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Regular Articles |
From the Department of Obstetrics and Gynecology,*
Division of Gynecologic Oncology, the Department of Anatomy and Cell
Biology,
the Department of
Pathology,
and the Holden Comprehensive
Cancer Center at the University of Iowa, University of Iowa Hospitals
and Clinics, Iowa City, Iowa
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ovarian cancer has the highest mortality rate of all gynecological malignancies, which reflects the fact that most patients are diagnosed with advanced cancer. Extensive metastatic deposits of tumor have been established within the peritoneal cavity at the time of initial diagnosis in most patients. The growth and spread of neoplasms depend, in part, on the formation of adequate vascular support.10 As proposed by Folkman,11 neovascularization of tumors is required to provide essential nutrients to the growing tumor beyond the limit of simple diffusion, and to allow for growth >2 mm. Based on this concept, several different anti-angiogenic therapies have been developed and are now in clinical trials.12,13 Although angiogenesis and the associated factors have been shown to occur in ovarian cancers14 and the degree of vascularity in ovarian cancer correlates with poor clinical outcome,15-19 the mechanisms involved in the generation of this vascularity have not been well defined.
In a recent study, Orre and Rogers20 found that the endothelial proliferation index in high-vessel density regions of ovarian cancers relative to benign tumors was significantly lower. Thus, we hypothesized that in a manner similar to melanomas, aggressive ovarian cancer cells may have the ability to generate matrix-rich, embryonic-like, patterned networks independent of endothelial cells. In this study, we show experimental and clinicopathological evidence for the presence of molecular vasculogenic mimicry by invasive human ovarian cancer cells. Ovarian cancer plasticity is reflected by their pluripotential ability to express a variety of phenotypes including formation of endothelial-like matrix-rich networks. These findings advance our present thinking concerning the vascularization of ovarian cancers, and identify some new, potential targets for therapeutic intervention.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The five established ovarian cancer cell lines used in this study were OVCAR3, SKOV3, 222, EG, and A2780-PAR. The derivation and sources of these cell lines have been reported previously.21 These cells were maintained and propagated in vitro by serial passage in RPMI 1640 supplemented with 15% fetal bovine serum and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA). The immortalized normal human ovarian surface epithelial cell lines (HIO 1120 and HIO 180) were a kind gift from Dr. Andrew Godwin at the Fox Chase Cancer Center, Philadelphia, PA. These lines were maintained in Medium 199/MCDB 105/15% fetal bovine serum with 0.1% gentamicin sulfate. All cell lines are routinely screened for Mycoplasma species (GenProbe detection kit; Fisher, Itasca, IL). All experiments were performed with 70 to 80% confluent cultures.
Invasion Assay
The membrane invasion culture system chamber was used to measure the in vitro invasiveness of all cell lines used in this study.22 Briefly, a polycarbonate membrane with 10 µmol/L pores (Osmonics, Livermore, CA) was uniformly coated with a defined basement membrane matrix consisting of human laminin/type IV collagen/gelatin and used as the intervening barrier to invasion. Both upper and lower wells of the chamber were filled with serum-free RPMI containing 1x MITO+ (Collaborative Biomedical, Bedford, MA). Single-cell tumor suspensions were seeded into the upper wells at a concentration of 1 x 105 cells per well. After a 24-hour incubation in a humidified incubator at 37°C with 5% CO2, cells that had invaded through the basement membrane were collected, stained, and counted by light microscopy.22 For chemo-invasion assays, conditioned media from normal skin fibroblasts (kindly provided by Dr. Gregory Goldberg, Washington University, St. Louis, MO) was added to the lower wells. Invasiveness was calculated as the percentage of cells that had successfully invaded through the matrix-coated membrane to the lower wells compared to the total number of cells seeded into the upper wells and corrected for cell proliferation.
Three-Dimensional Cultures and in Vitro Network Formation
Fifty µl of Matrigel (Collaborative Biomedical) was dropped onto glass coverslips and allowed to polymerize for 1 hour at room temperature, then 30 minutes at 37°C in a humidified 5% CO2 incubator. Tumor cells (7.5 x 105) were then seeded onto the gels and incubated at 37°C with 5% CO2 and humidity. For experiments on a collagen I matrix, 20 µl of undiluted rat-tail collagen I solution (Collaborative Biomedical) were dropped onto glass coverslips and polymerized for 5 minutes with 100% ethanol. The coverslips were then washed once with 1x phosphate-buffered saline (PBS), rehydrated with culture medium, then seeded with tumor cells. The cultures were maintained in RPMI 1640 supplemented with 15% fetal bovine serum, and 0.1% gentamicin sulfate. The culture medium was changed every 3 days.
Light Microscopy
Five-µm serial sections of paraffin-embedded, formaldehyde-fixed tissues were cut, stained with a hematoxylin and eosin (H&E) stain and examined by light microscopy using a Zeiss Axioskop 2 (Carl Zeiss, Inc., Thornwood, NY), Spot 2 camera (Diagnostic Instruments; Inc., Sterling Heights, MI) and Axiovision 2.0.5 software (Carl Zeiss, Inc.). Companion serial sections were stained with periodic acid-Schiff without a hematoxylin counterstain and viewed through a green filter to visualize any extracellularly derived structures.
Immunohistochemical Staining
Detection of laminin in the in vitro samples was performed using a polyclonal anti-laminin antibody (Life Technologies, Inc., Gaithersburg, MD) at 1:200 dilution in PBS containing 0.3% normal goat serum, and the Vectastain Elite avidin-biotin-peroxidase complex (ABC) kit and 3-amino-9-ethylcarbazole (AEC) substrate kit for peroxidase activity (Vector Laboratories, Inc., Burlingame, CA) with a slight modification of the manufacturer’s protocol. Briefly, after the samples were rinsed with PBS, an additional step of incubating the samples with 0.1% Triton X-100 in PBS for 5 minutes was added. After washing with PBS, these samples were treated with 0.3% H2O2 in PBS containing 0.3% normal goat serum for 5 minutes, washed, incubated for 20 minutes with normal goat blocking serum, then incubated with the anti-laminin antibody for 30 minutes. These samples were then washed with PBS, incubated for 30 minutes with the biotinylated secondary antibody, PBS washed, incubated with the Vectastain Elite ABC reagent for 30 minutes, PBS washed, then incubated with the AEC substrate for 10 to 30 minutes (or until adequate color development). The samples were then washed with PBS, rinsed with tap water, and mounted with an aqueous mounting solution (Aqua-Mount; Lerner Laboratories, Pittsburgh, PA) onto precleaned microscope slides. The AEC substrate turns red in the presence of peroxidase activity.
Immunostaining for laminin in tissue sections was performed using a monoclonal anti-laminin antibody (Sigma Chemical Co., St. Louis, MO) at either 1:100 or 1:250 dilution. Briefly, the samples were treated with proteinase K for 10 to 15 minutes, washed with deionized water, and rinsed with Tris-buffered saline with Tween-20 (0.05%) for 10 minutes. Subsequent steps (3% H2O2 for 5 minutes, avidin for 15 minutes, biotin for 15 minutes, protein block-serum-free (DAKO, Carpinteria, CA) for 7 minutes, laminin antibody for 45 to 52 minutes) were performed in the automated DAKO stainer (DAKO). Tris-buffered saline washes were performed between each step. The Vector Universal Elite ABC ready-to-use kit (Vector Laboratories) was used for secondary and tertiary reagents according to manufacturer’s instructions. For chromagen, VIP substrate (purple) (Vector Laboratories) was used for 7 minutes. Methyl-green counterstaining was performed for 15 to 20 minutes.
Inhibition of Network Formation
Inhibition of the network formation by tumor cells was accomplished using Metastat (CollaGenex Pharmaceuticals, Newton, PA) at 5 µg/ml added to the cultures daily beginning either on day 1, day 7, or day 14.23
Ribonuclease Protection Assay (RPA)
Total RNA was hybridized with the desired 32P-labeled multitemplate probe (hangio-1; BD Pharmingen, San Diego, CA) for 16 hours. After this, RNase treatment and purification of protected probes was performed according to the manufacturer’s guidelines. The samples were run on a 5% polyacrylamide gel containing urea at 50 W constant power until the leading edge of the bromophenol blue migrated 30 cm. The gel was dried under vacuum for 1 hour and exposed to Kodak Biomax MR X-ray film (Eastman-Kodak, Rochester, NY) at -70°C .
Electron Microscopy
For scanning electron microscopy and transmission electron microscopy, tissue cultures were fixed in cold 2.5% glutaraldehyde in 0.1 mol/L of sodium cacodylate buffer and postfixed in osmium. Specimens were then either embedded, sectioned, and stained by routine means for transmission electron microscopy, or critically point-dried, and sputter-coated with gold/palladium for scanning electron microscopy, as previously described.24
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The membrane invasion culture system assay was used to
measure the in vitro ability of cells to invade a basement
membrane matrix—an important step in the metastatic cascade. The
invasion results are summarized in Figure 1
. Both HIO 180 and HIO 1120 were poorly
invasive (2.3%), whereas OVCAR3, EG, A2780-PAR, and SKOV3 were
moderately invasive (3.9 to 5.1%), and the 222 cell line was the most
invasive (8.3%). With the addition of chemoattractant to the invasion
assay, the percent invasive ability of OVCAR3, SKOV3, EG, and 222
increased by 30 to 47%, compared with the poorly invasive cell lines
that remained basically unchanged.
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All of the cell lines were assayed for their ability to form tumor
cell-lined, matrix-rich networks on three-dimensional matrices,
consisting of Matrigel or collagen I, in the absence of endothelial
cells or fibroblasts. The invasive cancer cell lines formed networks on
both matrices (representative examples are shown in Figure 2, A–D
) except A2780-PAR that did not
form networks even after 3 weeks in culture. The tumor-formed networks
initiated formation within 3 days after seeding the cells onto the
matrix with optimal structure formation achieved by 3 weeks.
Microscopic analysis at regular intervals revealed that the networks
consisted of tubular structures that continued to evolve during the
3-week period with variations of tubular and sinusoidal structures
surrounding clusters of tumor cells. There were no significant
differences in the efficiency of network formation between the highly
invasive and moderately invasive cell lines. During formation, the
tubular networks became channelized or hollowed, and were stable
through 6 weeks after seeding the cells onto a matrix. However, the HIO
180 and HIO 1120 normal surface epithelial cells did not form networks
or channels on either matrix (more than 3 weeks in culture; Figure 2, E and F
).
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. Transmission
electron microscopy of the tubular structures revealed that the tubes
consisted of a matrix-rich central region (from the collagen gel) lined
by tumor cells externally (Figure 3C)
. Polarized tumor cells were
arranged with their basal aspects toward the tubular lumen, and their
microvillar apical surfaces facing the opposite direction. Tumor cells
lining the lumen were connected by electron-dense cellular junctions.
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To evaluate the putative existence of tumor-lined networks or
channels in ovarian cancer, nine ovarian cancers and four benign
ovarian tumors were histologically examined. Representative examples
are shown in Figure 4, A–D
, and the
results are summarized in Table 1
.
Endothelial-lined blood vessels were identified in all cases of
low-stage (stage I) and low-grade (grade I or II) tumors. However,
tumor cell-lined channels containing red blood cells were detected in
three of five high-grade (III), high-stage (III or IV) tumors.
Representative tumor-lined channels are shown in Figure 5, A and B
. Pan-cytokeratin staining of
high-grade cancers confirmed the presence of tumor-lined channels
(Figure 5C)
. Serial sections at 5 µmol/L were examined to ensure
morphological continuity through various thicknesses of the tissue.
Among tumors with evidence of tumor cell-lined channels, 10 high-power
fields were counted to estimate the proportion of vessels that were
nonendothelial-lined. The incidence of these channels ranged from 7 to
10% in the three most aggressive tumors that exhibited this
phenomenon.
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To explore the potential role(s) of key molecular determinants of
ovarian tumor cell plasticity with respect to the formation of tubular
networks, we focused on matrix metalloproteinases (MMPs) in the
formation and remodeling of the tumor-formed networks in
vitro, as well as laminin—an important basement membrane
component. Immunohistochemical analysis revealed that MMPs-1, -2, -9,
and MT1-MMP were primarily localized to the networks in
vitro (Figure 6, B–E)
. The
extracellular matrix molecule laminin also localized to these
structures (Figure 6F)
. Treatment of the cultures with Metastat, an
inhibitor of MMP activity, prevented network formation when added to
the culture on day 1 (Figure 6G)
, but did not destroy established
networks in separate cultures when added on day 7 (Figure 6H)
.
|
. In benign ovarian cysts, laminin
staining was seen in the subepithelial basement membrane and in the
walls of the blood vessels (Figure 7A)
. However, in high-grade invasive
ovarian cancer, laminin staining was seen in the matrix surrounding
spheroids of tumor cells as well as in the cytoplasm of cancer cells
(Figure 7B
and inset). The pattern formed by the laminin
immunohistochemical stain is reminiscent of a reticular meshwork.
Within these areas, in high-grade ovarian cancers, tumor cell-lined
channels were seen containing red blood cells (Figure 7C
and inset).
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. The human
umbilical vein endothelial cell line was used as an internal positive
control. All of the ovarian cancer cell lines that formed matrix-rich
networks expressed the thrombin receptor and three lines expressed
TIE2. EG and 222 showed strong expression of CD31 (PECAM) whereas SKOV3
showed weaker expression. In addition, EG and 222 expressed other
vascular-associated markers including endoglin and angiopoietin 1. All
of the cancer cell lines expressed vascular endothelial growth factor
(VEGF). The most invasive cell line, 222, also expressed the VEGF
receptor, FLT1 (VEGFR-1), and TIE-1. Although A2780-PAR and HIO-180
both express VEGF, neither showed significant expression of the other
vascular-associated markers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent findings from our laboratory have shown that aggressive melanomas, but not nonaggressive tumors, contain patterns of matrix-rich networks surrounding spheroids of tumors cells in the absence of tumor necrosis and classical angiogenesis.1,27 The presence of matrix-rich networks in aggressive primary and metastatic melanoma tumors from patients has been shown to correlate with a poor clinical outcome.28 In vitro, aggressive melanoma cells capable of generating similar matrix-rich networks with channels express inappropriate vascular molecular markers that include TIE-1 (an endothelial receptor kinase), and more than a dozen other endothelial- and vascular-associated genes, in addition to fibroblast- and epithelial-related genes. This molecular profile suggests a genetic reversion to an embryonic-like phenotype, which may be capable of forming a primitive network in aggressive tumors, as demonstrated experimentally in three-dimensional cultures in vitro-– referred to as vasculogenic mimicry.1 However, the physiological significance of these networks has yet to be elucidated.
Before this study, it was not known whether epithelial tumors such as ovarian cancer were capable of engaging in vasculogenic mimicry. However, it has recently been shown that human breast cancer metastasis occurs via angiogenic- and nonangiogenic-characterized pathways, suggesting an important role for alternative vascularization and dissemination of epithelial tumors.29 It has also been shown that aggressive breast cancer cells express vascular-associated markers.30 Most epithelial ovarian cancers are thought to metastasize through the process of exfoliation from the primary tumor followed by dissemination of cells throughout the peritoneal cavity, implantation, and subsequent growth. The majority of patients succumb to their disease, ultimately dying from bowel obstruction resulting from multiple metastatic tumor deposits. For this mode of tumor spread, the tumor cells must develop a vascular supply at the sites of metastasis and tumors capable of rapid vascularization would theoretically be able to rapidly increase their volume. In addition, we have previously shown that at least 22% of the epithelial ovarian cancer patients either present with or develop distant metastases, most likely through hematogenous spread.31 Our in vitro studies indicate that invasive ovarian cancer cells are able to develop matrix-rich tubular networks in a three-dimensional matrix without the presence of fibroblasts or endothelial cells. This pattern of tubular networks was not observed with the normal, immortalized human ovarian surface epithelial cells, but may represent a primitive microcirculatory network for tumors to support blood flow similar to embryonic microcirculatory vasculogenic networks. Alternatively, these networks may represent receptacles for vascular leakage previously reported in certain animal models.32 We are actively pursuing this question in appropriate ovarian cancer animal models.
In recent years, the vascular bed of human tumors has been characterized extensively by performing microvessel density counts.15-19,33,34 These studies have revealed that high microvessel density counts within vascular hot spots of tumors correspond with a poor prognosis for patients. Microvessel density studies using endothelial markers may reflect the vascular status of a tumor, but they may not be indicative of the nature of the vascularity. In a recent study, Eberhard and colleagues35 demonstrated that counting microvessels in tumor vascular hot spots resulted in a relatively uniform high microvessel density in the different tumor types, but showed marked differences in the endothelial proliferation index among the various tumor types. This suggested that there is marked heterogeneity of vasculature in human tumors. Similarly, Orre and Rogers20 found that the endothelial proliferation index in high-vessel density regions of ovarian cancers relative to benign tumors was significantly lower. Thus, it is tempting to speculate that these findings may be partially explained by the phenomenon of tumor cell vasculogenic mimicry in the paucity of endothelial cells.
Tumor vessels seem to differ from normal vessels in many ways. Human
colon tumors in mice were found to contain mosaic vessels that are
lined by tumor cells in many areas and endothelial cells in other
regions.9
In the present study, 
10% of tumor vessels
seemed to be nonendothelial-lined. Interestingly, mosaic vessels
comprised 15% of colon tumor vessels in a mouse
model.9
Another consideration with respect to red blood
cells in areas surrounded by tumor cells might be explained by a recent
report that tumor vessels are leaky because of disorganized and loosely
interconnected endothelial cells.32
There is also strong evidence that even in normal developmental
processes, nonendothelial cells can adopt a vascular-related
phenotype.6,8
For example, it has been shown that human
cytotrophoblasts can adopt an endothelial cell phenotype as they
actively participate in the dynamics of establishing the placenta and
primordial microcirculation, which has been designated "trophoblast
pseudo-vasculogenesis."6-8
Cytotrophoblasts were able
to mimic the molecular phenotype of cells of the vascular system by
expressing adhesion molecules characteristic of endothelial
cells.6
In our study, we have demonstrated that ovarian
cancer cells forming networks in vitro also display
molecular vasculogenic mimicry. Our findings show that pure populations
of ovarian cancer cells can express aberrant endothelial- and other
vascular-associated markers. As shown in Figure 8
, all of the invasive
ovarian cancer cells except A2780-PAR express several of the
traditional endothelial markers. These include several members of the
VEGF family that mediate angiogenic signals to the vascular endothelium
via high-affinity receptors that have been thought to be specific for
endothelial cells.36
Endoglin (CD105) is a receptor for
transforming growth factor-ß and has been used as an endothelial
cell-specific marker. It has been shown to be important for
developmental angiogenesis and plays a significant role in
capillary-like tube formation by endothelial cells in three-dimensional
collagen matrix cultures.37
The endothelial cell-cell
adhesion molecule, CD31, has also been shown to play a critical role in
migration and the process of tube formation by endothelial
cells.38,39
Thus, our findings suggest that invasive
ovarian cancer cells may express a deregulated, embryonic-like
phenotype and are capable of forming primitive tubular networks
in vitro, thereby exhibiting tumor plasticity.
The ability to invade extracellular matrices plays an important role in metastasis and in development of blood flow to tumors. Matrix metalloproteinases have been shown to play an active role in the neovascularization of tumors through their ability to degrade the extracellular matrix.40,41 Human cytotrophoblasts have also been shown to up-regulate the expression of MMP-9 in the process of invasion and when adopting a vascular phenotype.7,42 The thrombin receptor has been shown to mediate an increase in matrix metalloproteinases in human endothelial cells during the processes of tissue remodeling and vessel development.43 Thrombin receptor has also been shown to be expressed in human cancers and play a role in increasing breast cancer invasiveness.44,45 Interestingly, the ovarian cancer cells capable of generating tubular networks in vitro expressed both thrombin receptor and MMPs in our study. These findings indicate that secretion of MMPs may facilitate matrix-rich network formation, which was inhibited by treatment with an MMP inhibitor. Thus, MMP inhibitors might play an important role in the treatment of ovarian cancer in conjunction with other therapies. Our study also examined laminin, a major component of blood vessels that has been shown to be a critical glycoprotein in tube formation by endothelial cells in three-dimensional collagen gels.46,47 Laminin was expressed in the tubular structures formed by pure ovarian cancer cells.
Based on our cellular, molecular, and clinical findings, we speculate that the tumor-generated, laminin-containing networks may represent either a primitive microcirculatory-like network or a remodeled vascularized portion of a tumor and/or discrete areas of vascular leakage, which is strictly associated with the aggressive tumors, but not the low-stage/grade and benign tumors. Further experiments and imaging data are required to elucidate issues related to the physiological significance of these networks. The invasive ovarian cancer cells expressing a molecular vasculogenic phenotype may serve as an important indication of tumor plasticity in a growing tumor mass, signifying aggressive treatment management. Additionally, these data may offer alternative targets for therapeutic intervention.
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Footnotes |
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Supported by grant IN-1227 from the American Cancer Society administered through the Holden Comprehensive Cancer Center at the University of Iowa (to A. K. S.), the Reproductive Scientist Development Program through National Institutes of Health grant 5K12HD00849 and the American Board of Obstetrics and Gynecology (to A. K. S.); National Institutes of Health grants CA83137 (to R. E. B. S.) and CA59702 (to M. J. C. H.), and the Kate Daum Research Endowment and the H. B. Wallace Foundation Award (to M. J.C. H.).
Accepted for publication January 9, 2001.
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S. K. Lutgendorf, S. Cole, E. Costanzo, S. Bradley, J. Coffin, S. Jabbari, K. Rainwater, J. M. Ritchie, M. Yang, and A. K. Sood Stress-Related Mediators Stimulate Vascular Endothelial Growth Factor Secretion by Two Ovarian Cancer Cell Lines Clin. Cancer Res., October 1, 2003; 9(12): 4514 - 4521. [Abstract] [Full Text] [PDF]
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 R. M. Wenham, B. Calingaert, S. Ali, K. McClean, R. Whitaker, R. Bentley, J. M. Lancaster, J. Schildkraut, J. Marks, and A. Berchuck Matrix Metalloproteinase-1 Gene Promoter Polymorphism and Risk of Ovarian Cancer Reproductive Sciences, September 1, 2003; 10(6): 381 - 387. [Abstract] [PDF]
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W. Ruf, E. A. Seftor, R. J. Petrovan, R. M. Weiss, L. M. Gruman, N. V. Margaryan, R. E. B. Seftor, Y. Miyagi, and M. J. C. Hendrix Differential Role of Tissue Factor Pathway Inhibitors 1 and 2 in Melanoma Vasculogenic Mimicry Cancer Res., September 1, 2003; 63(17): 5381 - 5389. [Abstract] [Full Text] [PDF]
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A. R. Hess, E. A. Seftor, R. E. B. Seftor, and M. J. C. Hendrix Phosphoinositide 3-Kinase Regulates Membrane Type 1-Matrix Metalloproteinase (MMP) and MMP-2 Activity during Melanoma Cell Vasculogenic Mimicry Cancer Res., August 15, 2003; 63(16): 4757 - 4762. [Abstract] [Full Text] [PDF]
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X. Chen, Z. Ai, M. Rasmussen, P. Bajcsy, L. Auvil, M. Welge, L. Leach, S. Vangveeravong, A. J. Maniotis, and R. Folberg Three-Dimensional Reconstruction of Extravascular Matrix Patterns and Blood Vessels in Human Uveal Melanoma Tissue: Techniques and Preliminary Findings Invest. Ophthalmol. Vis. Sci., July 1, 2003; 44(7): 2834 - 2840. [Abstract] [Full Text] [PDF]
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S. M. Rybak, E. Sanovich, M. G. Hollingshead, S. D. Borgel, D. L. Newton, G. Melillo, D. Kong, G. Kaur, and E. A. Sausville "Vasocrine" Formation of Tumor Cell-lined Vascular Spaces: Implications for Rational Design of Antiangiogenic Therapies Cancer Res., June 1, 2003; 63(11): 2812 - 2819. [Abstract] [Full Text] [PDF]
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L. Rosano, F. Spinella, D. Salani, V. Di Castro, A. Venuti, M. R. Nicotra, P. G. Natali, and A. Bagnato Therapeutic Targeting of the Endothelin A Receptor in Human Ovarian Carcinoma Cancer Res., May 15, 2003; 63(10): 2447 - 2453. [Abstract] [Full Text] [PDF]
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 K. D. Miller, C. J. Sweeney, and G. W. Sledge The Snark is a Boojum: the continuing problem of drug resistance in the antiangiogenic era Ann. Onc., January 1, 2003; 14(1): 20 - 28. [Abstract] [Full Text] [PDF]
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 Y.-J. Lee, N. Nagai, C.-H. Siar, K. Nakano, H. Nagatsuka, H. Tsujigiwa, C.-H. Roan, and M. Gunduz Angioarchitecture of Primary Oral Malignant Melanomas J. Histochem. Cytochem., November 1, 2002; 50(11): 1555 - 1562. [Abstract] [Full Text] [PDF]
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C. Liu, H. Huang, F. Donate, C. Dickinson, R. Santucci, A. El-Sheikh, R. Vessella, and T. S. Edgington Prostate-specific Membrane Antigen Directed Selective Thrombotic Infarction of Tumors Cancer Res., October 1, 2002; 62(19): 5470 - 5475. [Abstract] [Full Text] [PDF]
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A. K. Sood, M. S. Fletcher, L. M. Gruman, J. E. Coffin, S. Jabbari, Z. Khalkhali-Ellis, N. Arbour, E. A. Seftor, and M. J. C. Hendrix The Paradoxical Expression of Maspin in Ovarian Carcinoma Clin. Cancer Res., September 1, 2002; 8(9): 2924 - 2932. [Abstract] [Full Text] [PDF]
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O. Straume and L. A. Akslen Importance of Vascular Phenotype by Basic Fibroblast Growth Factor, and Influence of the Angiogenic Factors Basic Fibroblast Growth Factor/Fibroblast Growth Factor Receptor-1 and Ephrin-A1/EphA2 on Melanoma Progression Am. J. Pathol., March 1, 2002; 160(3): 1009 - 1019. [Abstract] [Full Text] [PDF]
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A. K. Sood, M. S. Fletcher, and M. J. C. Hendrix The Embryonic-Like Properties of Aggressive Human Tumor Cells Reproductive Sciences, January 1, 2002; 9(1): 2 - 9. [Abstract] [PDF]
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 M. J. C. Hendrix, E. A. Seftor, P. S. Meltzer, L. M. G. Gardner, A. R. Hess, D. A. Kirschmann, G. C. Schatteman, and R. E. B. Seftor Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry PNAS, June 12, 2001; (2001) 131209798. [Abstract] [Full Text] [PDF]
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M. J. C. Hendrix, E. A. Seftor, P. S. Meltzer, L. M. G. Gardner, A. R. Hess, D. A. Kirschmann, G. C. Schatteman, and R. E. B. Seftor Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry PNAS, July 3, 2001; 98(14): 8018 - 8023. [Abstract] [Full Text] [PDF]
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