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Title: Morphological evidence for an invasion-independent metastasis pathway exists in multiple human cancers
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Title: Morphological evidence for an invasion-independent metastasis pathway exists in multiple human cancers
Physical Description: Book
Language: English
Creator: Sugino, Takashi
Yamaguchi, Tomiko
Ogura, Go
Saito, Atsuko
Hashimoto, Takeaki
Hoshi, Nobuo
Yoshida, Sayaka
Goodison, Steve
Suzuki, Toshimitsu
Publisher: BMC Medicine
Publication Date: 2004
 Notes
Abstract: BACKGROUND: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.METHODS: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.RESULTS: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.CONCLUSIONS: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.
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Research article

Morphological evidence for an invasion-independent metastasis
pathway exists in multiple human cancers
Takashi Sugino*1, Tomiko Yamaguchi', Go Ogural, Atsuko Saito2,
Takeaki Hashimoto1, Nobuo Hoshi1, Sayaka Yoshida3, Steve Goodison4 and
Toshimitsu Suzuki1


Address: 'Department of Pathology, Fukushima Medical University School of Medicine, Fukushima, 960-1295, Japan, 2Pathology Division,
Jusendo General Hospital, Koriyama, 963-8002, Japan, 3Department of Surgery, Fukushima Medical University School of Medicine, Fukushima,
960-1295, Japan and 4Department of Pathology, University of Florida, Jacksonville, FL 32209-6511, USA
Email: Takashi Sugino* sugino@fmu.ac.jp; Tomiko Yamaguchi yamat@fmu.ac.jp; Go Ogura g-ogura@fmu.ac.jp;
Atsuko Saito a.saito@jusendo.or.jp; Takeaki Hashimoto thashi@fmu.ac.jp; Nobuo Hoshi nobuo@fmu.ac.jp;
Sayaka Yoshida ysayaka@fmu.ac.jp; Steve Goodison steve.goodison@jax.ufl.edu; Toshimitsu Suzuki tmt@fmu.ac.jp
* Corresponding author



Published: 05 April 2004 Received: 03 December 2003
8MC Medicine 2004, 2:9 Accepted: 05 April 2004
This article is available from: http://www.biomedcentral.com/1741-7015/2/9
2004 Sugino et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.



Abstract
Background: 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.
Methods: 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.
Results: 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.
Conclusions: 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.



Background proteolysis of components of the blood vessel wall and
Cancer metastasis is most often described as a series of survival during transport in the bloodstream. After reach-
sequential processes that involve the following steps: ing the target organ, adhesion to endothelial lining
growth of new blood vessels into the primary tumor, local occurs, followed by extravasation through the vessel wall
invasion of the extracellular matrix, intravasation through and subsequent proliferation at the secondary site.

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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 intra-
vasation of tumor nests that are surrounded by blood ves-
sels, transportation of tumor emboli enveloped with
endothelial cells and intravascular tumor growth in the
lung without penetration of the vascular wall at the sec-
ondary site. Our comparative studies between highly met-
astatic 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, intravascu-
lar 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 meta-
static 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 [4] 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 can-
cers 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-inde-
pendent metastatic pathway.

Methods
Tissue procurement
An immunohistochemical study was performed using for-
malin-fixed, paraffin-embedded tissue specimens
obtained from the collections of Fukushima Medical Uni-
versity 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.

Immunohistochemistry
Monoclonal antibodies to CD34 (clone QBEND10;
Immunotech, Hamburg, Germany) and CD31 (clone
JC70; Dako, Grostrup, Denmark) were used for endothe-
lial cell staining on 3- m paraffin-embedded sections
according to the manufacturer's instructions. Immunohis-
tochemical 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 perox-
ide in methanol for 30 min, slides were incubated with
primary antibodies overnight at 4C, washed with PBS,
and then incubated with secondary biotin-labeled anti-
bodies for 30 min at room temperature. Antibody locali-
zation was visualized with peroxidase-conjugated
streptavidin for 30 min at room temperature, followed by
the diaminobenzidene reaction. The slides were counter-
stained with hematoxylin.

Assessment of tumor vascularity
Tumor vascularity was assessed using two parameters.
Microvessel density was measured according to standard
procedures [7]. Briefly, immunochemically-stained slides
were first observed at low power magnification (x 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 (x200) 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 sec-
ond parameter assessed was the mean area of tumor
blood vessels. The vascular area was evaluated in three
low-power fields (x40) on each case. The areas of micro-
vessels (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.

Statistical analysis
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.

Results
A wide variety of human cancers generate tumor emboli
which become enveloped with endothelial cells
Results compiled from the analysis of 10 cases per cancer
type, and of 10 different types of human cancers, are listed
in Table 1. Intravascular tumor emboli were categorized as
two groups: emboli covered with endothelial cells and


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Table I: Endothelial covering on intravascular tumor emboli
from human cancers.


Origin of cancer


Thyroid
Liver
Kidney
Stomach
Colon
Breast
Pancreas
Lung
Uterus
Esophagus


Histological type
(number of cases)

FTC (10)
HCC (10)
RCC (10)
ADC (10)
ADC (10)
ADC (10)
ADC (10)
SQCC (4), ADC (6)
SQCC (5), ADC (5)
SQCC (10)


Endothelial covering


10
10
9
I
2
3
2
2


FTC, follicular thyroid carcinoma; HCC, hepatocellular carcinoma;
RCC, renal cell carcinoma; ADC, adenocarcinoma; SQCC, squamous
cell carcinoma.




naked emboli with no evident endothelial association.
The majority of intravascular tumor emboli present in all
cases of renal cell carcinoma (RCC) (Figure 1A), hepato-
cellular carcinoma (HCC) (Figure 1B) and follicular thy-
roid carcinoma (FTC) (Figure IC) were coated with a
CD31, and CD34-positive endothelial lining. The vascula-
ture of these three types of cancers formed well-developed
sinusoidal structures enveloping tumor nests (Figure 1,
AIII, Bill, CIII). These tumors exhibited an intravasation
feature in which tumor nests enveloped with sinusoidal
vessels entered the collecting drainage vein. Most of the
tumor emboli present in the other seven types of human
cancers were naked, homogeneous structures with no
endothelial covering evident (Figure 2A). Many of those
with naked emboli contained small veins filled with
organized fibrous tissue coating the tumor emboli, per-
haps due to vascular injury or tumor-associated thrombo-
sis. However, in all types of cancers tested, there were cases
with tumor emboli that were directly covered with
endothelial cells entirely (Figure 2B), or partially (Figure
2C), and with only scanty intervening fibrous tissue evi-
dent. The tumors possessed a well-developed sinusoidal
vasculature surrounding and enveloping the tumor nests
(Figure 2D).

Sinusoidal vascular development may facilitate
intravasation of tumor cells in an invasion-independent
manner
In order to assess accurately tumor vascularity, we meas-
ured both vascular density (Figure 3A,3B) and the per-
centage of vascular area (Figure 3C,3D) in the primary
tumors. Both the median mean vascular density and the
percentage of vascular area were significantly greater in
the cases that exhibited endothelium-coated tumor
emboli (Figure 3A,3C). Vascular densities in FTC and RCC


samples were much greater than in any other cancers.
HCC samples did not show high vascular density in spite
of having well-developed vasculature (Figure 3B). Con-
versely, the percentage of vascular area in all three cancers
developing sinusoidal vasculature (FTC, HCC and RCC)
was significantly higher than that observed in the other
seven cancer types (Figure 3D). Moreover, in seven cancer
types, exclusive of FTC, HCC and RCC which have well-
developed sinusoidal vasculature in most cases, the
median mean percentage of vascular area was also signifi-
cantly higher in cases with endothelial-coated tumor
emboli (P < 0.01), whereas the mean vascular density did
not detect the difference (P = 0.06).

Discussion
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 pen-
etrate the vascular wall barrier at either the primary or sec-
ondary 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 (MCH 66), 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 pro-
pensities 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 intravas-
cular 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 hepato-
cellular carcinoma.

Endothelial cell association with tumor emboli in FTC has
been described previously [5,6,10], but neither the mech-
anism 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 vas-
cular endothelial cells.

The observed correlation of the morphology of tumor vas-
culature with the presence of endothelium-coated tumor
emboli in this study suggests a possible mechanism for an
alternative pathway of tumor cell dissemination and
metastasis. Such a mechanism is depicted in Figure 4 and
has been previously proposed and discussed by ourselves


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A: Renal cell carcinoma


B: Hepatocellular carcinoma


C: Follicular thyroid carcinoma


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.fl:IIlj.


Figure I
Representative photomicrographs of primary tumor vasculature and examples of tumor emboli within efferent veins. (A)
Renal cell carcinoma, (B) hepatocellular carcinoma and (C) follicular thyroid carcinoma. An endothelial layer covering intra-
vascular tumor emboli can be seen using immunostaining with anti-CD31 antibody (All, Bll, Cll). The vasculature in the primary
tumors of the three carcinomas formed sinusoidal structures surrounding tumor cell nests (anti-CD31 staining, Alll, Bill, Clll).
Al, BI and Cl are H&E staining. Original magnifications: (All, Bll, Cll) x400, all others x200.





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Representative photomicrographs of intravascular tumor. Endothelial cells were detected and visualized using immunostaining
with (A, C) anti-CD3 I and (B, D) anti-CD34 antibodies. (A) An example of a naked tumor embolus, that is, no evident
endothelial covering, within an efferent vein in a case of breast carcinoma. (B) In a case of esophageal carcinoma, a tumor
embolus within a vein is entirely covered with endothelial cells. (C, D) Tumor emboli originating from a breast carcinoma are
enveloped by endothelial cells. (E) The same case as D. The tumor possessed well-developed sinusoidal vasculature. Original
magnifications: (A, E), x200; (B-D), x400.



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Figure 3
Tumor vascularity in cancer cases with and without evidence of the presence of endothelium-coated tumor emboli. Vascular
density was determined by counting the number of vessels per unit of HPF (high power field) at magnification x400. (A) Mean
vascular density of total cases and seven cancer types, excluding FTC, HCC and RCC. Standard deviation (SD) is indicated by
error bars. (B) Vascular density of individual cases for each type of cancer. The mean vascular densities are indicated by the
horizontal lines. (C) Mean percentage of vascular area and SD of all cases examined and for the seven cancer types. (D) Per-
centage of vascular area of individual cases for each type of cancer. The mean percentages of vascular area are indicated by the
horizontal lines. Solid bars and circles, cancer cases with endothelium-coated tumor emboli; open bars and circles, cases with
uncoated emboli. FTC, follicular thyroid carcinoma; HCC, hepatocellular carcinoma; RCC, renal cell carcinoma. *Significant dif-
ference between cancer cases with endothelium-coated tumor emboli and with naked ones (P < 0.0 1).


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Figure 4
Schematic representation of an invasion-independent path-
way of intravasation by human cancer cells. Tumor cell nests
become surrounded by sinusoidal blood vessels (SV) and
enter the collecting drainage vein (DV) as endothelial-coated
tumor cell emboli.




and others [4,111. A common feature of the vasculature in
the primary tumors carrying endothelium-coated tumor
emboli was the presence of sinusoid structures surround-
ing tumor nests. In fact, FTC, RCC and HCC are known as
hypervascular tumors, that is, harboring large vascular
channels which have the appearance of sinusoidal capil-
laries [12-14]. In these cancers, re-organized tissue struc-
tures consisting of tumor nests and surrounding
sinusoidal vessels presumably become the embolus 'unit',
which is subsequently disseminated. Therefore, this path-
way may depend not on tissue destruction by cancer cell
invasiveness but on remodeling of tissue architecture
through tumor-stroma interactions.

Our results indicate that an invasion-independent metas-
tasis 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 devel-
opment, or the endothelial association with the accompa-
nying 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 accu-
rately. Instead of vascular density, measurements ofvascu-
lar area or microvessel fractal dimension are used to
quantify intratumoral vascularity, especially for the assess-
ment of dilated and complex vasculature such as sinusoi-
dal 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 demon-
strate 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.

Conclusions
Our analyses demonstrate that conditions for an invasion-
independent dissemination pathway exist in a wide vari-
ety of human cancers. Our analysis of morphology and
vascularity suggest that this metastatic pathway can be
initiated by the development of sinusoidal tumor vascula-
ture. 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. Further-
more, as observed in our mammary metastasis model [4],
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 clin-
ico-pathological and prognostic significance of an inva-
sion-independent metastasis pathway will probably lead
to the development of new treatment agents and strategies
for cancer metastasis.

Competing interests
None declared.

Authors' contributions
TS conceived the study, participated in its design and
coordination, and drafted the manuscript. TY assisted
with the immunohistochemical staining. GO, AS, TH,
NH, SY, SG and TS participated in study design and
evaluations, critical discussion and manuscript prepara-
tion. All authors read and approved the final manuscript.

Acknowledgements
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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References
I. Fidler IJ, Balch CM: The biology of cancer metastasis and impli-
cations for therapy. Curr Probl Surg 1987, 24:129-209.
2. Chambers AF, Groom AC, MacDonald IC: Dissemination and
growth of cancer cells in metastatic sites. Nat Rev Cancer 2002,
2:563-572.
3. Sugino T, Kawaguchi T, Suzuki T: Sequential process of blood-
borne lung metastases of spontaneous mammary carcinoma
in C3H mice. IntJ Cancer 1993, 55:141-147.
4. Sugino T, Kusakabe T, Hoshi N, Yamaguchi T, Kawaguchi T, Goodi-
son S, Sekimata M, Homma Y, Suzuki T: An invasion-independent
pathway of blood-borne metastasis: a new murine mam-
mary tumor model. Am j Pathol 2002, 160:1973-1980.
5. Rosai J, Carcangui ML, DeLellis RA: Tumors of the thyroid gland.
Atlas of Tumor Pathology. Edited by: Rosai J and Sobin L H.
Washington DC, Armed Forces Institute of Pathology; 1992:49-63.
6. Thompson LD, Wieneke JA, Paal E, Frommelt RA, Adair CF, Heffess
CS: A clinicopathologic study of minimally invasive follicular
carcinoma of the thyroid gland with a review of the English
literature. Cancer 2001, 91:505-524.
7. Fox SB: Microscopic assessment of angiogenesis in tumors.
Methods in Molecular Medicine: Angiogenesis Protocols Volume 46. Edited
by: Murray J C. Totowa, NJ, Humana Press, Inc.; 2001:29-46.
8. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S: Meta-
static potential correlates with enzymatic degradation of
basement membrane collagen. Nature 1980, 284:67-68.
9. Egeblad M, Werb Z: New functions for the matrix metallopro-
teinases in cancer progression. Nat Rev Cancer 2002, 2:161-174.
10. Franssila KO, Ackerman LV, Brown CL, Hedinger CE: Follicular
carcinoma. Semin Diagn Pathol 1985, 2:101-122.
I I. Nakashima T, Kojiro M, Kawano Y, Shirai F, Takemoto N, Tomimatsu
Y, Kawasaki H, Okuda K: Histologic growth pattern of hepato-
cellular carcinoma: relationship to orcein (hepatitis B sur-
face antigen)-positive cells in cancer tissue. Hum Pathol 1982,
13:563-568.
12. Sabo E, Boltenko A, Sova Y, Stein A, Kleinhaus S, Resnick MB: Micro-
scopic analysis and significance of vascular architectural
complexity in renal cell carcinoma. Clin Cancer Res 2001,
7:533-537.
13. Wong NA, WillottJ, Kendall MJ, Sheffield EA: Measurement ofvas-
cularity as a diagnostic and prognostic tool for well differen-
tiated thyroid tumours: comparison of different methods of
assessing vascularity. Clin Pathol 1999, 52:593-597.
14. Macchiarini P, Fontanini G, Hardin MJ, Squartini F, Angeletti CA:
Relation of neovascularisation to metastasis of non-small-
cell lung cancer. Lancet 1992, 340:145-146.
15. Gasparini G: Prognostic and predictive value of intra-tumoral
microvessel density in human solid tumours. Tumour angiogen-
esis Edited by: Bicknell R, Lewis C E and Ferrara N. Oxford, U. K.,
Oxford Univ. Press; 1997:29-44.
16. Weidner N: Tumoral vascularity: What does it tell us about
the growth and spread of cancer? Tumor angiogenesis and micro-
circulation Edited by: Voest E E and D'Amore P A. New York, U. S. A.,
Marcel Dekker, Inc.; 2001:465-486.
17. Weidner N, Folkman J, Pozza F, Bevilacqua P, Allred EN, Moore DH,
Meli S, Gasparini G: Tumor angiogenesis: a new significant and
independent prognostic indicator in early-stage breast
carcinoma. j Natl Cancer Inst 1992, 84:1875-1887.
18. Bochner BH, Cote RJ, Weidner N, Groshen S, Chen SC, Skinner DG,
Nichols PW: Angiogenesis in bladder cancer: relationship
between microvessel density and tumor prognosis. j Natl Can- Publish with BioMed Central and every
cer Inst 1995, 87:1603-1612. scientist can read your work free of charge
19. Pavlopoulos PM, Konstantinidou AE, Agapitos E, Kavantzas N, Nikol-
opoulou P, Davaris P: A morphometric study of neovasculariza- "BioMed Central will be the most significant development for
tion in colorectal carcinoma. Cancer 1998, 83:2067-2075. disseminating the results of biomedical research in our lifetime."
20. Simpson JF, Ahn C, Battifora H, Esteban JM: Endothelial area as a Sir Paul Nurse, Cancer Research UK
prognostic indicator for invasive breast carcinoma. Cancer
1996, 77:2077-2085. Your research papers will be:
available free of charge to the entire biomedical community
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