Vasculature generally represents only a small fraction of the tumor mass and, at any one time, includes a mixture of blood vessel types, making their formation difficult to study. Therefore, we developed a reductionist model, based on the generally accepted belief that tumors induce new blood vessels by overexpressing VEGF. We used an adenovirus-expressing murine VEGF-A164, the most common VEGF-A isoform, to deliver VEGF locally to the tissues of immunoincompetent nude mice, and were able to induce large numbers of each of the 6 types of tumor blood vessels in the absence of tumor cells.10,11,26,59,63 Because adenoviral vectors are not integrated into the genome, their encoded proteins are expressed for only a limited period. As a result, VEGF levels fall exponentially to ineffectual levels within a few days. Therefore, in contrast to tumors in which VEGF levels remain elevated indefinitely, Ad–VEGF-A164 delivers a single, self-limited pulse of VEGF. This has allowed us to elucidate some of the steps and mechanisms by which VEGF induces new blood vessel formation and furthermore to isolate surrogate forms of the different tumor blood vessel types in nearly pure form for molecular characterization.64,65
Lacking basement membrane and pericyte support, MVs are unstable and differentiate into several types of daughter vessels10,11,26,59,63 (Figs. 2 and 3; Table 1). The first of these are glomeruloid microvascular proliferations (GMPs), structures that resemble renal glomeruli (hence the name). Glomeruloid microvascular proliferations result from MV collapse, accompanied by accumulation of pericytes and macrophages and extensive synthesis of new basement membrane.59,71,72 They are found in many human cancers but are particularly abundant in glioblastoma multiforme.73 Over time, GMPs devolve into normal-appearing capillaries (Fig. 3).
Mother vessels can also differentiate into vascular malformations (VMs), stabile vessels that retain their large size by acquiring a coat of smooth muscle cells and perivascular collagen. Vascular malformations can be distinguished from feeding arteries (FAs) by their thinner and often asymmetric muscular coat and from draining veins (DVs) by their smaller lumens. Vascular malformations resemble the nonmalignant VMs that are found in skin, brain, and so on, perhaps suggesting a mechanism by which VMs form in those circumstances. As their structure implies, VMs are not hyperpermeable to plasma proteins. Also unlike MVs and GMPs, VMs persist indefinitely, long after adenoviral vector-induced VEGF-A164 expression has ceased. Thus, VMs have attained independence from exogenous VEGF, although it is likely that their endothelial cells are supported by paracrine VEGF secretion from the smooth muscle cells that envelop them.
Three additional possibilities deserve consideration to explain the limited success of anti-VEGF/VEGFR therapy. One possibility is that some tumors live on the edge and are able to tolerate an inadequate blood supply. A second possibility is that some tumors do not need to induce new blood vessels because they co-opt preexisting normal blood vessels.76–78 We have found this to be the case in mouse tumors growing in the lung. Primary subcutaneous B16 melanomas induce an abundant, MV-rich vasculature; however, when metastatic to the lung, they grow to large size without generating new blood vessels (unpublished data). This is perhaps not surprising in that the lung provides an oxygen-rich environment with an abundant supply of capillaries capably designed for efficient gas and nutrient exchange.
To test this hypothesis more rigorously, we made use of our reductionist model, administering drugs at early and successively later times after injection of Ad–VEGF-A164 into the skin of nude mice. The results with rapamycin,84 a drug active downstream of the VEGF–VEGFR-2 pathway, and with aflibercept and other drugs targeting VEGF or its receptors, have been consistent.82,83 All of these drugs prevented the formation of MVs, the majority “early”-type blood vessel found in freshly transplanted mouse tumors; they also caused the regression of already formed MVs and GMPs when administered a few days later. However, their antivascular activities were progressively less effective when treatment was delayed further. None had a significant effect on VMs, FAs, or DVs. These findings, then, are consistent with the limited success that anti-VEGF/VEGFR therapy has against human tumors; such therapy may be expected to prevent further angiogenesis and prune early tumor vessels (MVs, GMPs) but have little or no effect on vessels of the late type. They are also supportive of the work of Carmeliet and Jain85,86 and Goel and colleagues,85,86 who demonstrated that anti-VEGF therapy causes tumor blood vessel “normalization.” Jain and colleagues found that anti-VEGF/VEGFR therapy reduces the hyperpermeability characteristic of tumor blood vessels, along with the consequent resulting edema and interstitial pressure. This result would be expected if these drugs were primarily targeting MVs, the hyperpermeable tumor blood vessel subset.
Finally, the tumor vasculature is heterogeneous, consisting of at least 6 distinct blood vessel types. Although all 6 types are induced by VEGF, many, including VMs, FAs, and DVs, have lost their dependency on VEGF and therefore are not susceptible to anti-VEGF/VEGFR therapy. Feeding arteries would be ideal targets because they are relatively few and provide the blood supply to all of the other 5 vessel types. By analogy, plumbers find it more efficient to shut off the water supply at its entry into the house, rather than turning off the faucets in each room. That this approach may have potential comes from studies using photodynamic therapy to occlude the FAs supplying mouse ear tumors87 and also from uterine artery embolization (myolysis), an approach that is being used to ablate uterine fibroids.88 These examples, of course, are special cases, and it remains to be determined whether FAs can be successfully targeted in other cancers.
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