Tumor vessels have thin cytoplasmic projections extending across the vessel lumen. These projections resemble tip-like filopodia observed during intussusceptive growth but they may overlap with one another and form loose connections. The origin of these sprouts may be the oxygen seeking tip cells at the leading edge of sprouting vessels in hypoxic regions of the tumor microenvironment.
Carmeliet and colleagues recently proposed a molecular basis for these abnormal sprouts Mazzone et al. For example, tip cells of tumor vessels in mice haploinsufficient for the oxygen sensor PHD2 appear quiescent and adjust their shape and phenotype to restore oxygen supply. If one considers that the vasculature is a supply line for oxygen and nutrients but also a conduit for the removal of waste products, then abnormalities in the blood vessels themselves are a major contributor to the abnormal microenvironment in tumors.
Not only do cancer cells thrive in this environment, but selection pressure created by these microenvironmental bottlenecks may actually contribute to their propagation Merlo et al. VEGF alone is a potent vasodilator that can promote fluid leakage and high interstitial pressures, abnormal branching morphogenesis, and small gaps and fissures in the vasculature Nagy et al.
Thus, chronic VEGF stimulation in tumors promotes sprouting and excessive branching of tip cells leading to irregularities in the TEC monolayer and loss of barrier function. Irregularities in the TEC lining surrounding these vessel protrusions impairs blood flow resulting in hypoxia and hypoperfusion. Tumor vessels are also squeezed and compressed by overlying tumor cells, which creates biomechanical tension, strain, and changes in blood flow Padera et al.
This chaotic pattern of blood flow can alter endothelial shape, size, and differentiation perhaps through aberrant expression of flow-mediated transcription factors De Val and Black Notably, endothelial dysfunction in tumors is not a dead end.
All cell types found within the perivascular niche either in direct contact with the endothelium or dependent on soluble vascular-derived factors e. For many years, it was assumed that TECs were similar to their normal counterparts irrespective of the obvious morphological abnormalities of tumor blood vessels in situ. In fact, the theoretical success of antiangiogenic specifically antiendothelial strategies in cancer depends to some extent on TECs remaining stabile and not altering their phenotype over time.
However, recent studies have shown that TEC are more complex and labile than expected challenging the assumption that TEC are normal. Collectively, these studies show morphological, pathophysiological, cytogenetic, epigenetic, and gene expression changes in the TEC pool.
Despite this new knowledge, overall our understanding of TEC biology has been hampered by several technical limitations, which are described below. However, these markers are shared by endothelial cells from virtually all vascular beds capillary, venous, arterial, and lymphatic ; thus, heterogeneity in the isolated population of TECs is unavoidable.
Diphtheria toxin has been successfully used to eliminate human tumor cells from cultures of mouse TECs Arbiser et al. The most proficient way to ensure purity of isolated TECs is to prepare clonal populations, but this can be challenging because ECs plated at clonal density will often undergo senescence. Second, the conditions for culturing TECs in vitro are not well defined.
TECs may be adapted to the tumor microenvironment e. There may be unique media requirements and combinations of growth and attachment factors. For example, in one study, the use of oncofetal fibronectin was necessary to maintain the phenotype of TECs isolated from Lewis lung carcinoma LLC Allport and Weissleder Despite the challenges, several groups have isolated and characterized i.
These studies typically fall into two categories: those that isolated and characterized TECs that were never cultured, and those that used isolated and culture expanded TECs for their analyses. Although there is some value to these approaches, the use of surrogates cannot substitute for isolating TECs from the native tumor microenvironment. Croix published the first comprehensive genetic screening of human TECs from malignant colorectal tissues St Croix et al.
TEMs 1 and 5 were also found in some normal vascular beds Seaman et al. In one study, TEM1 was detected in fibroblasts and perivascular cells but not endothelium MacFadyen et al.
Another study showed ubiquitous TEM1 expression in developing tissues Opavsky et al. TEM7 expression was found in the brain Lee et al.
TEM8 the anthrax toxin receptor is also expressed in the vasculature of the developing corpus luteum St Croix et al. Despite TEM8 expression in some normal tissues, its utility as a marker or target in tumor vessels is still being investigated. Aird has nicely collated the transcriptional profiling results of TECs from multiple studies and multiple tumor types Aird The majority of genes were limited to one tumor type or to invasive tumors.
Thus, there may be tumor stage and tumor-type-specific differences in the TEC pool. In support of this possibility, Hoffman used phage display and found peptides that homed to the neovasculature of premalignant lesions but not of malignant skin tumors Hoffman et al.
Similarly, tumor stage and tumor site—specific orthotopic vs. High-throughput screens to identify unique proteins expressed on the surface of TECs have also been used.
One approach uses colloidal silica nanoparticles to coat the luminal surface of the endothelium followed by subcellular fractionation and subtractive proteomic mapping. Schnitzer and colleagues used this technique and found amino peptidase-P and annexin A1 as selective targets for the lung tumor vasculature Oh et al. Furthermore, I-labelled annexin A1 antibodies accumulated in the lung tumor vasculature, destroyed tumors and prolonged survival in tumor-bearing rats.
The miRNA miR is elevated in the endothelium of human tumors and hemangioma where it is proposed to function as an angiogenic switch by suppressing pRasGAP leading to Ras activation and angiogenesis stimulation Anand et al. Taken together, high-throughput protein and gene arrays and more recently miRNA screens have been used to identify unique molecular signatures in the tumor vasculature.
These studies collectively show diverse patterns of altered gene expression from different tumor types and stages of progression see Table 1. However, no unique marker or factor in the tumor vasculature has proven suitable as a durable target for antiangiogenesis therapy.
A time line of tumor endothelial cell TEC isolation and characterization: 41 studies of isolated tumor endothelial cells grouped by year. For example, some studies listed in the table use human tumor cell lines implanted in mice, but the isolated TECs are of mouse origin. This table includes only those studies where TECs were interrogated following immunomagnetic separation, laser capture microdissection, in silico subcellular fractionation, or phage display.
Isolation of the first primary cultures of ECs from normal umbilical veins dramatically increased our fundamental understanding of EC biology Gimbrone et al. Incredibly, it would be almost 30 years before the first cultures of TECs were isolated and culture expanded. One of the advantages of culture expansion is that functional in vitro assays can be performed. For example, in one of the first reports of successful isolation and culture of human TECs, Allesandri and coworkers showed that human TECs maintain their phenotype in culture, they express growth factor receptors and they are stimulated by typical EC mitogens Alessandri et al.
Silencing Pax2 resulted in a decrease in phospho-Akt and decreased resistance to vincristine. A good example is the expression of Thy-1 an adhesion receptor for leukocytes in tumor vessels in situ and in isolated TECs that could mediate interactions with proinflammatory cells or tumor cells Dudley et al. Constitutive expression of adhesion molecules in tumor vessels could mediate the increased tropism and trafficking of proinflammatory cells that are also proangiogenic.
Adhesion molecule expression in TECs may be induced by contact with tumor cells Haddad et al. Only a few laboratories have reported successful isolation and culture expansion of mouse TECs from tumor bearing mice either xenografts or spontaneous tumors. Similar to human TECs, there are problems with contamination by tumor cells and other cell types found in the tumor stroma making it difficult to obtain pure TEC cultures. Amin reported that isolated and culture expanded TECs from implanted melanoma and breast tumors express ErbBs 1, 2, and 4 and are stimulated to proliferate by EGF Amin et al.
A principle of antiangiogenesis therapies in cancer is that the tumor endothelium is normal and will not change over time or develop drug resistance. However, Hida reported abnormal centrosomes and aneuploid chromosomes in TECs isolated from human melanoma and liposarcoma implanted in mice Hida et al. These chromosomal abnormalities were not clonal nor were they derived from human genetic material incorporated into mouse chromosomes. On the other hand, Streubel detected primary and secondary translocations in ECs that were identical to those found in follicular lymphoma suggesting either a tumor cell of origin for TECs or sharing of DNA between tumor cells and ECs Streubel et al.
Horizontal transfer of genetic material via apoptotic bodies between tumor cells and ECs has been reported Ehnfors et al. Loss of function of gatekeeper genes in the stroma e. Our laboratory has reported unusual patterns of differentiation in TECs isolated from spontaneous prostate tumors in TRAMP transgenic adenocarcinoma of the mouse prostate mice.
For example, prostate tumor TECs unexpectedly differentiate to form bone and cartilage Dudley et al. Ectopic microvascular calcification was also detected along the capillary lumens of human prostate cancers. Breast tumors are also characterized by microcalcifications in the vasculature Tse et al.
Unexpected patterns of differentiation in tumor vessels may arise as vascular cells and other stromal cells coevolve with tumor cells and switch their phenotype Polyak et al. For example, lineage switches and coexpression of lymphatic, endothelial, and fibroblast markers have been observed in the blood vessels of other malignancies Breiteneder-Geleff et al.
Pathophysiological conditions in the tumor microenvironment including aberrant expression of growth and differentiation factors may control the fate, differentiation, and mesenchymal transition of TECs Verfaillie For example, Ghosh showed that isolated TECs fail to reorient their actin cytoskeleton when exposed to uniaxial cyclic strain and they display greater traction forces in response to variations in ECM elasticity in vitro Ghosh et al. TECs may be reprogrammed or adapted to these conditions, which could account for some of the structural and functional abnormalities in the tumor vasculature.
The range of morphological, cellular, and molecular abnormalities reported specifically in TECs is broad and diverse. As already noted, VEGF-A alone is sufficient to induce most of the morphological changes tortuosity, excessive branching, and leakiness observed in the tumor vasculature.
But can these abnormalities facilitate tumor growth and progression to metastases? One can easily imagine how fragile, leaky vessels or gaps and holes in the vasculature might allow tumor cells to enter the circulation and disseminate to distant sites. For example, deficient pericyte coverage, which can lead to vessel leakiness and hemorrhage, is associated with increased metastases in human cancers and in mouse tumor models Yonenaga et al.
As a corollary, treatment modalities that reverse these abnormalities in the vasculature might prevent metastasis. In a mouse model, a single dose of Avastin anti-VEGF antibodies decreased microvessel density, vessel permeability and interstitial pressure while intratumoral perfusion was improved Dickson et al.
Recent clinical studies support the concept that combining Avastin with chemotherapy can lead to improved outcomes in patients with advanced rectal cancer Willett et al. Heterotypic interactions and cross talk between TECs and other cell types, particularly leukocytes mobilized from the circulation, might also be affected by abnormalities in the tumor vasculature.
As an example, some adhesion molecules may be decreased in the tumor vasculature allowing tumors to escape immune surveillance because of impaired interactions between T-lymphocytes and the blood vessel wall Griffioen et al.
Indeed, the penetration and efficacy of primed T cells for tumor immunotherapy was enhanced when proinflammatory agents that up-regulated ICAM and VCAM in the vasculature were coadministered Garbi et al. Thus, a perivascular positioning of leukocytes at the periphery of tumor vessels is common Dudley et al. These proinflammatory cells express most of the endothelial survival and matrix remodeling factors required for angiogenesis.
In one sense then, inflammation or activation of TEC enables the conscription of a diverse population of auxiliary cells that play a catalytic role during angiogenesis. Furthermore, proinflammatory cells, particularly macrophages, not only stimulate angiogenesis, but they may also enable metastasis Qian and Pollard Because the endothelium acts a gatekeeper controlling the egress of proinflammatory cells into the tissue or tumor , blocking specific interactions between TECs and the immune cell infiltrate may indirectly impair angiogenesis and metastasis.
Although many of the chemokines that control leukocyte tropism are tumor cell—derived, TECs may be a direct source for many of these chemotactic factors Butler et al.
Where does the tumor endothelium come from? The answer to this question has been more difficult to answer than expected. For a long time, the ECs lining tumor vessels were thought to arise only by mitoses, sprouting, or simple cooption of preexisting capillaries, whereas vasculogenesis occurred only during embryonic development.
However, a distorted variation of each of these processes probably generates new endothelium in tumors. There may also be additional, unexpected sources for TECs.
For example, Hendrix suggests that stem-like tumor cells may transdifferentiate to form endothelium Hendrix et al. A mesenchymal stem cell, with properties of endothelial cells, was shown to form pericytes and endothelium in hemangioma Khan et al. The turnover of the endothelium in normal tissues is low. It is estimated that only 0. In tumors, EC turnover greatly accelerates and may be 20— times the rate in normal tissues Hobson and Denekamp In a seminal paper, Folkman showed that normal adult tissues implanted in the chick chorioallantoic membrane CAM did not promote neovascularization whereas implanted tumors rapidly stimulated the growth of new blood vessels from the host Ausprunk et al.
For the next 20 years, angiogenesis was considered the sole or predominate source of new endothelium in tumors. A number of subsequent studies identified similar cells circulating in blood that were localized to sites of angiogenesis in ischemic tissues and tumors Shi et al. Thus, postnatal vasculogenesis was proposed as an alternative route for new tumor blood vessels.
However, hematopoietic cells e. Their proximity to the blood vessel and expression of markers shared with bona fide endothelium has created confusion and discrepancies over the identification of circulating ECs in solid tumors; especially in rodents Kerbel et al. The functional differences distinguishing hematopoietic cells from bona fide endothelium are becoming clear.
Yoder suggests that only the endothelial colony forming cells ECFCs can form vessel lumens, whereas hematopoietic cells generally do not Yoder These same criteria should be applied for identifying ECFCs in tumors using rodent models. However, these assays provide no functional information about the nature of the recruited circulating cell type s.
Culture expansion and characterization e. Recently, the vessel wall itself has been proposed as a source for vascular endothelium because it contains subpopulations of ECs with properties similar to blood-derived ECFCs Ingram et al. Thus, in contrast to a bone marrow origin, there may be a local reservoir of highly proliferative endothelium proximal to the tumor site.
No studies to date have determined whether VW-EPCs might form the majority of the angiogenesis response in tumors, if there are any unique properties in VW-EPCs that could be exploited as an antiangiogenesis strategy, or if VW-EPCs might mediate vascular rebound often observed following antiangiogenic therapies in the clinic Bergers and Hanahan ; Ellis and Hicklin However, an unexpected complication is that cell-to-cell heterogeneity, acquired resistance, and a multisource origin for TECs might impinge on the success of antiendothelial strategies.
Although the success and selectivity of antiangiogenesis therapies in tumors depend to some extent on there being differences in TECs compared to their counterparts, too much variation in the TEC pool can have the opposite effect.
One way around this problem is to target multiple cell types simultaneously, including those now known to play auxiliary roles during tumor blood vessel formation. Pericytes, fibroblasts, and other mesenchymal-lineage cells in the stroma may be valuable indirect targets for antiangiogenesis in tumors Loeffler et al. The role of hematopoietic lineage cells in tumor angiogenesis and their potential as targets for antiangiogenic therapies is briefly discussed below.
An early clue that hematopoietic cells might facilitate angiogenesis comes from studies in the AMLdeficient embryos. These mice lack definitive hematopoiesis and show impaired angiogenesis in the head and pericardium that can be rescued by addition of hematopoietic cells expressing ANG-1 Takakura et al. Later and in tumors, marrow-derived inflammatory cells including neutrophils, macrophages, and mast cells were shown to provide the majority of the proangiogenic factor MMP9 during angiogenesis Coussens et al.
In the last 10 years, a number of studies have confirmed the role of proinflammatory cells in tumor angiogenesis and have identified the molecular pathways linking inflammation and cancer Mantovani et al. Macrophages in particular appear to promote tumor invasion, cancer initiation, and angiogenesis Qian and Pollard There appear to be tumor-specific and distinct populations of myeloid cells recruited to tumors that release angiogenic factors involved in matrix remodeling or that stimulate endothelial cells directly Coffelt et al.
For example, selective depletion of neutrophils Nozawa et al. Furthermore, their activity in tumors might mediate resistance to antiangiogenic therapies by stimulating vascular rebound. Finally, hematopoietic cells may stimulate tumor blood vessel formation in other, unexpected ways. For example, macrophages may participate during nascent vessel formation by providing guidance and mechanical cues that mediate anastomosis between branching tip cells Fantin et al.
Since Dr. For example, tumor vessels have proven to be more complex and labile than expected and it was not predicted that TECs might be cytogenetically abnormal or derived from multiple sources Fig. Furthermore, there have been unexpected consequences of VEGF inhibition including an up-regulation of compensatory angiogenic pathways Crawford et al. Other obstacles include heterogeneity in the vascular bed and tumor-type or stage-specific differences in TEC that could ultimately impinge on the effectiveness of the antiangiogenic therapies designed to target them Bergers et al.
There may be unique gene expression profiles in blood vessels from different regions of the tumor microenvironment or even in individual TECs of the same vessel Chi et al. In a striking example, it was recently suggested that some tumor vessels may lose their dependence on VEGF signaling altogether theoretically rendering them refractory to VEGF inhibition Nagy et al.
Our ability to isolate and better characterize TEC from different tumors or during different stages of tumor progression should be a valuable approach for finding new targets, beyond VEGF and its receptors, in vascular cells.
On the other hand, an innovative approach that includes eliminating alternative cell types e. Thus, TEC may be moving targets and their phenotypic diversity or multisource origin might impinge on the effectiveness of the drugs intended to target them, but there are other approaches to consider toward the goal of effective and durable antiangiogenic strategies in cancer.
A reductionist versus contemporary view of tumor endothelium. In a reductionist view, all of the endothelial cells lining a tumor blood vessel are homogeneous and perhaps derived by spouting or simple cooption of nearby vessels. In the contemporary view, TECs are heterogeneous and derived from multiple sources. A composite tumor blood vessel is shown in which the endothelium may be derived by cooption or sprouting, from endothelial progenitors localized in the vessel wall, from tumor cells masquerading as endothelial cells, from marrow-derived endothelial progenitors, and from unexpected sources including transdifferentiated myeloid and mesenchymal lineage cells.
Plasticity and a multisource origin may contribute to tumor blood vessel abnormalities and allow tumor vessels to evade antiangiogenic therapies. I thank Dr. Juan M. Melero-Martin for reviewing this manuscript and Kristin Johnson for her excellent assistance with the figures.
Additional Perspectives on Angiogenesis available at www. National Center for Biotechnology Information , U. Cold Spring Harb Perspect Med. Andrew C. Author information Copyright and License information Disclaimer. Correspondence: Email: ude. This article has been cited by other articles in PMC. Angiogenesis In the adult, new blood vessels arise from preexisting ones by angiogenesis. Vasculogenesis In contrast to angiogenesis, vasculogenesis occurs mainly during development when progenitor cells angioblasts committed to the vascular lineage differentiate to form an immature vascular plexus in the embryo Jin and Patterson Intussusceptive Angiogenesis An alternative and rapid mechanism for a new vessel to form is through intussusceptive angiogenesis IA.
Open in a separate window. Figure 1. Defective Endothelial Monolayer In normal tissues the ECs form a continuous and uniform monolayer with few cytoplasmic projections. Abnormal Sprouts Tumor vessels have thin cytoplasmic projections extending across the vessel lumen. What Are the Causes of These Abnormalities? Table 1. Human kidney tumor St. Croix et al. Human colon tumor Unger et al.
Human brain tumor Allport et al. Mouse lung tumor Hida et al. Mouse liposarcoma and melanoma Miebach et al. Human brain tumor Amin et al. Mouse breast tumor Seaman et al. Mouse multiple tumors Amin et al. Mouse melanoma Xiong et al. Human liver tumor Johnson et al. Mouse prostate tumor Hoffman et al. Mouse skin tumors Streubel et al.
Human B cell lymphoma Charalambous et al. Human brain tumor Grange et al. Human breast tumor Pen et al. Human brain tumor Dudley et al. Mouse prostate tumor Issa et al. Human pancreatic tumor You et al.
Human glossal lymphangioma Joyce et al. Mouse pancreatic tumor Madden et al. Human brain tumor Grover et al. Human prostate tumor Buckanovitch et al. Human ovarian tumor Bhati et al. Human breast tumor Jayasinghe et al. Human colon tumor Bussolati et al. Human kidney tumor Parker et al. Human breast tumor Fonsato et al. Human kidney tumor Lu et al. Human ovarian tumor Wurdinger et al. Human brain tumor Mazzone et al. Mouse multiple tumors Oh et al.
Rat lung tumor Bussolati et al. Human kidney tumor Hellebrekers et al. Human colon tumor Ghosh et al. Mouse prostate tumor van Beijnum et al. Human colon tumor Schellerer et al.
Human colon tumor Wu et al. Human liver tumor Doublier et al. Human kidney tumor Nummer et al. Human pancreatic tumor. Tumor Models in Mice Only a few laboratories have reported successful isolation and culture expansion of mouse TECs from tumor bearing mice either xenografts or spontaneous tumors.
Figure 2. Contribution of bone marrow—derived cells to blood vessels in ischemic tissues and tumors. Matrix metalloproteinase-9 is required for tumor vasculogenesis but not for angiogenesis: Role of bone marrow—derived myelomonocytic cells. Mechanisms of endothelial cell heterogeneity in health and disease. Molecular heterogeneity of tumor endothelium. Importantly, a pharmacological ASK1 inhibitor prevents tumor-induced vascular leakage, macrophage infiltration, and tumor growth in two mouse models.
Since transcoelomic metastasis is also associated with many other cancers, such as pancreatic and colon cancers, our study provides ASK1 as a therapeutic target for the treatment of ovarian cancer and other transcoelomic metastasis cancers. Keywords: Cancer; Macrophages; Oncology; Therapeutics; endothelial cells. Abstract We have recently reported that tumor-associated macrophages TAMs promote early transcoelomic metastasis of ovarian cancer by facilitating TAM-ovarian cancer cell spheroid formation.
Publication types Research Support, N.
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