634/theoncologist.2010-0432. [DOI] [PMC free article] [PubMed] [Google Scholar] - 9.Galluzzi L, Vacchelli E, Bravo-San Pedro JM, Buque A, Senovilla L, Baracco EE, et al. Classification of current anticancer immunotherapies. Oncotarget. 2014;5(24):12472–12508. doi: 10.18632/oncotarget.2998. [DOI] [PMC free article] [PubMed] [Google Scholar] - 10.Quail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 2013;19(11):1423–1437. doi: 10.1038/nm.3394. [DOI] [PMC free article] [PubMed] [Google Scholar] - 11.Klemm F, Joyce JA. Microenvironmental regulation of therapeutic response in cancer. Trends Cell Biol. 2015;25(4):198–213. doi: 10.1016/j.tcb.2014.11.006. [DOI] [PMC free article] [PubMed] [Google Scholar] - 12.Junttila MR, de Sauvage FJ. Influence of tumour micro-environment heterogeneity on therapeutic response. Nature. 2013;501(7467):346–354. doi: 10.1038/nature12626. [DOI] [PubMed] [Google Scholar] - 13.St Croix B, Rago C, Velculescu V, Traverso G, Romans KE, Montgomery E, Lal A, Riggins GJ, Lengauer C, Vogelstein B, Kinzler KW. Genes expressed in human tumor endothelium. Science. 2000;289(5482):1197–1202. doi: 10.1126/science.289.5482.1197. [DOI] [PubMed] [Google Scholar] - 14.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. [PubMed] [Google Scholar] - 15.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 USA. 1992;89(22):10832–10836. doi: 10.1073/pnas.89.22.10832. [DOI] [PMC free article] [PubMed] [Google Scholar] - 16.Rmali KA, Puntis MC, Jiang WG. Prognostic values of tumor endothelial markers in patients with colorectal cancer. World J Gastroenterol. 2005;11(9):1283–1286. doi: 10.3748/wjg.v11.i9.1283. [DOI] [PMC free article] [PubMed] [Google Scholar] - 17.Rouleau C, Curiel M, Weber W, Smale R, Kurtzberg L, Mascarello J, Berger C, Wallar G, Bagley R, Honma N, Hasegawa K, Ishida I, Kataoka S, Thurberg BL, Mehraein K, Horten B, Miller G, Teicher BA. Endosialin protein expression and therapeutic target potential in human solid tumors: sarcoma versus carcinoma. Clin Cancer Res. 2008;14(22):7223–7236. doi: 10.1158/1078-0432.CCR-08-0499. [DOI] [PubMed] [Google Scholar] - 18.Carson-Walter EB, Winans BN, Whiteman MC, Liu Y, Jarvela S, Haapasalo H, Tyler BM, Huso DL, Johnson MD, Walter KA. Characterization of TEM1/endosialin in human and murine brain tumors. BMC Cancer. 2009;9:417. doi: 10.1186/1471-2407-9-417. [DOI] [PMC free article] [PubMed] [Google Scholar] - 19.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. doi: 10.1016/j.modgep.2006.07.006. [DOI] [PubMed] [Google Scholar] - 20.MacFadyen JR, Haworth O, Roberston D, Hardie D, Webster MT, Morris HR, Panico M, Sutton-Smith M, Dell A, van der Geer P, Wienke D, Buckley CD, Isacke CM. Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium. FEBS Lett. 2005;579(12):2569–2575. doi: 10.1016/j.febslet.2005.03.071. [DOI] [PubMed] [Google Scholar] - 21.Christian S, Ahorn H, Koehler A, Eisenhaber F, Rodi HP, Garin-Chesa P, Park JE, Rettig WJ, Lenter MC. 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. doi: 10.1074/jbc.M009604200. [DOI] [PubMed] [Google Scholar] - 22.Christian S, Winkler R, Helfrich I, Boos AM, Besemfelder E, Schadendorf D, Augustin HG. Endosialin (Tem1) is a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells. Am J Pathol. 2008;172(2):486–494. doi: 10.2353/ajpath.2008.070623. [DOI] [PMC free article] [PubMed] [Google Scholar] - 23.Bagley RG, Rouleau C, St Martin T, Boutin P, Weber W, Ruzek M, Honma N, Nacht M, Shankara S, Kataoka S, Ishida I, Roberts BL, Teicher BA. Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248. Mol Cancer Ther. 2008;7(8):2536–2546. doi: 10.1158/1535-7163.MCT-08-0050. [DOI] [PubMed] [Google Scholar] - 24.Nanda A, Karim B, Peng Z, Liu G, Qiu W, Gan C, Vogelstein B, St Croix B, Kinzler KW, Huso DL. Tumor endothelial marker 1 (Tem1) functions in the growth and progression of abdominal tumors. Proc Natl Acad Sci USA. 2006;103(9):3351–3356. doi: 10.1073/pnas.0511306103. [DOI] [PMC free article] [PubMed] [Google Scholar] - 25.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. doi: 10.1023/B:CLIN.0000017168.83616.d0. [DOI] [PubMed] [Google Scholar] - 26.Facciponte JG, Ugel S, De Sanctis F, Li C, Wang L, Nair G, Sehgal S, Raj A, Matthaiou E, Coukos G, Facciabene A. Tumor endothelial marker 1-specific DNA vaccination targets tumor vasculature. J Clin Invest. 2014;124(4):1497–1511. doi: 10.1172/JCI67382. [DOI] [PMC free article] [PubMed] [Google Scholar] - 27.Rice J, Ottensmeier CH, Stevenson FK. DNA vaccines: precision tools for activating effective immunity against cancer. Nat Rev Cancer. 2008;8(2):108–120. doi: 10.1038/nrc2326. [DOI] [PubMed] [Google Scholar] - 28.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. doi: 10.1002/eji.1830191209. [DOI] [PubMed] [Google Scholar] - 29.Widera G, Austin M, Rabussay D, Goldbeck C, Barnett SW, Chen M, Leung L, Otten GR, Thudium K, Selby MJ, Ulmer JB. Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol. 2000;164(9):4635–4640. doi: 10.4049/jimmunol.164.9.4635. [DOI] [PubMed] [Google Scholar] - 30.Vanderlugt CL, Miller SD. Epitope spreading in immune-mediated diseases: implications for immunotherapy. Nat Rev Immunol. 2002;2(2):85–95. doi: 10.1038/nri724. [DOI] [PubMed] [Google Scholar] - 31.Huang AY, Gulden PH, Woods AS, Thomas MC, Tong CD, Wang W, Engelhard VH, Pasternack G, Cotter R, Hunt D, Pardoll DM, Jaffee EM. 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 USA. 1996;93(18):9730–9735. doi: 10.1073/pnas.93.18.9730. [DOI] [PMC free article] [PubMed] [Google Scholar] - 32.McWilliams JA, Sullivan RT, Jordan KR, McMahan RH, Kemmler CB, McDuffie M, Slansky JE. Age-dependent tolerance to an endogenous tumor-associated antigen. Vaccine. 2008;26(15):1863–1873. doi: 10.1016/j.vaccine.2008.01.052. [DOI] [PMC free article] [PubMed] [Google Scholar] - 33.Malarkannan S, Serwold T, Nguyen V, Sherman LA, Shastri N. The mouse mammary tumor virus env gene is the source of a CD8+ T-cell-stimulating peptide presented by a major histocompatibility complex class I molecule in a murine thymoma. Proc Natl Acad Sci USA. 1996;93(24):13991–13996. doi: 10.1073/pnas.93.24.13991. [DOI] [PMC free article] [PubMed] [Google Scholar] - 34.Feltkamp MC, Smits HL, Vierboom MP, Minnaar RP, de Jongh BM, Drijfhout JW, ter Schegget J, Melief CJ, Kast WM. 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. doi: 10.1002/eji.1830230929. [DOI] [PubMed] [Google Scholar] - 35.Scappaticci FA, Fehrenbacher L, Cartwright T, Hainsworth JD, Heim W, Berlin J, Kabbinavar F, Novotny W, Sarkar S, Hurwitz H. Surgical wound healing complications in metastatic colorectal cancer patients treated with bevacizumab. J Surg Oncol. 2005;91(3):173–180. doi: 10.1002/jso.20301. [DOI] [PubMed] [Google Scholar] - 36.Petrou P, Georgalas I, Giavaras G, Anastasiou E, Ntana Z, Petrou C. Early loss of pregnancy after intravitreal bevacizumab injection. Acta Ophthalmol. 2010;88(4):e136. doi: 10.1111/j.1755-3768.2009.01572.x. [DOI] [PubMed] [Google Scholar] - 37.Mennuni C, Ugel S, Mori F, Cipriani B, Iezzi M, Pannellini T, Lazzaro D, Ciliberto G, La Monica N, Zanovello P, Bronte V, Scarselli E. Preventive vaccination with telomerase controls tumor growth in genetically engineered and carcinogen-induced mouse models of cancer. Cancer Res. 2008;68(23):9865–9874. doi: 10.1158/0008-5472.CAN-08-1603. [DOI] [PubMed] [Google Scholar] - 38.Ugel S, Scarselli E, Iezzi M, Mennuni C, Pannellini T, Calvaruso F, Cipriani B, De Palma R, Ricci-Vitiani L, Peranzoni E, Musiani P, Zanovello P, Bronte V. Autoimmune B-cell lymphopenia after successful adoptive therapy with telomerase-specific T lymphocytes. Blood. 2010;115(7):1374–1384. doi: 10.1182/blood-2009-07-233270. [DOI] [PubMed] [Google Scholar] - 39.Krysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P. Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer. 2012;12(12):860–875. doi: 10.1038/nrc3380. [DOI] [PubMed] [Google Scholar] - 40.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. doi: 10.1111/j.0105-2896.2004.00145.x. [DOI] [PubMed] [Google Scholar] - 41.Fenoglio D, Traverso P, Parodi A, Kalli F, Zanetti M, Filaci G. Generation of more effective cancer vaccines. Hum Vaccin Immunother. 2013;9(12):2543–2547. doi: 10.4161/hv.26147. [DOI] [PMC free article] [PubMed] [Google Scholar] - 42.Rosenberg SA, Restifo NP, Yang JC, Morgan RA, Dudley ME. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer. 2008;8(4):299–308. doi: 10.1038/nrc2355. [DOI] [PMC free article] [PubMed] [Google Scholar] - 43.Gattinoni L, Powell DJ, Jr, Rosenberg SA, Restifo NP. Adoptive immunotherapy for cancer: building on success. Nat Rev Immunol. 2006;6(5):383–393. doi: 10.1038/nri1842. [DOI] [PMC free article] [PubMed] [Google Scholar] - 44.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. doi: 10.1016/j.jim.2010.09.001. [DOI] [PMC free article] [PubMed] [Google Scholar] - 45.Li C, Chacko AM, Hu J, Hasegawa K, Swails J, Grasso L, El-Deiry WS, Nicolaides N, Muzykantov VR, Divgi CR, Coukos G. Antibody-based tumor vascular theranostics targeting endosialin/TEM1 in a new mouse tumor vascular model. Cancer Biol Ther. 2014;15(4):443–451. doi: 10.4161/cbt.27825. [DOI] [PMC free article] [PubMed] [Google Scholar] - 46.André N, Carré M, Pasquier E. Metronomics: Towards personalized chemotherapy? Nat Rev Clin Oncol. 2014;11(7):413–431. doi: 10.1038/nrclinonc.2014.89. [DOI] [PubMed] [Google Scholar] - 47.Chacko AM, Li C, Nayak M, Mikitsh JL, Hu J, Hou C, Grasso L, Nicolaides NC, Muzykantov VR, Divgi CR, Coukos G. Development of 124I immuno-PET targeting tumor vascular TEM1/endosialin. J Nucl Med. 2014;55(3):500–507. doi: 10.2967/jnumed.113.121905. [DOI] [PMC free article] [PubMed] [Google Scholar] - 48.Diaz LA, Jr, Coughlin CM, Weil SC, Fishel J, Gounder MM, Lawrence S, Azad N, O’Shannessy DJ, Grasso L, Wustner J, Ebel W, Carvajal RD. A first-in-human phase I study of MORAb-004, a monoclonal antibody to endosialin in patients with advanced solid tumors. Clin Cancer Res. 2015;21(6):1281–1288. doi: 10.1158/1078-0432.CCR-14-1829. [DOI] [PMC free article] [PubMed] [Google Scholar] - 49.Chinnasamy D, Yu Z, Theoret MR, Zhao Y, Shrimali RK, Morgan RA, Feldman SA, Restifo NP, Rosenberg SA. 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. doi: 10.1172/JCI43490. [DOI] [PMC free article] [PubMed] [Google Scholar] - 50.Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, Shen A, Teachey DT, Levine BL, June CH, Porter DL, Grupp SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med. 2014;371(16):1507–1517. doi: 10.1056/NEJMoa1407222. [DOI] [PMC free article] [PubMed] [Google Scholar] - 51.Galluzzi L, Senovilla L, Zitvogel L, Kroemer G. The secret ally: immunostimulation by anticancer drugs. Nat Rev Drug Discov. 2012;11(3):215–233. doi: 10.1038/nrd3626. [DOI] [PubMed] [Google Scholar] - 52.Kang TH, Mao CP, Lee SY, Chen A, Lee JH, Kim TW, Alvarez RD, Roden RB, Pardoll D, Hung CF, Wu TC. Chemotherapy acts as an adjuvant to convert the tumor microenvironment into a highly permissive state for vaccination-induced antitumor immunity. Cancer Res. 2013;73(8):2493–2504. doi: 10.1158/0008-5472.CAN-12-4241. [DOI] [PMC free article] [PubMed] [Google Scholar] - 53.Moserle L, Jimenez-Valerio G, Casanovas O. Antiangiogenic therapies: going beyond their limits. Cancer Discov. 2014;4(1):31–41. doi: 10.1158/2159-8290.CD-13-0199. [DOI] [PubMed] [Google Scholar] - 54.Choi HJ, Armaiz Pena GN, Pradeep S, Cho MS, Coleman RL, Sood AK. Anti-vascular therapies in ovarian cancer: moving beyond anti-VEGF approaches. Cancer Metastasis Rev. 2015;34(1):19–40. doi: 10.1007/s10555-014-9538-9. [DOI] [PMC free article] [PubMed] [Google Scholar] - 55.Kerbel RS, Yu J, Tran J, Man S, Viloria-Petit A, Klement G, Coomber BL, Rak J. Possible mechanisms of acquired resistance to anti-angiogenic drugs: implications for the use of combination therapy approaches. Cancer Metastasis Rev. 2001;20(1–2):79–86. doi: 10.1023/A:1013172910858. [DOI] [PubMed] [Google Scholar] - 56.Huang J, Soffer SZ, Kim ES, McCrudden KW, New T, Manley CA, Middlesworth W, O’Toole K, Yamashiro DJ, Kandel JJ. Vascular remodeling marks tumors that recur during chronic suppression of angiogenesis. Mol Cancer Res. 2004;2(1):36–42. [PubMed] [Google Scholar] - 57.Ferrara N. Role of myeloid cells in vascular endothelial growth factor-independent tumor angiogenesis. Curr Opin Hematol. 2010;17(3):219–224. doi: 10.1097/MOH.0b013e3283386660. [DOI] [PubMed] [Google Scholar] - 58.Shojaei F, Wu X, Zhong C, Yu L, Liang XH, Yao J, Blanchard D, Bais C, Peale FV, van Bruggen N, Ho C, Ross J, Tan M, Carano RA, Meng YG, Ferrara N. Bv8 regulates myeloid-cell-dependent tumour angiogenesis. Nature. 2007;450(7171):825–831. doi: 10.1038/nature06348. [DOI] [PubMed] [Google Scholar] - 59.Ugel S, Delpozzo F, Desantis G, Papalini F, Simonato F, Sonda N, Zilio S, Bronte V. Therapeutic targeting of myeloid-derived suppressor cells. Curr Opin Pharmacol. 2009;9(4):470–481. doi: 10.1016/j.coph.2009.06.014. [DOI] [PubMed] [Google Scholar] - 60.Giatromanolaki A, Bates GJ, Koukourakis MI, Sivridis E, Gatter KC, Harris AL, Banham AH. The presence of tumor-infiltrating FOXP3+ lymphocytes correlates with intratumoral angiogenesis in endometrial cancer. Gynecol Oncol. 2008;110(2):216–221. doi: 10.1016/j.ygyno.2008.04.021. [DOI] [PubMed] [Google Scholar] - 61.Gupta S, Joshi K, Wig JD, Arora SK. Intratumoral FOXP3 expression in infiltrating breast carcinoma: its association with clinicopathologic parameters and angiogenesis. Acta Oncol. 2007;46(6):792–797. doi: 10.1080/02841860701233443. [DOI] [PubMed] [Google Scholar] - 62.Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang LP, Gimotty PA, Gilks CB, Lal P, Zhang L, Coukos G. Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and T(reg) cells. Nature. 2011;475(7355):226–230. doi: 10.1038/nature10169. [DOI] [PubMed] [Google Scholar] - 63.Talpur R, Jones DM, Alencar AJ, Apisarnthanarax N, Herne KL, Yang Y, Duvic M. CD25 expression is correlated with histological grade and response to denileukin diftitox in cutaneous T-cell lymphoma. J Invest Dermatol. 2006;126(3):575–583. doi: 10.1038/sj.jid.5700122. [DOI] [PubMed] [Google Scholar] - 64.Naylor AJ, Azzam E, Smith S, Croft A, Poyser C, Duffield JS, Huso DL, Gay S, Ospelt C, Cooper MS, Isacke C, Goodyear SR, Rogers MJ, Buckley CD. The mesenchymal stem cell marker CD248 (endosialin) is a negative regulator of bone formation in mice. Arthritis Rheum. 2012;64(10):3334–3343. doi: 10.1002/art.34556. [DOI] [PMC free article] [PubMed] [Google Scholar]