Review Article

Endothelial progenitor cells for cancer gene therapy

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Abstract

Endothelial progenitor cells (EPCs) are promising for cancer therapy because they specifically target tumors. They have the capacity to home to, invade, migrate within and incorporate into tumor structures. They are easily expanded and can be armed with therapeutic payloads protected within the progenitor cells. Once in the tumor, armed EPCs can be triggered to induce cell death in surrounding tumor cells while being transiently protected from premature demise. In preclinical studies, therapeutic EPCs attenuated tumor growth and increased survival. Enhancing homing, self-protection and collateral tumor cell damage will increase the efficacy of EPCs for cancer gene therapy.

EPCs in physiological vasculogenesis and angiogenesis

During development, the vasculature is formed by vasculogenesis. In this process, endothelial progenitors differentiate to endothelial cells (ECs) that form a primary capillary network. Developmental vasculogenesis depends on endothelial progenitor cells (EPCs) that are derived from a common precursor of both the hematopoietic system and the vascular system called the hemangioblast.1 Vascular endothelial growth factor (VEGF) and the receptor VEGFR2 are pivotal for embryonic vasculogenesis as shown by the phenotype of both VEGFR2 and VEGF knockout mice, who die in utero owing to lack of endothelial and blood cells.2, 3

In the adult, new vessels are built solely in healing wounds, the cycling endometrium and in growing tumors. Postnatal vessel formation was thought to proceed exclusively by sprouting from existing vessels (angiogenesis). However, it was shown that EPCs from the bone marrow participate in normal and pathological vessel formation in the adult (vasculogenesis).4, 5 Adult EPCs proliferate rapidly and share other characteristics of embryonic angioblasts. They are recruited from the bone marrow by stimuli emanating from angiogenic sites. At these sites, the EPCs differentiate into mature ECs that—to some degree—incorporate into vessels. Thus, vasculogenesis appears not to be restricted to embryogenesis but to be operative in adults also. From this the concept has emerged that angiogenesis and vasculogenesis may be complementary mechanisms for vessel formation in the adult. It remains to be determined how much EPCs contribute to new vessel formation. Preclinical and clinical studies have shown that transplantation of ex vivo expanded EPCs enhances tissue vascularization after ischemic events in the hind limb, retina and myocardium.6

Cell surface markers define adult EPCs and allow their isolation. The combined expression of CD133 and VEGFR2 is widely thought to characterize early functional EPCs,7, 8 although this notion has recently been questioned.9 While VEGFR2 is expressed on both mature ECs and their progenitors, CD133 distinguishes EPCs from mature ECs sloughed from injured vessel walls. CD133 expression also sets EPCs apart from CD34/CD14+ cells of myelomonocytic lineage that can ‘transdifferentiate’ into endothelial-like cells, which appear to be effective for re-endotheliazation when given intravenously (i.v.) after vascular injury.10 In addition to CD133 and VEGFR2 other markers and characteristics are also associated with EPCs. These include CD34, CD31, VE-cadherin, von Willebrand factor, CD105, UEA lectin staining and uptake of acetylated low-density lipoprotein.4, 11

EPCs in tumor angiogenesis

Bone marrow-derived cells play a role in the neovascularization of tumors. In cancer patients, the number of circulating EPCs is increased.12, 13, 14 Importantly, in various animal tumor models, transplanted EPCs incorporated functionally into tumor-associated vessels. Following transplantation of marked bone marrow into tumor-bearing mice, marked cells were found in the neovasculature of the developing tumors.15, 16, 17 When Id-mutant mice that did not grow tumors owing to defective angiogenesis were transplanted with wild-type bone marrow tumor, growth was restored.18 The majority of the newly formed tumor vessels were derived from the genetically marked donor, mainly during the early stages of tumor growth. Other studies also highlighted the important contribution of EPCs during the early phase of tumor neovascularization.19, 20 Treatment of tumor-bearing mice with vascular disrupting agents recruited EPCs to the tumors.21 In patients with hepatocellular carcinoma, CD 133+ EPCs incorporated into tumor vessels.22 In contrast to these studies, no contribution of bone marrow-derived cells to tumorigenesis was found by others.23, 24 Additional studies could not detect bone marrow-derived EPCs in the tumor vasculature but defined bone marrow-derived cells of hematopoietic origin that contributed indirectly to tumor neovascularization,25, 26 such as by generation of vascular mural cells.26 These partially conflicting data suggest that while bone marrow-derived cells take part in the cellular make-up of tumor vessels, their relative contribution to tumor ECs, periendothelial cells and nonstructural but proangiogenic cells has yet to be determined.27

Vascular endothelial growth factor is a critical factor promoting mobilization of EPCs.28, 29 VEGF also promotes maturation of EPCs and their recruitment to the sites of neovascularization. Placental growth factor—which signals primarily through VEGFR1—also contributes to vessel growth in tumors, possibly by recruiting bone marrow-derived cells.30 VEGF and placental growth factor are released by many tumors. Inhibition of VEGF31, 32 inhibits tumor growth. The simultaneous inhibition of both receptors impairs mobilization of bone marrow-derived cells to the tumor vasculature and retards tumor growth.18, 30, 33 Recent data show that stromal-derived factor 1 (SDF-1/CXCL12) contributes to the recruitment of EPCs to tumors and their retention in the tumor vessels.27, 34, 35

Antiapoptotic default mode of EPCs

As in other cellular vehicles, apoptosis has to be attenuated in EPCs during ex vivo expansion, during homing to the tumor, and after activation of their proapoptotic payload. Little is known about apoptosis in EPCs; thus, information has to be extrapolated from ECs. VEGF and VEGFR2 play a pivotal role in maintaining EC integrity as demonstrated by targeted disruption of these genes.3, 36, 37 The antiapoptotic effect of VEGF and also of angiopoietin-1 (Ang-1; via activation of its receptor Tie2) is mainly mediated through activation of the survival-promoting PI3K/Akt signaling pathway.38, 39 By diverse mechanisms, the activation of PI3K/Akt inhibits the apoptosis of ECs40, 41, 42, 43 VEGF upregulates antiapoptotic proteins such as survivin, XIAP and Bcl-2.44, 45, 46 VE-cadherin47 promotes EC survival, as does CD31 (platelet EC adhesion molecule (PECAM-1)).48 aυβ3 triggers antiapoptotic nuclear factor-κB signaling after binding to ligands attached to the extracellular matrix.49 However, the receptor switches to proapoptotic activity when triggered by ligands not anchored to the extracellular matrix.50 This occurs during detachment of therapeutic EPCs from their culture matrix and during homing to the tumor.51 EPCs derived from blood express high levels of the antioxidative enzymes MnSOD, catalase and glutathione peroxidase leading to decreased oxidative stress-induced apoptosis.52 Therapeutic EPCs may therefore be protected to some degree against radical-producing proapoptotic payloads.

Taken together, the default mode of ECs and EPCs is antiapoptotic. In addition, ex vivo expanded EPCs are subjected to the antiapoptotic factors VEGF or basic fibroblast growth factor, and to the antiapoptotic extracellular matrices collagen and fibronectin. In concert, these mechanisms allow for ex vivo expansion, arming and homing of therapeutic EPCs with minimal spontaneous apoptosis. Furthermore, they delay the onset of cell death when the payload of the EPC is activated.

EPCs for tumor therapy

The need of endothelial progenitors for tumor vasculogenesis is the fundamental reason why these cells are being considered for therapeutic targeting of the tumor vasculature. In the adult, new vessels are only formed in wounds during wound healing and in the uterus during the female ovulatory cycle. Thus, it was reasoned to utilize the natural tropism of circulating EPCs to deliver and selectively express genes at tumor sites.

Sources of EPCs

Investigators have isolated or generated EPCs from many sources: mouse embryos, mouse or human embryonic stem cells, fetal liver, human umbilical cord blood, postnatal bone marrow and peripheral blood. Embryonic stem cell-derived embryonic EPCs differentiate into mature ECs and contribute to adult vasculogenesis following transplantation.53 The advantage of embryonic EPCs is their unlimited proliferative capacity and the ease of genetic manipulation. These cells have the potential for systemic cancer gene therapy, as shown in a proof-of-principle study.54 However, mature embryonic stem cell derivatives are immunogenic and ethical considerations may limit their generation. Thus, additional sources of EPCs have been explored.

Umbilical cord blood is a rich source of stem and progenitor cells. Cord blood-derived progenitors have many advantages compared to progenitors generated from adult bone marrow. They possess a larger capacity to form colonies, proliferate more and longer, and have longer telomeres.55 Of note, harvesting cord blood does not require invasive methods, in contrast to the procurement of bone marrow. Transplanted cord blood-derived EPCs efficiently enhance postnatal neovascularization in vivo.56

Peripheral blood is a ubiquitous source of circulating autologous EPCs. To enrich for potential EPCs, peripheral blood mononuclear cells positive for CD34,4, 11 CD133 or VEGFR2 have been isolated, as has the fraction of peripheral blood mononuclear cells that adheres to plastic, and have been induced to differentiate into EPCs. These cells are cultured for a short period of time, typically 7 days or less, on fibronectin- or collagen-coated dishes in the presence of growth factors, including VEGF, basic fibroblast growth factor and insulin-like growth factor.4, 11 Upon endothelial differentiation, EPCs decrease expression of CD133 and begin to express markers of mature ECs, such as VEGFR2, VE-cadherin, PECAM-1, CD31, von Willebrand factor and CD105. It is controversial whether these cells are true EPCs. Evidence is mounting that these cells are of hematopoietic lineage with characteristics of monocytes/macrophages and myeloid cells.57 In contrast, the so-called blood late outgrowth ECs (BOECs) that emerge after long-term culture of adherent peripheral blood mononuclear cells under conditions described above58 have been shown by clonal analysis to represent true EPCs.57 These cells are highly proliferative.

Another cell population from postnatal bone marrow with extensive proliferation potential and the capacity to differentiate into different cell types of the mesodermal lineage has been reported.59 These stem cells, termed multipotent adult progenitor cells, differentiate into ECs in the presence of VEGF in vitro and contribute to vasculogenesis in vivo. The intravenous injection of undifferentiated multipotent adult progenitor cells resulted in new vessel formation in transplanted tumors, indicating that multipotent adult progenitor cells can differentiate into ECs in vivo. The authors suggested that multipotent adult progenitor cells are precursors of EPCs and proposed them as a source of ECs for therapeutic approaches.

Ex vivo expansion of EPCs

A sufficient number of EPCs has to be generated to yield the large number of EPCs required for therapeutic applications. As described above, EPCs in the bone marrow and in circulation are rare. The number of circulating EPCs can be increased by injecting chemotactic VEGF, placental growth factor or granulocyte-macrophage colony-stimulating factor.60 Statins can also mobilize a larger number of EPCs.61 Of note, the vasculogenic rebound of tumors recovering from cytotoxic therapy involves the mobilization of EPCs.62 This raises the possibility of harvesting EPCs after chemotherapy. However, even if these measures were to be applied, ex vivo expansion of EPCs is needed to produce sufficient numbers of EPCs. BOECs are ideally suited for this as they can be expanded to yield a very large number of EPCs.58

Genetic modification of EPCs

Ex vivo expanded EPCs can be readily manipulated genetically. Transduction of EPCs and their putative precursors with retrovirus16, 63 and lentivirus25 vectors is feasible. These vectors stably integrate into the genome, allowing for long-term transgene expression. This may be necessary for certain therapeutic approaches using EPCs as cellular vehicles. In addition, replication-defective retro- and lentiviral vectors do not express viral proteins. This decreases clearance of the transduced EPCs following administration to the host.

Endothelial progenitor cells can also be modified with nonintegrating vectors, such as those based on adenovirus and herpes simplex virus. Because of their safety, such vectors are advantageous if an antitumor effect can be achieved within a short time.

EPCs as cellular vehicles for cancer gene therapy

Several features make EPCs attractive cellular vehicles for systemic tumor gene therapy. Isolation of EPCs is uncomplicated and ex vivo expansion can generate the number of EPCs required for cancer gene therapy. Transduction with viral vectors carrying therapeutic payloads is efficient. Of particular importance is the ability of EPCs to home not only into the tumor proper but also into the tumor's vasculature.

Tumor vessels are promising targets for anticancer therapeutics. Most tumors depend on recruiting new blood vessels for growth and metastasis. Thus, tumor vessels constitute a common target for the treatment of different tumor types, independent of antigenic and genetic differences. Except for wound healing and the cycling uterine endometrium, neovascularization in adults is restricted to growing tumors. Finally, tumor vessels are accessible to systemically applied therapeutic EPCs. This might circumvent some hurdles that aggravate targeting of solid tumors, such as poor tissue penetration. Tumor vessels are an Achilles’ heel of tumors, however, only a minority of systemically administered EPCs incorporate into tumor vessels15, 16, 54, 59, 63 (Figure 1a). This limits the ability of therapeutic EPCs to destroy tumor vessels. However, EPCs also home, and quite efficiently so, to the tumor proper. This opens a second venue to attack the tumor. Therefore, as far as the ability to target a tumor is concerned, the relative contribution of EPCs to the formation of tumor vessels is not crucial.

Figure 1
figure1

Homing and bystander effect of tumorcytotoxic endothelial progenitor cells (EPCs). (a) A substantial number of systemically applied EPCs home into the tumor proper, and some EPCs incorporate into tumor vessels. Hypoxic areas of tumors preferentially attract EPCs. (b) The transient resistance of therapeutic EPCs to their cytotoxic effectors results in a strong bystander effect on surrounding tumor cells (left), whereas susceptibility of EPCs to their payload attenuates the effect on surrounding tumor cells (right).

Given the importance of homing for the efficacy of EPC-mediated cancer gene therapy, investigations focusing on EPC homing have been published. EPCs derived from cord blood and i.v. injected into mice bearing orthotopic human gliomas specifically homed to the gliomas, with few EPCs being sequestered in normal brain or in other organs, except for the spleen.64 The homing efficacy of EPCs was superior to that of human umbilical vein ECs. Similarly, subcutaneously growing human lung carcinomas recruited EPCs generated from human cord blood.65

Arming EPCs with apoptotic payloads

EPCs with suicide genes

Suicide genes metabolize nontoxic prodrugs to cancer drugs. As in vivo gene transfer efficacy is too low to reach every tumor cell, tumor eradication relies on a strong bystander effect,66 the collateral cytotoxicity of the suicide gene-expressing cells on neighboring tumor cells.

Suicide gene-expressing ECs, bone marrow cells and EPCs have been explored as cellular vehicles for preclinical systemic cancer therapy. The rationale is that the therapeutic cells exert a bystander effect on surrounding tumor cells (Figure 1b).

Mice inoculated intraperitoneally with human ovarian cancer cells mixed 1:1 with human umbilical vein ECs expressing thymidine kinase (TK) showed a moderate survival benefit following ganciclovir treatment.67 CD34-positive cells transduced by herpes simplex virus vector encoding TK migrated to skin autografts in a primate model and accelerated graft regression after treatment with ganciclovir.68 CD34-positive human cells expressing TK mixed 1:1 with human ovarian cancer cells exerted a bystander effect in vitro and also in vivo, where tumor take was abrogated.69 Injection of human umbilical vein ECs expressing nitroreductase into subcutaneous murine tumors reduced their growth after administration of the prodrug CB1954.70 These studies showed that cells of endothelial lineage can, in principle, be employed as cellular vehicles for cancer gene therapy.

Investigators have transplanted mice with murine bone marrow cells expressing the TK gene under the control of the endothelial promoter/enhancer Tie2.25 Murine tumors were then generated subcutaneously The tumors markedly recruited Tie2 expressing bone marrow cells to a perivascular location, with few cells incorporating into the tumor vessels. After administration of ganciclovir, tumor bystander cells were killed, resulting in decreased tumor growth. Others transplanted sublethally irradiated mice with ex vivo expanded human EPCs derived from CD34+ cells.63 Human glioma cells were then inoculated subcutaneously. When tumors were procured, 10–25% of the tumor-associated vessels contained EPC-like cells. In contrast, far fewer donor EPCs incorporated into the tumor vessels of nonirradiated mice. Thus, systemically administered EPCs might have to compete with endogenous EPCs for incorporation into the tumor, necessitating suppression of endogenous EPCs by irradiation. Experimental lung metastases of murine melanoma cells were generated in sublethally irradiated mice following transplantation with human CD34+ cells expressing TK. There was a clear antitumor effect upon administration of ganciclovir. Taken together, these two studies showed the potency of EPCs as cancer-targeting vehicles in a transplant setting.

It remained to be demonstrated that ex vivo expanded, systemically applied therapeutic EPCs could target established tumors outside the transplant setting. It was shown that murine embryonic EPCs administered i.v. preferentially home to hypoxic areas of lung metastases.54 As embryonic EPCs do not express major histocompatibility complex I proteins and are not killed by nonactivated NK cells, they were able to evade immunological rejection. When modified to express the cytosine deaminase gene, these embryonic EPCs exerted a bystander effect on tumor cells upon administration of 5-FC, thus prolonging the life of tumor-bearing mice.

Going one step further, the antitumor efficacy of human EPCs expanded ex vivo was determined. Blood outgrowth ECs injected i.v. into tumor-bearing mice cleared from lung, liver and spleen with time while being retained in lung metastases and subcutaneously growing tumors.71, 72 Most of the homed BOECs took up an extravascular position, whereas some integrated into tumor vessels71 and persisted.72 Others found specificity of BOECs for tumors to be less pronounced.73 VEGF and placental growth factor mediated both migration and invasion of BOECs into tumor spheroid masses in vitro,71 and infiltration by BOECs involved the action of matrix metalloproteinases.74 When armed with a suicide gene, BOECs exerted a bystander effect on tumor cells in vitro and in vivo.71 Surprisingly, i.v. administration of these armed BOECs into mice bearing multiorgan metastases did not prolong survival. In addition to homing efficacy, other parameters impacted upon the efficacy of BOECs. These include the ultimate susceptibility of BOECs to suicide gene-induced cell death, their paracrine proliferative effect on tumor cells, and their low proliferation rate compared to tumor cells.

Antiangiogenic EPCs

Antiangiogenic approaches for the treatment of cancer are being actively investigated. It has been proposed that delivering these agents by gene transfer has the advantage of generating high drug concentrations limited to the tumor, thus avoiding systemic toxicity. When unfractionated bone marrow cells expressing a truncated form of VEGFR2 were transplanted into mice, some of these cells incorporated in the vasculature of tumors generated post-transplant.16 The therapeutic protein was expressed for a prolonged period of time and decreased the vascularity and growth of the tumors.

The i.v. injection of endostatin-expressing human BOECs into mice bearing subcutaneously. Lewis lung carcinoma resulted in decreased tumor growth.72 Of note, the injection of nonmodified BOECs increased the vessel density of tumors, although tumor growth was not increased.

Immunomodulatory ECs

Murine lung ECs expressing interleukin-2 injected i.v. homed to experimental lung metastases, decreased tumor burden and prolonged survival of the mice.75

EPCs as packaging cells for viral vectors

Tumor-targeting EPCs that produce oncolytic virus would avoid systemic exposure to viral vectors and protect them from immune inactivation, nonspecific adhesion or other causes of particle loss during transit to the tumor. Virus producing, tumor targeting cells would be particularly advantageous if the number of cell carriers incorporating into the tumor is low compared to the tumor mass, as each cell at the tumor site can release multiple virus particles, thereby amplifying the therapeutic effect.

Genetically modified BOECs have been shown to produce viral vectors.73, 76 The tail vein injection of BOECs infected with an adenoviral/retroviral chimeric system resulted in transduction of subcutaneously. growing B16 cells overexpressing VEGF.73 Attenuated measles viruses of the Edmonston B strain (MV-Edm) are oncolytic and have shown efficacy against experimental gliomas,77 among other tumors. However, host immune response and the infiltrative nature of gliomas limit their efficacy. BOECs infected by MV-Edm allowed replication of MV-Edm while surviving long enough after infection to serve as vehicles for MV-Edm (BOEC/MV-Edm).76 MV-Edm within BOECs were protected from neutralizing MV antibodies. After intravenous and peritumoral injection, BOEC/MV-Edm migrated to orthotopic U87 gliomas and delivered the viruses to the tumors. At the tumor, MV-Edm released by the BOECs infected glioma cells and then spread from tumor cell to tumor cell. This caused glioma cell death that decreased tumor size and, in the case of peritumoral injection, prolonged the survival of mice.

Clinical translation

Endothelial progenitor cells generated from autologous blood, such as BOECs, will most likely be the first choice as the source of therapeutic EPCs. Ubiquitously available autologous blood is easily procured. EPCs are readily expanded and manipulated, and they do not pose problems of immunological intolerance. However, survival benefit by tumorcytotoxic EPCs has been moderate in the preclinical studies published so far, and no cures have been reported yet. Thus, several points have to be addressed to increase the efficacy of EPC-based tumor gene therapy.

First, to achieve therapeutic efficacy, a sufficient number of EPCs has to reach the tumor site. So far, there are no data on tumor homing of systemically injected autologous EPCs in humans. One is thus confined to mouse studies that may be confounded by yet-to-be defined structural incompatibilities between human EPCs and mouse vasculature. As discussed above, while being substantial, the number of exogenous EPCs recruited into tumors is limited, and the number of EPCs incorporating into tumor vessels is low. Several parameters potentially limit recruitment of EPCs. For once, therapeutic EPCs may not sufficiently express all the receptors mediating recruitment. BOECs, for example, while expressing VEGFR2 and -1, do not express CXCR4 and thus cannot respond to stromal-derived factor-1, a potent attractor of hematopoietic stem cells. Furthermore, ‘true’ progenitor status may be required for efficient homing and incorporation. BOECs, for example, lack progenitor status in as much as they do not express the progenitor-associated surface markers CD133 and c-kit. Thus, directed differentiation or genetic manipulation of the EPCs may be necessary to increase their homing capacity, as may be regional delivery. It remains to be established whether myelosuppression improves homing by decreasing recruitment of endogenous EPCs that potentially compete with therapeutic EPCs. Administering cancer-targeting EPCs following chemotherapy, irradiation or vascular disrupting agents could increase homing by exploiting the vascular rebound often observed after these therapies. Looking at EPC recruitment from the tumor's side, inter- and intratumoral heterogeneity has to be taken into account. Hypoxia, for example, is unequally distributed within a tumor and EPCs predominantly home into hypoxic areas54 (Figure 1a). Additional investigations are required to define tumors or tumor environments that depend on the recruitment of EPCs and to delineate the molecular mechanisms determining their recruitment.

Second, although it appears that ex vivo expanded EPCs are temporarily protected against apoptosis, prolonged protection against their cytotoxic payload would be beneficial to increase their efficacy (Figure 1b). Further research is needed to determine how to prolong protection of EPCs, such as, by transfer of genes encoding antiapoptotic molecules, without jeopardizing the elimination of EPCs at the end of therapy. Terminal elimination is required to prevent the proangiogenic effects of surviving EPCs leading to increased growth of remaining tumor tissue78 and to preclude the remote possibility of malignant transformation of the highly proliferative EPCs.

Third, the most effective tumorcytotoxic effector to be delivered by the EPCs has to be carefully chosen. Even with efficient homing to tumors and effective intratumoral migration, therapeutic EPCs will be outnumbered by tumor cells by a large margin. Thus, a pronounced bystander effect is necessary. The choice of the optimal tumorcytotoxic effector for EPCs remains yet to be determined, given the limited number of published studies, the different entities, localizations and stages of the tumors treated, and the differences in generation and application of the EPCs. On theoretical grounds, viral, immunomodulatory or antiantiogenic approaches should be superior, given that they exert farther-reaching bystander effects than suicide genes or death ligands.

Addressing these challenges may lead to a cellular vehicle that provides efficient, specific, protected and safe systemic delivery of cytotoxic moieties into tumors.

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Acknowledgements

We thank Nora Hipp for clerical assistance and Helgard Knauß for artwork. This work was supported in part by a grant from the Deutsche Forschungsgemeinschaft (to CB).

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Correspondence to C Beltinger.

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Debatin, K., Wei, J. & Beltinger, C. Endothelial progenitor cells for cancer gene therapy. Gene Ther 15, 780–786 (2008) doi:10.1038/gt.2008.36

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Keywords

  • endothelial progenitor cells
  • cellular vehicle
  • cancer

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