Tie2-expressing monocytes: regulationof tumor angiogenesis and therapeuticimplicationsMichele De Palma1, Craig Murdoch2, Mary Anna Venneri1, Luigi Naldini1,3 andClaire E. Lewis4

1 Angiogenesis and Tumor Targeting Research Unit and San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific

Institute, Via Olgettina, 58-20132 Milan, Italy2 Department of Oral & Maxillofacial Surgery, University of Sheffield School of Clinical Dentistry, Claremont Crescent, Sheffield S10

2TA, UK3 San Raffaele Vita-Salute University, Via Olgettina, 58-20132 Milan, Italy4 Tumor Targeting Group, Academic Unit of Pathology, The Sir Henry Wellcome Laboratories for Medical Research, University of

Sheffield Medical School, Sheffield S10 2RX, UK

Opinion TRENDS in Immunology Vol.28 No.12

Tumor-infiltrating myeloid cells are involved in crucialprocesses during tumor development. A subset ofmonocytes that express the angiopoietin receptor Tie2play an important role in tumor angiogenesis. Selectivedepletion of these Tie2-expressing monocytes (TEMs) intumor-bearing mice inhibits tumor angiogenesis andgrowth, suggesting that they might regulate angiogenicprocesses in tumors by providing paracrine support tonascent blood vessels. TEMs have also been identified inhuman blood and tumors. We discuss here the thera-peutic opportunities emanating from the discovery ofTEMs, which include the identification of new antitumortargets, monitoring TEMs as surrogate markers forclinical responses in cancer patients, and the possibleuse of TEMs as cellular vehicles for gene delivery totumors.

Role of bone marrow-derived cells in tumorangiogenesisDuring physiological angiogenesis, new blood vessels areformed through a well-orchestrated series of events, whichinclude endothelial cell (EC) proliferation, their migrationtoward angiogenic stimuli, recruitment of perivascularsupport cells, functional lumen formation, and blood flow[1,2]. During development and organogenesis, the acti-vation of molecular and cellular programs that controlangiogenesis is tightly regulated by an assortment ofpositive and negative mediators (pro- and antiangiogenicsignals) whose balanced equilibrium is kept in check underhomeostatic conditions. Similar to growing tissues, devel-oping tumors build new blood vessels in response to theincreasing demand for nutrients and oxygen experiencedby the tumor mass [3,4]. However, the chaotic proangio-genic program driving tumor angiogenesis makes thetumor-associated vasculature inherently unstable andpoorly functional [2,3,5].

Corresponding authors: Naldini, L. (naldini.luigi@hsr.it);Lewis, C.E. (claire.lewis@sheffield.ac.uk).

Available online 5 November 2007.

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Developing tumors build intratumoral blood vessels bycoopting neighbouring, preexisting blood vessels, whichare activated and expand within the growing tumor massas a result of the brisk proliferation of ECs under theinfluence of tumor-derived proangiogenic signals. It hasbeen proposed that bone marrow (BM)-derived endothelialprogenitor cells (EPCs) also contribute to blood vesselformation in tumors [6]. EPCs are thought to reside inthe BM as rare CD45�AC133+VEGFR-2+ cells [6,7], whichare highly clonogenic and can be mobilized to the periph-eral circulation as circulating ECs (CECs) in response tospecific angiogenic stimuli, such as the plasma or BMelevation of vascular endothelial growth factor (VEGF),CXCL12 (also known as stromal-derived factor-1 [SDF-1]),and matrix-metalloproteinase-9 (MMP-9). CECs arethought to participate in tumor neovascularization byincorporating into newly formed blood vessels as bona fideECs [6,8]. However, there is much debate on the origin,phenotype, and differentiation potential of EPCs andCECs[8–11], and there is currently no consensus on their abilityto incorporate into the tumor vasculature and supportangiogenesis. Indeed, estimates of the BM contributionto the tumor endothelium have been highly variable;different studies showed that as low as 0.01% and up to50%–90% of the tumor vessels contained BM-derived ECs,with the majority of the reports showing figures in thelower range [12–16]. Some studies have suggested thatthe vascular incorporation of BM-derived ECs might heav-ily depend on the tumor type or its developmental stage[17–20]. However, even studies employing similar tumormodels have reported contradictory results [12,13,16]. Thepossible reasons for this lack of consensus have beendiscussed elsewhere [8,21,22]. Although in our studieswe did not find evidence for the vascular incorporationof BM-derived EPCs [13,23], we showed that BM-derivedhematopoietic cells that are recruited to the tumors(Figure 1) play important roles in the angiogenic process.

Several tumor-secreted factors can recruit hematopoieticcells, which, in turn, stimulate angiogenesis by secreting

d. doi:10.1016/j.it.2007.09.004

Figure 1. Recruitment of BM-derived cells to tumors. (a–c) Immunofluorescence

and confocal analysis of TS/A mammary carcinomas injected subcutaneously in

BM-transplanted mice. Mice were transplanted 6 weeks earlier with BM cells

obtained from CMV/ACTb-GFP transgenic mice, which ubiquitously express GFP

[13]. Photos show several BM-derived, GFP-positive (green) cells present in highly

vascularized tumor regions. Note that the GFP-positive cells do not colocalize with

CD31-positive (red) blood vessels and virtually all express the pan-hematopoietic

marker CD45 (red). The arrow in (c) shows a small blood vessel containing

intraluminal GFP-positive platelets. Nuclei are labelled with TO-PRO-3 (blue). The

scale bar in (a) is 150 mm; those in (b) and (c) are 75 mm. (d) Immunofluorescence

staining and confocal analysis of human gliomas grown in the mouse brain. Mice

were transplanted 6 weeks earlier with BM cells transduced ex vivo by a lentiviral

vector constitutively expressing GFP from the phosphoglycerate kinase promoter

[23]. A CD31-positive (red) blood vessel at the tumor periphery is surrounded by

several BM-derived, GFP-positive cells. Nuclei are labelled with TO-PRO-3 (TP3,

blue). The scale bar is 20 mm.

520 Opinion TRENDS in Immunology Vol.28 No.12

several proangiogenic mediators. CCL2 (also known asmonocyte chemoattractant protein-1 [MCP-1]), colony-sti-mulating factor-1 (CSF-1), VEGF, placental growth factor(PlGF), CXCL12, CXCL8 (also known as interleukin-8[IL-8]), and MMP-9 have all been implicated in the recruit-ment of BM-derived cells to tissues undergoing remodellingand angiogenesis, such as tumors. The mechanisms bywhich these factors mobilize hematopoietic cells from theirBM niches and recruit them to tumors and ischemic tissueshave been reviewed elsewhere [24–28].

Once recruited to the tumor microenvironment, hema-topoietic cells might exert a paracrine effect on the angio-genic process by different means: (i) the direct stimulationof ECs, leading to EC proliferation and their acquisition ofa migratory and invasive phenotype [1,2]; (ii) the release ofboth soluble and matrix-bound proangiogenic factors [29–31]; (iii) the assistance of ECs during cell migration andvessel morphogenesis [1,2,32]; (iv) the suppression of anti-tumor immunity [33]. Several hematopoietic cell subsets ofthe myeloid lineage have been shown to have proangio-genic activity in tumors (for a recent review, see [22]).These include mast cells, neutrophils, and the so-calledCD11b+Gr-1+ myeloid-derived suppressor cells, which area heterogeneous population of myeloid cells comprisingneutrophils, monocytes, and myeloid progenitors. These

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tumor-infiltrating myeloid cells produce a wide array ofproangiogenic molecules and matrix-remodelling factors(including VEGF, basic fibroblast growth factor [bFGF],platelet-derived growth factor [PDGF], CXCL8, urokinase-type plasminogen activator [uPA], and MMPs) that mightdirectly activate EC proliferation and/or induce a proinva-sive program in ECs that facilitates their migration withinthe extracellular matrix (ECM) [1,2]. The bioavailability ofcertain proangiogenic factors is, however, limited in thetumor microenvironment, because they are either seques-tered to the ECM or tethered to the cell membrane. Macro-phages and other myeloid cells produce MMPs (and otherextracellular proteases), which regulate release of thesefactors, rendering them available for interaction with cog-nate receptors on ECs, and thus activating the develop-ment of tumor-associated vasculature [30,31]. Severalstudies have reported that, in tumors and ischemic tissues,some monocytes/macrophages cluster around nascentblood vessels, where they could function as pathfindersfor proliferating ECs [13,22,23]. In this regard, recentstudies have shown that some molecular signals knownto control the wiring of the axonal network (such as sema-phorins, netrins, and slits) aremore broadly involved in thecontrol of cell migration [32]. ECsmight use such attractiveand repulsive signals to regulate the patterning and direc-tional growth of the vascular network, and it is possiblethat some tissue-derived axon-guidance factors are alsoexpressed by tumor-infiltrating myeloid cells. There is alsoincreasing evidence that tumor-infiltrating myeloid cellscan antagonize the antiangiogenic activities of T cells intumors [33].

Tie2-expressing monocytes and tumor angiogenesisAlthough our studies have shown that putative BM-derived EPCs do not incorporate directly into tumor bloodvessels, they highlighted the important contribution ofhematopoietic-lineage cells in the angiogenic process[13,23]. In particular, we noted that one subset of mono-cytes plays an essential role in this phenomenon, thoseexpressing the angiopoietin receptor Tie2 (Box 1). The Tie2tyrosine kinase receptor has been shown previously to beexpressed only by endothelial and hematopoietic stem cells(HSCs) [34]. Tie2-expressing monocytes (TEMs) circulateat low frequency in the mouse peripheral blood and havebeen observed in several mouse tumor models, includingsubcutaneous tumor grafts, spontaneous insulinomasdeveloping in RIP1-Tag2 transgenic mice, and humangliomas growing in the mouse brain [13,23]. In thesemodels, TEMs constitute a subpopulation of the tumor-infiltrating, CD11b+ myeloid cells, but they can be distin-guished from the majority of tumor-associated macro-phages (TAMs) by their surface marker profile(Tie2+CD11b+) and their preferential localization in highlyvascularized, viable tumor areas. In most cases, TEMswere found in close proximity to, but not incorporated into,nascent tumor vessels. This feature suggested a potentialrole in the regulation of tumor angiogenesis. To investigatethis, transgenic mice were generated that expressthe conditionally toxic gene, Herpes Symplex Virus thymi-dine kinase (tk) under the control of Tie2 transcriptionregulatory elements. In this mouse model, proliferating

Box 1. Properties of Tie2-expressing monocytes, TEMs

Surface markers� Mouse: CD45+ CD11b+ Gr-1low/� [23]

� Human: CD45+ CD11b+ CD16+ CD14low L-Selectin� CCR2� [35,36]

Mouse tissues and tumor models in which TEMs have been

observed� Bone marrow and peripheral blood [23]

� Regenerating liver, granulation tissue [13]

� Subcutaneous TS/A and N202 mammary tumors, Lewis lung

carcinoma (LLC), and B16 melanoma [13,23]

� Orthotopic human glioblastoma and U87 glioma [23]

� RIP1-Tag2 spontaneous insulinomas [23]

� Mostly absent in intact tissues and organs [13,23]

Features in mouse tumors� A minor fraction of the tumor-infiltrating CD11b+ myeloid cells

[13,23]

� More abundant at the tumor periphery and scarce in necrotic,

nonviable inner tumor areas [13]

� Often have perivascular location, but distinct from pericytes [23]

Role in tumor angiogenesis� When selectively depleted in mice, angiogenesis and tumor

growth are impaired [13,23]

� Do not incorporate in the endothelium of blood vessels [13,23]

� Enhance angiogenesis when coinjected with tumor cells in mice

[23,35]

Modulation by the tumor microenvironment (human TEMs)� Migrate toward Ang-2 in vitro [35,36]

� Hypoxia upregulates Tie2 expression in monocytes in vitro [36]

� Hypoxia and/or Ang-2 downregulate the expression of IL-12 and

TNF-a in monocytes [36]

Opinion TRENDS in Immunology Vol.28 No.12 521

Tie2-expressing cells can be selectively killed by admin-istration of the pro-drug ganciclovir (GCV). HSCs isolatedfrom these mice (or wild-type hematopoietic progenitorstransduced ex vivo by aTie2-tk lentiviral vector) were thentransplanted into wild-type mice, to obtain chimeric micewith TEMs sensitive to GCV-mediated cell killing(Figure 2). A few weeks after the transplant, mice wereinoculated with either subcutaneous mammary tumors ororthotopic human gliomas and were administered GCV toeliminate TEMs during the early stages of tumor growth.In this model, GCV treatment ablated the recruitment ofTEMs to the developing tumors and markedly inhibitedtumor angiogenesis and slowed tumor growth [23]. Oneinteresting finding was that TEM elimination did notaffect the overall number of TAMs and granulocytes,making it improbable that TEMs comprise precursors ofTAMs. Rather, these studies suggested that TEMsrepresent a distinctmonocyte subset with inherent proan-giogenic activity. Because TEMs neither differentiatedinto ECs nor incorporated into the endothelial layer oftumor blood vessels, we postulated that their proangio-genic activity could consist of a paracrine stimulation ofthe angiogenic process. This hypothesis was supported bythe observation thatTEMswere seen in someperivascularspaces in tumors, where they expressed high levels ofbFGF, a proangiogenic molecule [23]. Moreover, TEMseither isolated from mouse tumors or peripheral bloodhad marked proangiogenic activity in transplantationassays [23].

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These studies have now been extended to show theexistence of TEMs in human blood and cancer cells[35,36]. In human cancer specimens, TEMs were a minorproportion of the bulk of tumor-infiltrating leukocytes,whichmostly comprise TAMs and granulocytes [34]. TEMswere found both in perivascular and avascular, viable(hypoxic) areas of human tumors and were largely missingin non-neoplastic tissues adjacent to tumors [35]. Inaddition to their presence in tumors, we have shown thatTEMs can be detected at low frequency in the humanperipheral blood [35,36].

Similar to murine cells, human TEMs isolated fromperipheral blood also have proangiogenic activity. Indeed,when coinjected with human glioma cells subcutaneouslyin nude mice, Tie2+CD14+ cells markedly promoted tumorvascularization [35]. Interestingly, circulating humanTEMs do not express CCR2, the receptor for CCL2/MCP-1, a chemokine that regulates the recruitment ofmonocytes to inflamed tissues and tumors. Although therecruitment of TEMs to inflamed tissues has yet to beinvestigated, it is possible that non-MCP-1 circuits governthe recruitment of TEMs to tumors. TEMs might beattracted to tumors in a CCR2-independent manner, bysignals produced by the cancer cells or stromal componentsof the tumor, such as the blood vessels themselves. Onepossible, but as yet unproven, mechanism for this wouldinvolve Ang-2, a proangiogenic cytokine known to be upre-gulated by tumor blood vessels. TEMs might be recruitedinto tumors by Ang-2 because it is a powerful chemoat-tractant for TEMs in vitro [35,36].

Our recent studies have also demonstrated that Ang-2and tumor microenvironmental signals such as hypoxiamodulate the angiogenic activity of TEMs. Ang-2 is upre-gulated in hypoxic areas of human tumors [37]; thus, it ishighly probable that TEMs are exposed to both hypoxiaandAng-2 in such sites. Exposure to both hypoxia and Ang-2 markedly suppresses the release of the potent antiangio-genic cytokine IL-12 by human TEMs [36], suggesting thatthe tumor modulates these cells so that they can no longermount this form of antiangiogenic response. This, togetherwith the pronounced effects of hypoxia on the release ofproangiogenic factors by macrophages [38], would contrib-ute to the rapid revascularization of hypoxic tumor areas.Interestingly, the combined action of Ang-2 and hypoxiaalso inhibits the release of tumor necrosis factor-a (TNF-a)by TEMs [36]. This is important because TNF-a exerts aprofound proapoptotic effect on both tumor cells and ECs[39]; thus, its downregulation in sites of angiogenesismight help to drive metastasis and angiogenesis.

From the findings discussed above, it is clear that onequestion that still needs to be addressed is whether or notTEMs are a committed, nonredundant population of proan-giogenic monocytes, or rather represent a functional,proangiogenic state of tumor-infiltrating monocytes,possibly induced by microenvironmental factors. Dataare available that support both concepts. Indeed, TEMscan be isolated from both mouse and human blood thatpossess high proangiogenic activity (as compared to theirTie2-negative counterpart), suggesting that TEMs existas a circulating reservoir of committed, proangiogenicmonocytes [23,35]. However, hypoxia has been shown to

Figure 2. TEM depletion in mice inhibits tumor angiogenesis. A schematic of the TEM elimination experiments described in the main text. Lineage-negative bone marrow-

derived cells are transduced ex vivo by a lentiviral vector encoding for the Herpes Symplex Virus thymidine kinase gene and GFP (through an internal ribosome entry site

[IRES] element). The two genes are expressed by transcription regulatory sequences from the Tie2 gene. Six weeks after the transplant of the transduced cells, mice are

inoculated with subcutaneous tumors (Lewis lung carcinoma is shown; tumor blood vessels are stained by an anti-CD31 antibody) and either treated with GCV (ganciclovir)

or left untreated. GCV treatment depleted TEMs and markedly inhibited tumor angiogenesis in the mice (see [13] and [23] for technical details).

522 Opinion TRENDS in Immunology Vol.28 No.12

upregulate Tie2 expression in blood-derived monocytesand macrophages, causing them to suppress their antian-giogenic activity [25]. It is known that hypoxia upregulatesTie2 expression on ECs [40]; thus, it is tempting to specu-late that Tie2 might also function as a molecular switch toregulate the recruitment and/or enhance the proangiogenicactivity of monocytes in tumors. Future studies are neededto clarify these important issues.

Possible role of TEMs in other diseasesWe investigated the recruitment of TEMs to sites of angio-genesis other than tumors. We analysed the recruitment ofgene-marked TEMs in mice that underwent partial hepa-tectomy 7–10 days earlier, and we found that TEMs werepresent in the granulation tissue surrounding the regen-erating hepatic lobules [13]. Some TEMs were found inproximity to newly formed vessels, similar to tumor TEMs,suggesting that they might also contribute to promotingangiogenesis during liver regeneration. Other reports haveshown that Tie2+ cells distinct from ECs participate indisease conditions characterized by pathological vascularremodelling, such as arthritis [41] and thrombus recana-lization [42]. It is tempting to speculate that, in variouspathological forms of angiogenesis, certain tissue-derivedfactors upregulated by tissue damage and remodelling andby vascular activation – such as Ang-2 [43] – might

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promote the recruitment of TEMs from circulation andlocally enhance their proangiogenic activity.

Therapeutic opportunities: targeting TEMs or exploitingthem as gene-delivery vehicles in anticancer therapyThe future identification of specific molecules expressed byTEMs in tumors could facilitate the design of novel antic-ancer therapies that selectively target these cells, therebyremoving their proangiogenic contribution in such tissueswhile preserving their potential role in regulating tissuehomeostasis and immunity (Box 2). The identification ofgene expression by TEMs in tumors and the tumor micro-environmental factors that regulate TEM recruitment and/or function probably highlight such new targets. Drugsdeveloped to target TEMs in tumors could have sustainedtherapeutic efficacy, because these cells (unlike tumorcells) are nonproliferating and genetically stable, andtherefore probably don’t develop drug resistance [44].

As mentioned previously, TEMs are present in theperipheral blood of both healthy donors and cancerpatients [35,36], although studies comparing their fre-quency have yet to be performed. It would also be inter-esting to correlate this with clinical/tumor parameters tosee if TEMs could be used as surrogate markers to monitorclinical responses of cancer patients receiving chemother-apy or antiangiogenic drugs.

Box 2. Therapeutic implications and unanswered questions

Therapeutic opportunities

� Identification of specific molecular targets on TEMs to develop

new anticancer drugs that:

� Deplete them in cancer patients

� Inhibit their activities in tumors

� Monitoring TEMs as biomarkers of angiogenesis and clinical

responses in cancer patients treated by cytoablative or antiangio-

genic agents

� Exploiting TEMs as gene-delivery vehicles for the transport of gene

therapy to tumors via:

� Bone marrow transplantation with autologous vector-transduced

cells [13]

� Adoptive transfer of ex vivo transduced cells

Unanswered questions� What are the bone marrow precursors of TEMs? Do TEMs

represent a developmentally distinct hematopoietic lineage? What

is their lineage relationship with other proangiogenic myeloid cell

types found in tumors, such as TAMs?

� To what extent do tumor microenvironmental factors other than

hypoxia modulate TEMs’ function?

� Do TEMs respond to angiopoietins in vivo?

� What are the proangiogenic factors secreted by TEMs in tumors?

� Do TEMs have protumoral activities other than being proangio-

genic?

� Do TEMs have a role during development and organogenesis?

� Do TEMs have a role in maintaining vascular homeostasis?

� Is there a correlation between disease type, stage, and progres-

sion and the frequency of TEMs in cancer patients?

� Will TEMs provide a safe and effective means to deliver antitumor

or antiangiogenic molecules to tumors?

Opinion TRENDS in Immunology Vol.28 No.12 523

Monocytes/macrophages that infiltrate tumors (or theirprecursors in the BM) might have utility as cellularvehicles for the delivery of gene therapy to tumors. Tothis aim, genetically modified human macrophages havebeen used to target therapeutic genes and viruses tohypoxic areas of tumor masses in vitro [45,46], and HSCs,obtained from the BMor alternative sources, have alreadybeenused in the setting of transplantation [13,46]. Indeed,recent developments in methods to reduce the toxicity ofBM transplantation have opened up the possibility ofusing this approach to treat solid tumors [45,47,48], andapproaches based on transplantation of genetically modi-fied autologous HSCs could achieve therapeutic success.Interestingly, we have previously shown that BM trans-plantation of HSCs lentivirally transduced to expressgenes under the influence of Tie2 transcription elementsresults in selective gene expression in TEMs in bothperipheral blood and tumors in mice [13,23]. BecauseTEMs display remarkable tumor-homing ability, we arecurrently investigating whether this TEM-basedapproach can be used to target therapeutic genes totumors.

AcknowledgementsC.M. and C.E.L. would like to thank Yorkshire Cancer Campaign andBreast Cancer Campaign for grant support of their work in this area.L.N. acknowledges the grant support from Associazione Italiana per laRicerca sul Cancro (AIRC 52–2005), European Union (Tumor-HostGenomics, LSHC-CT-2005–518198), and Telethon (TIGET). M.D.P. wassupported by AIRC. None of the authors have a financial interest in thiswork.

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