Dynamism of tumour vasculature in the early phase of cancer progression: outcomes from oesophageal cancer research
Youichi Kumagai, Masakazu Toi, and Haruhiro Inoue
- Pathology: microvessel density and endothelial regulators
- Oncogenes, tumour suppressor genes, and neovascularisation
- Mechanism of neovascularisation in oesophageal cancer
- Other types of cancer
- Future perspectives for diagnostic and therapeutic application
- Search strategy and selection criteria
The vascular structure of cancer changes during tumour progression. A particularly dramatic change occurs during the early phase of progression when in situ tumour is transformed to invasive cancer. Recent advances in morphological investigations have made it possible to visualise and characterise the microvascular-network alterations. In addition, laboratory studies have also revealed the molecular profile—the changing levels of expression of different proteins—of cancer progression, which has helped to advance understanding of the mechanism of carcinogenesis. In this review, we discuss recent outcomes of research of oesophageal cancer and consider the responses of the vascular network and the development of new blood vessels during the early phase of cancer progression. Such considerations will be useful not only for understanding vascular biology but also for exploring novel diagnostic, therapeutic, and preventive approaches targeting early stage or latent phase human cancer.
The structure and organisation of blood vessels is dynamic and undergoes considerable change during the progression from neoplasia to invasive cancer. Understanding of the changes involved, and the underlying mechanisms, will contribute to our knowledge of cancer development and progression, and may offer new therapeutic targets for treatment or prevention of disease.
Recent advances in laboratory techniques have enabled detailed examination of vascular networks, both in normal and tumour microenvironments. The Microfil technique, in which an opaque silicone compound is injected into the microcirculation and then ‘cured’ to form a three-dimensional cast of the vascular network, has enabled us to visualise fine microvessels in the oesophageal mucosa (figure 1 ). We injected the silicone rubber compound through the oesophageal artery and observed the resulting cast under a stereoscopic microscope. To investigate the deeper layers of the oesophageal mucosa, we cleared the tissue with methyl salicylate and used a light microscope for further study. 1 In normal oesophageal mucosa, submucosal vessels that pierce the muscle layer are connected to the arborescent vascular network. Intrapapillary capillaries arise from the fourth branch of the aborescent vessels into the epithelial papillae and form single loops called intrapapillary capillary loops (IPCLs). Individual IPCLs are located at intervals of about 100 mm, which corresponds to the maximum distance oxygen can diffuse from a vessel. The aborescent vessels are dense above and below the muscularis mucosa, but the network becomes thin as the vessels rise to the surface of the mucosa and form IPCLs. In mucosal cancer (m1; figure 2 ) the IPCLs dilate and elongate and the aborescent vascular network becomes less transparent than normal mucosa. However, the number and density of aborescent vessels increases in the lamina propria mucosa and submucosa. These changes are similar to those observed in inflammation. As the cancer invades deeper into the mucosa (m2; figure 2), the papillae stretch further and the IPCLs become more dilated and elongated. Close to the lamina propria mucosae (m3; figure 2), the IPCL structure starts to collapse and the surface of the vessels becomes rough. The morphological structure of m3 lesions is obviously different from m1 or m2 lesions (figure 3 ). Although the m3 microvessel architecture still contains IPCL structures, when tumours progress to submucosal invasion (sm) they lose the IPCLs and the pre-existing vasculature is replaced by a neotumour vasculature, which is leaky and has an irregular-shaped fragile structure ( figure 4 ). Experimental brain tumours have similar vascular regression features. Early tumours implanted in the brain initially grow by recruiting existing host vessels, which causes massive tumour cell loss. However, the remaining tumour cells are rescued by robust angiogenesis at the tumour margin. 2
Figure 1. Border between mucosal cancer and normal squamous epithelium after penetration of Microfil. The thin arrows show the position of normal epithelium, while the dense aborecent vascular network is highlighted with the broad arrows.
Figure 2. Depth of invasion of superficial oesophageal carcinoma based on the subclassification critieria of the Japanese Society for Esophageal Diseases. m1, invasion as far as mucosal epitheliumin (including lesion in which slight invasion of the lamina propria mucosae cannot be ruled out); m2, mucosal cancer with a degree of invasion between type m1 and m2; m3, mucosal cancer beginning to invade the lamina muscularis mucosae; sm, submucosal cancer.
Figure 3. Submucosal oesophageal cancer injected with Microfil before formalin fixation. Tumour vessels that exist at the top of the protruding lesion are indicated by the broad arrow, while intrapapillary capillary loops are indicated by the thin arrows.
Figure 4. Stucture of oesophageal cancer. The 3 vertical panels on the left of the figure show a cross section of an oesophageal cancer specimen injected with Microfil, the middle 3 panels show the histology, and the 3 drawings on the right side of the figure show the changes to the vascular architecture during cancer progression. The top 3 horizonal panels show normal squamous epithelium, the middle 3 panels show m2 cancer and the lower 3 panels show the degree of invasion in a m3 cancer. Normal and modified intrapapillary capillary loops are indicated with thin arrows while tumour vessels are pointed to with broad arrows.
Many pathological studies have shown that in oesophageal carcinoma the microvessel density (MVD)—which can be established by use of antibodies to endothelial cells—increases in proportion to disease progression, tumour size and stage, and depth of invasion. 3–15 For example, Kitadai and colleagues found higher MVD in tumours invading the muscularis propria than in submucosal and mucosal tumours. 7 Several other studies have shown that increasing MVD is associated with lymphatic spread, which occurs as the tumour invades the submucosal layer. 7,9,10,12 Koide and co-workers noted that MVD is inversely associated with apoptotic index, but is not significantly associated with proliferation index as calculated by Ki-67 immunostaining. 5 Most prognostic analyses have confirmed that MVD is of value as a prognostic indicator ( table 1 ).
VEGF, vascular endothelial factor; TP, thymidine phosphorylase; MVD, microvessel density; FVIII RAg, factor VIII-related antigen; NS, not significant.
|Table 1. Studies that investigated the prognostic contribution of microvessel density, VEGF, and TP|
Several studies have investigated the expression of endothelial regulators such as vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP). 3,5–7,10,13–23 VEGF is mainly found in the cytoplasm of tumour cells, but can also be detected in stromal cells, including tumour-associated macrophages (TAMs), other immune cells, and fibroblastic cells, although in the latter cell type the level of expression is lower. 14 VEGF expression significantly correlates with the depth of tumour invasion; 5,7,8,13,15,20 it is only faintly expressed in epithelial ‘m’ tumours, but is frequently upregulated in submucosal ‘sm’ tumours ( figure 5 ). 14 More than half of tumours invading the submucosa or muscularis propria overexpress VEGF. There is agreement that VEGF expression in tumour cells correlates with MVD, and with poor prognosis. 5,8,13,15,20 These findings indicate that VEGF expression may be switched on as cancers begin to invade the lamina propria layer and, therefore, measurements of VEGF expression could be of significant prognostic value in invasive cancers. Similarly, TP expression is low in m1 lesions, 6,10,20 and increases with increasing extent of tumour invasion. Around 50–70% of tumours that invade the muscularis propria also overexpress TP, 3,6,10,20 whereas no significant increase in expression occurs in mucosal tumours. In most studies, a positive association between TP expression and increasing MVD has been found. 3,6,10 TAMs also have raised TP expression and this feature has been shown to correlate with MVD, so it is likely that tumour cells and TAMs co-operate to form the tumour vasculature. 24,25 VEGF-C is involved in the control of lymphangiogenesis 18 and is not detected in mucosal tumour, but is frequently detected in tumours invading the submucosa. This finding is consistent with the notion that about 10% of m3 lesions metastasise to lymph nodes, whereas m1 and m2 lesions do not. 26 Various metalloproteinases (MMPs) and other proteases such as trypsin are involved in neovascularisation and tumour-induced proteolysis. These enzymes tend to be expressed at higher levels in tumours that have invaded to the submucosa or deeper ( figure 5) 27–31 Viewing these findings as a whole, it seems that major endothelial regulators do not become upregulated during localised cancer growth in the mucosal layer, but production increases as the cancer invades the muscularis mucosa. It is at this stage that tumour vasculature starts to grow.
Figure 5. The correlation between surface vasculature and the expression of endothelial regulators. IPCL, intrapapillary capillary loop; LGD, low-grade dysplasia; HGD, high-grade dysplasia; COX2, cyclo-oxygenase 2; VEGF, vascular endothelial growth factor; TP, thymidine phosphorylase; MMP7, matrix metalloproteinase 7; MMP9, matrix metalloproteinase 9; MT1-MMP, membrane type-1 matrix metalloproteinase.
Several oncogenes and tumour suppressor genes, including HER2 and p53, play a significant part in promoting or suppressing neovascularisation. 32–38 For example, abnormalities in p53 are thought to facilitate neovascularisation by downregulation of production of thrombospondin-1 (a potent negative endothelial regulator). 39 In squamous-cell carcinoma of the oesophagus, p53 mutations have been detected at all stages of carcinogenesis—from precancerous lesions, such as hyperplasia or dysplasia, to invasive cancer ( figure 5). 35 There is no significant difference between the frequency of p53 mutations in mucosal and submucosal tumours. Amplification of MDM2, which diminishes the function of normal p53, had also been detected in about 20% of squamous oesophageal cancers, but no association with the depth of tumour invasion has been found in this case. 36 By contrast, a recent report by Biramijamal and colleagues included an interesting finding that high concentrations of cyclo-oxygenase-2 (COX2), which is known to induce inflammatory and angiogenic prostaglandins, is significantly associated with p53 mutations in the tumour. 37 Therefore, abnormalities in p53 may provide an environment conducive to neovascularisation. Recent studies have confirmed that HER2 overexpression is involved in the upregulation of VEGF in various types of tumour cells in vitro. 40 HER2 gene amplification has been observed in about 20–30% of invasive carcinomas, whereas it is only slightly upregulated in mucosal tumours. This finding indicates that HER2 might play a part in the late phase of cancer progression. 38 There is little evidence to suggest that mutations in other angiogenesis-related genes such as SRC and RAS are found in squamous carcinoma of the oesophagus.
Similar to COX2, inducible nitric oxidase synthase (iNOS) is overexpressed in both mucosal and invasive oesophageal cancer. 41–43 Production of these two enzymes is raised in chronically inflamed tissues, including precancerous lesions, and they are thought to have several functions. COX2 inhibits apoptosis and promotes angiogenesis whereas iNOS is also involved in various stages of neovascularisation from vasodilatation to vascular remodelling. 44 Therefore, the microenvironment of precancerous and mucosal lesions may already be angiogenic and predisposed to carcinogenesis. In addition, increased expression of hypoxia inducible factor-1 (HIF1) and HIF2 has been reported in 51% and 13% of early oesophageal cancers, respectively. 45 Although high VEGF expression is not necessarily apparent in mucosal tumours, hypoxia seems to be present in all cases. This finding is of importance because microenvironmental stress, particularly hypoxia, is known to cause genetic instability, which leads to uncontrolled cell growth, invasion, and stromal reactions.
The changes in vascular structure during the early phase of cancer progression resemble those that occur in non-cancerous situations such as wound healing. Initially, pre-existing capillaries elongate and dilate before new immature vessels arise and proliferate. The new vessels subsequently become organised and differentiate into mature vessels. In mucosal cancers, the pre-existing vasculature seems to change in a similar way. VEGF-D has been suggested to be an important factor in the elongation of capillaries. 46 With tumour progression, expansion of the existing vasculature ceases, and a new vessel network, which lacks organisation and full control over pressure or permeability, begins to take over. Physiological stress and capillary wall tension can trigger apoptosis in endothelial cells and vascular smooth muscle cells. And further studies have identified a mechanism for stretch-induced endothelin-B-receptor-mediated apoptosis in vascular smooth muscle cells. 47 Other possible mechanisms involve microcoagulation caused activated platelets in circulating mononuclear cells, 48 or the release of intrinsic negative endothelial-cell regulators, including breakdown products of the extracellular matrix. 49 These events seem to trigger a microenvironmental crisis, which facilitates neovascularisation and tumour progression.
New vessels in submucosal tumours generally consist of pre-existing endothelial cells and endothelial progenitor cells (EPCs), which are largely recruited from bone marrow. 50 Since VEGF is thought to be the key molecule responsible for sprouting from local vessels and recruitment of EPCs, new endothelial cells may accumulate if VEGF expression is switched on as the tumour invades the muscularis mucosa. Patients with high circulating concentrations of VEGF have a poorer prognosis than those with low concentrations, 51 which suggests that VEGF induces EPC recruitment and, presumably, mobilisation from bone marrow. However, the details of this process have yet to be uncovered.
During tumour progression, other types of cancer go through similar changes in MVD and expression of endothelial-cell regulators. Dilated vessels are frequently detected in non-invasive breast carcinoma lesions and MVD is considerably lower in these tumours than in invasive ones. Although overexpression of VEGF or TP can be detected even in non-invasive ductal lesions with a comedo phenotype (perhaps a result of hypoxic conditions), these substances are expressed at far higher levels in invasive tumours. 52 In bladder cancer, for example, expression of VEGF and TP seems to change during the invasion phase of tumour progression. In superficial tumours, VEGF expression exceeds that of TP, but in invasive tumours, TP is more highly expressed. 53,54 In cervical cancer, VEGF and TP are both overepressed from the early stage of invasion and MVD correlates with expression of TP and VEGF. 55,56 In T1 lung cancer, VEGF was overexpressed whereas in T2 disease TP overexpression was detected. 57,58 An interesting direction for further study would be investigation of the morphological changes which correspond with this changing molecular profile.
Advances in endoscope technology, particularly the development of the ultra-high magnification endoscope, have made it possible to visualise IPCLs of the oesophagus. 59,60 It is already possible to follow the changes in IPCLs in early-stage oesophageal cancer and to estimate the depth of infiltration of cancer according to careful observation of the structural changes of the fine vascular network in the mucosa ( figure 6 ). 60,61 Kumagai and colleagues documented the high accuracy of predicting the depth of infiltration by endoscopic diagnosis. 60
Figure 6. Ultrahigh-magnifying-endoscopic observation of oesophageal cancer before treatment. m1, each intrapapillary capillary loop shows changes in dilation, weaving, and tone; m2, the intrapapillary capillary loops are elongated and more dilated than the m1 cancer; m3, intrapapillary capillary loop are partially destroyed and the tumour vessels are rearranged at the tumour surface; sm, the intrapapillary capillary loop shows complete destruction with rearrangement of the tumour vasculature at the surface of the tumour.
An understanding of vascular phenotype will be of use for assessing tumour stage and especially for differentiating between superficial and invasive tumours. This distinction is particularly important for pretreatment because there must be considering of subsequent chemotherapy, radiotherapy, or surgery, including endoscopic surgery. Endoscopic observation of vasculature phenotype is also useful for monitoring therapeutic effect.
Various therapeutic approaches targeting the neovasculature have been investigated for oesophageal cancer. 62 One example is inhibition of COX2, which has previously been investigated for chemoprevention of several other cancers including colon cancer. 63,64 Inhibition of COX2 results in the suppression of neovascularisation and regression of solid tumours, especially those in the early stages. Several reports suggest that COX2 expression is upregulated in oesophageal cancer and that inhibition of this enzyme results in a significant reduction in tumour growth. 65 Retinoid compounds and vitamin D3 are also under investigation as potential chemopreventive agents. 66 Active metabolites of these agents inhibit VEGF-induced endothelial-cell sprouting and elongation in vitro and also inhibit the formation of networks of elongated endothelial cells in 3-dimensional collagen gels. These findings have led to optimism about the use of these compounds for the control of superficial tumours. Other approaches, such as combining antiangiogenic agents with radiotherapy, are also being tested experimentally. 67 Multiple occurrences, which can be either simultaneous or sequential, are quite frequent in oesophageal cancer so chemoprevention would be an extremely beneficial therapeutic development. Furthermore, the observation of vascular changes might offer a means of monitoring these preventive approaches. The precise understanding of tumour vasculature will undoubtedly lead to the efficient development of novel therapeutic approaches not only for oesophageal cancer, but for many other types of solid tumour.
Published and unpublished data for this review were identified by searches of Medline, the National Cancer Institute Register of Cancer Trials (http://cancertrials.nci.nih.gov/ ), and references from relevant articles. Search terms included: “angiogenesis”, “apoptosis”, “cell adhesion”, “cell cycle”, “chemokine”, “cyclo-oxygenase”, “cytokine”, “hypoxia”, “macrophage”, “NO”, “oncogene”, “p53”, and “proliferation”. We also contacted researchers working in the field. Prospective and retrospective cohort studies on the characterisation of neovascularisation in oesophageal cancer were included.
Conflict of interest
We thank Hiroko Bando for her kind help.
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a YK and MT are at the Department of Surgery, Tokyo Metropolitan Komagome Hospital Honkomagome, 3-18-22, Bunkyo-ku, Tokyo, Japan. b HI is at the Digestive Disease Center, Showa University Northern Yokohama Hospital, 35-1, Chigasaki-chuo, Tsuzuki-ku, Yokohama, Japan. Correspondence: Dr Masakazu Toi, Tokyo Metropolitan Komagome Hospital, Honkomagome 3-18-22, Bunkyo-ku, Tokyo, Japan 113-0021. Tel: +81 3 3823 2101. Fax: +81 3 3824 1552