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Morphological evidence for an invasion-independent metastasis pathway exists in multiple human cancers
© Sugino et al; licensee BioMed Central Ltd. 2004
Received: 03 December 2003
Accepted: 05 April 2004
Published: 05 April 2004
We have previously described an alternative invasion-independent pathway of cancer metastasis in a murine mammary tumor model. This pathway is initiated by intravasation of tumor nests enveloped by endothelial cells of sinusoidal vasculature within the tumor. In this study, we examined whether evidence for the invasion-independent pathway of metastasis is present in human cancers.
Archival specimens of 10 common types of human cancers were examined for the presence of sinusoidal vasculature enveloping tumor nests and subsequently generated endothelial-covered tumor emboli in efferent veins.
A percentage of tumor emboli in all cancers was found to be enveloped by endothelial cells, but these structures were particularly prevalent in renal cell carcinomas, hepatocellular carcinomas and follicular thyroid carcinomas. A common feature of the vasculature in these tumors was the presence of dilated sinusoid-like structures surrounding tumor nests. A high mean vascular area within tumors, an indication of sinusoidal vascular development, was significantly related to the presence of endothelial-covered tumor emboli.
These results suggest that an invasion-independent metastatic pathway is possible in a wide variety of human cancers. Further investigation of this phenomenon may present new therapeutic strategies for the amelioration of cancer metastasis.
Cancer metastasis is most often described as a series of sequential processes that involve the following steps: growth of new blood vessels into the primary tumor, local invasion of the extracellular matrix, intravasation through proteolysis of components of the blood vessel wall and survival during transport in the bloodstream. After reaching the target organ, adhesion to endothelial lining occurs, followed by extravasation through the vessel wall and subsequent proliferation at the secondary site. Accordingly, it is widely believed that active invasion by cancer cells is essential to the metastatic process [1, 2].
However, we have recently reported a murine mammary tumor model for blood-borne metastasis that does not require invasion of the vascular wall at either the primary tumor or the target organ [3, 4]. The process involves intravasation of tumor nests that are surrounded by blood vessels, transportation of tumor emboli enveloped with endothelial cells and intravascular tumor growth in the lung without penetration of the vascular wall at the secondary site. Our comparative studies between highly metastatic and non-metastatic cells suggest that tumor cell intravasation, induced by high angiogenic activity and sinusoidal remodeling of tumor blood vessels, is a key step in the invasion-independent metastatic pathway [3, 4].
Microscopic observations of routine surgical specimens suggest that the invasion-independent pathway may occur in some types of human cancers. For example, intravascular tumor emboli from follicular carcinoma of the thyroid have been observed as being enveloped with endothelial cells [5, 6]. However, there has not been any report describing the meaning of this phenomenon in the metastatic process. Moreover, little attention has been paid to the existence of tumor embolus-associated endothelia in other human cancers.
The aim of this study was to take the findings we observed in our murine model of metastasis  and to evaluate whether evidence for an invasion-independent metastatic pathway exists in human cancers. Interestingly, analysis of archival tissue samples obtained from several types of cancers did reveal the presence of endothelium-coated tumor cell emboli in primary tumor vasculature. Furthermore, the immunochemical assessment of tumor vasculature identified a positive correlation between the status of sinusoidal vasculature and the presence of endothelium-coated tumor emboli, indicating a link between tumor angiogenesis and the facilitation of an invasion-independent metastatic pathway.
An immunohistochemical study was performed using formalin-fixed, paraffin-embedded tissue specimens obtained from the collections of Fukushima Medical University and Jusendo General Hospital. Ten types of human cancers were chosen for examination. Primary tumor specimens originated from the following organ systems: thyroid, liver, kidney, stomach, colon, breast, pancreas, lung, uterus and esophagus. Ten cases from each cancer type were selected for immunohistochemical analysis. Samples that had tumor emboli present in the afferent veins were selected. Archival tissues and anonymized data were used in accordance with national and local human material investigative protocols.
Monoclonal antibodies to CD34 (clone QBEND10; Immunotech, Hamburg, Germany) and CD31 (clone JC70; Dako, Grostrup, Denmark) were used for endothelial cell staining on 3-μm paraffin-embedded sections according to the manufacturer's instructions. Immunohistochemical staining was performed using an indirect streptavidin-biotin immunoperoxidase method (SAB-PO (M) kit, Nichirei Corp., Tokyo, Japan). After blocking of endogenous peroxidase activity in 0.3% hydrogen peroxide in methanol for 30 min, slides were incubated with primary antibodies overnight at 4°C, washed with PBS, and then incubated with secondary biotin-labeled antibodies for 30 min at room temperature. Antibody localization was visualized with peroxidase-conjugated streptavidin for 30 min at room temperature, followed by the diaminobenzidene reaction. The slides were counterstained with hematoxylin.
Assessment of tumor vascularity
Tumor vascularity was assessed using two parameters. Microvessel density was measured according to standard procedures . Briefly, immunochemically-stained slides were first observed at low power magnification (×100) to identify areas with the highest density of microvessels. In each case, the three most vascularized areas were selected, and microvessels in these areas were counted at high power magnification (×200) in a grid area of 0.19 mm2. Three areas of high vascular density were counted on each section, and the vascular density was determined. The second parameter assessed was the mean area of tumor blood vessels. The vascular area was evaluated in three low-power fields (×40) on each case. The areas of microvessels (exclusive of arteries and veins) were measured in each field using a computerized image analyzer (Image-Pro Plus, Media Cybernetics, Silver Spring, Maryland, USA). The percentage of total vascular area in each tumor parenchyma was determined.
The student's t test was used to compare groups of mean values of vascular density and percentage of vascular area; P < 0.01 was considered statistically significant.
A wide variety of human cancers generate tumor emboli which become enveloped with endothelial cells
Endothelial covering on intravascular tumor emboli from human cancers.
Origin of cancer
Histological type (number of cases)
SQCC (4), ADC (6)
SQCC (5), ADC (5)
Sinusoidal vascular development may facilitate intravasation of tumor cells in an invasion-independent manner
The invasive property of cancer cells is generally believed to be one of the most essential factors in the multi-step process of metastasis, enabling the metastatic cells to penetrate the vascular wall barrier at either the primary or secondary site of growth [8, 9]. However, we have previously proposed an alternative metastatic model whereby tumors gain access to the host vasculature in a mechanism independent of active invasion. We developed a murine model of mammary tumor metastasis (MCH66), in which tumor cell nests became surrounded by sinusoidal blood vessels and entered the circulation as endothelium-coated tumor cell emboli [3, 4]. The isolation from our murine model of monoclonal cell lines that have differential propensities to achieve this mode of intravasation, indicates that there is a tumor cell-specific genetic component to this phenomenon. In the present study, we examined whether a similar phenomenon exists in human cancers by monitoring the association of endothelia with intravascular tumor emboli and sinusoidal development of tumor vasculature enveloping tumor nests – major features of the invasion-independent metastasis pathway. Indeed, cases with both indices were detected in all cancer types examined, but with particularly high incidence in renal cell carcinoma, follicular thyroid carcinoma and hepatocellular carcinoma.
Endothelial cell association with tumor emboli in FTC has been described previously [5, 6, 10], but neither the mechanism nor clinical significance of this phenomenon has been investigated. To our knowledge, there have been no reports describing whether tumor emboli from HCC, RCC or any other cancers are consistently associated with vascular endothelial cells.
Our results indicate that an invasion-independent metastasis pathway is related to both angiogenesis and vascular remodeling. Microvessel density is assumed to reflect the level of tumor angiogenesis. Many studies have reported a correlation of vascular density with the occurrence of metastasis and poor prognosis [15–19]. In this study, a positive correlation of mean vascular density with the presence of endothelium-coated tumor emboli suggests that angiogenic factors may be responsible for inducing this metastatic pathway. In contrast to the mean value of groups of cancer cases, vascular density in individual cases, especially of HCC that is reportedly hypervascular, did not necessarily reflect their sinusoidal vascular development, or the endothelial association with the accompanying tumor emboli. The reason for this may be the often peculiar vascular structure, consisting of dilated and fused sinusoidal vessels, which prove difficult to quantify accurately. Instead of vascular density, measurements of vascular area or microvessel fractal dimension are used to quantify intratumoral vascularity, especially for the assessment of dilated and complex vasculature such as sinusoidal vessels in FTC and RCC [13, 14, 20]. In this study, intratumoral vascular area was more tightly correlated than vascular density in individual cases with endothelium-coated tumor emboli. Our data demonstrate that the invasion-independent metastatic pathway can be controlled not only by increasing the number of blood vessels attracted to the tumor, but also by vascular remodeling into a more sinusoidal structure.
Our analyses demonstrate that conditions for an invasion-independent dissemination pathway exist in a wide variety of human cancers. Our analysis of morphology and vascularity suggest that this metastatic pathway can be initiated by the development of sinusoidal tumor vasculature. It is also possible that the alternative dissemination pathway leads to an improved metastatic efficiency. The defensive morphology of endothelium-associated tumor emboli may protect the tumor cells from hemodynamic forces and from immune surveillance systems. Furthermore, as observed in our mammary metastasis model , proliferation in a distant secondary site may be facilitated by the pre-requisite intimate association with stromal components. Further studies analyzing the molecular mechanisms of this type of dissemination, and the clinico-pathological and prognostic significance of an invasion-independent metastasis pathway will probably lead to the development of new treatment agents and strategies for cancer metastasis.
This work was supported in part by a Grant-in-Aid for Scientific Research (C) (no. 14570126) from the Ministry of Education, Science, Sports and Culture, Japan. We thank Natsuko Takahashi for excellent technical assistance.
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