Cancer Research Meeting Calendar Jordan
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gesierich, S.
Right arrow Articles by Zöller, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gesierich, S.
Right arrow Articles by Zöller, M.
[Cancer Research 66, 7083-7094, July 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Systemic Induction of the Angiogenesis Switch by the Tetraspanin D6.1A/CO-029

Sabine Gesierich1, Igor Berezovskiy1, Eduard Ryschich2 and Margot Zöller1,3

1 Department of Tumor Progression and Immune Defence, German Cancer Research Centre; 2 Department of Surgery, University of Heidelberg, Heidelberg; and 3 Department of Applied Genetics, Faculty of Chemistry and Bioscience, University of Karlsruhe, Karlsruhe, Germany

Requests for reprints: Margot Zöller, Department of Tumor Progression and Tumor Defence, German Cancer Research Centre, Im Neuenheimer Feld, 280 D-69120 Heidelberg, Germany. Phone: 49-6221-422454; Fax: 49-6221-424760; E-mail: m.zoeller{at}dkfz.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Expression of the tetraspanin CO-029 is associated with poor prognosis in patients with gastrointestinal cancer. In a pancreatic tumor line, overexpression of the rat homologue, D6.1A, induces lethally disseminated intravascular coagulation, suggesting D6.1A engagement in angiogenesis. D6.1A-overexpressing tumor cells induce the greatest amount of angiogenesis in vivo, and tumor cells as well as exosomes derived thereof strikingly increase endothelial cell branching in vitro. Tumor cell–derived D6.1A stimulates angiogenic factor transcription, which includes increased matrix metalloproteinase and urokinase-type plasminogen activator secretion, pronounced vascular endothelial growth factor expression in fibroblasts, vascular endothelial growth factor receptor expression, and strong D6.1A up-regulation in sprouting endothelium. Thus, D6.1A initiates an angiogenic loop that, probably due to the abundance of D6.1A in tumor-derived exosomes, reaches organs distant from the tumor. Most importantly, because of the strong D6.1A up-regulation on sprouting capillaries, angiogenesis could be completely inhibited by a D6.1A-specific antibody, irrespective of whether or not the tumor expresses D6.1A. Tetraspanins have been suggested to be involved in morphogenesis. This is the first report that a tetraspanin, CO-029/D6.1A, promotes tumor growth by its capacity to induce systemic angiogenesis that can effectively, and with high selectivity for sprouting endothelium, be blocked by a D6.1A-specific antibody. (Cancer Res 2006; 66(14): 7083-94)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
The term angiogenesis defines the process of new capillary formation from preexisting vasculature that occurs in physiologic as well as pathologic conditions (1). Tumor cells essentially depend on angiogenesis to grow beyond a threshold size of a few cubic millimeters (2). Angiogenesis is the result of an intricate balance between proangiogenic and antiangiogenic factors, and the dominance of proangiogenic factors is called the angiogenic switch (3). One of the main angiogenic switch inducers is hypoxia (4), whereas the most well-known proangiogenic factor is vascular endothelial growth factor (VEGF; ref. 5), and matrix metalloproteinases (MMP) are essentially required to create space for sprouting capillaries by degradation of the basal membrane as well as for the liberation of angiogenic factors and for inducing the transcription of angiogenic factors (6, 7). Recent studies in knockout and transgenic mouse models have provided further evidence that tumor angiogenesis is not only guided by the tumor cell itself, but is also closely tied to the tumor microenvironment (8).

Tetraspanins are a large family of proteins grouped according to structural relatedness. The key feature of tetraspanins is their potential to associate with each other and with a multitude of molecules from other protein families (9–11), the most prominent partners being integrins (12). The tetraspanin, D6.1A (rat)/CO-029 (human), associates with {alpha}3ß1 and {alpha}6ß1 and, after disassembly of hemidesmosomes, with {alpha}6ß4. It also associates with the tetraspanins, CD9 and CD81, and the immunoglobulin superfamily member, prostaglandin F2{alpha} receptor regulatory protein, a type II phosphoinositide-4-kinase, EpCAM, and CD44v4-v7 (13–15).

According to their association with different molecules, tetraspanins are assumed to function as adaptors that assemble protein complexes in defined membrane microdomains, called tetraspanin-enriched microdomains, that provide a link to specific signal-transducing molecules (16). This mode of activity might explain why tetraspanins are said to take part in a wide range of diverse functions (9, 11), such as B and T cell activation, platelet aggregation, migration, proliferation (10, 17, 18), and tumor cell progression (18, 19). With respect to the latter, high expression of CD9 (20, 21) and CD82 (22) is mostly associated with a favorable prognosis. CD151, D6.1A, and its human homologue, CO-029, are supposed to exert tumor-promoting activities (23–26). A possible mechanistic basis for the prometastatic functions of tetraspanins could be their association with certain integrins and the accompanying increase in cell motility (22, 23). Furthermore, tetraspanin interactions with platelets and leukocytes were suggested to provide tumor cells with a survival advantage in the hostile environment which they encounter during metastatic spread (27). CD151 has also been reputed to be involved in cellular morphogenesis, promoting the formation of cord-like structures, considered as a preform of sprouting vessels (28). Finally, tumor cells can grow in mosaics with endothelial cells, and mosaic growth depends on tetraspanin expression (29). Taking into account that coagulopathy and thrombosis are frequent complications, particularly in patients with lung and pancreatic cancer (30), it is important to note that CD9, CD63, and CD151 are expressed by platelets. CD9 and CD63 associate with {alpha}IIbß3, which supports activated platelet adhesion to neutrophils and platelet aggregation (31). In CD151 knockout mice, platelet spreading is impaired, aggregation is reduced, and the bleeding time is prolonged. This is likely due to the absence of an outside-in signal provided by CD151, leading to the activation of {alpha}IIbß3 (32).

D6.1A overexpression on a pancreatic rat adenocarcinoma line is associated with the formation of hemorrhagic ascites (15) and can induce a severe consumption coagulopathy, such that rats become moribund due to disseminated intravascular coagulation rather than the tumor burden (13). These features prompted us to search for the possible involvement of D6.1A in angiogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Rats and tumors. BDX rats, bred at the animal facilities of the German Cancer Research Centre, were kept under specific pathogen–free conditions, fed sterilized food and water ad libitum. Rats were used for experiments at the age of 6 to 10 weeks. BSp73AS (AS) cells, a pancreatic adenocarcinoma line of the BDX rat strain (33), were transfected with D6.1A cDNA (AS-D6.1A; ref. 13). For intravital microscopy, AS cells were transfected with the enhanced green fluorescent protein (EGFP) cDNA–containing pEGFP-N1 vector (Becton Dickinson, Heidelberg, Germany). AS-D6.1A cells were transfected with the EGFP cDNA inserted in the pcDNA3.1Hygro vector (Invitrogen, Karlsruhe, Germany). Transfected lines were selected in RPMI 1640, 10% FCS, and 500 µg/mL of G418. The selection medium for double-transfected lines contained an additional 50 µg/mL of hygromycin. A BDX fibroblast line, generated by NiSO4 treatment, and RAEC, a Wistar rat–derived aortic endothelial cell line (Cell-lining, Berlin, Germany) was maintained in RPMI 1640 and 10% FCS. Confluent cultures were trypsinized and split.

Antibodies. The following monoclonal and polyclonal antibodies were used: mouse anti-D6.1A (D6.1; ref. 34), mouse anti-{alpha}6ß4 (B5.5; ref. 34), rabbit anti-CD151 (14); anti-CD9 and anti-CD44 (Ox50; European Collection of Animal Cell Cultures); anti-{alpha}1, anti-{alpha}2, anti-{alpha}3, anti-{alpha}4, anti-{alpha}5, anti-{alpha}6, anti-ß1, anti-ß2, anti-ß3, anti-ß4, anti-CD31, anti–tumor necrosis factor-{alpha} (TNF{alpha}), anti-IL4 (BD PharMingen, Heidelberg, Germany); anti-TIMP-1, anti-TIMP-2 (Biozol, Munich, Germany); anti-MMP-2, anti-MMP-9, anti-MMP-13 (Dianova, Hamburg, Germany); anti-VEGFR1, anti-VEGFR2 (Biotrend, Köln, Germany); anti–urokinase-type plasminogen activator receptor (uPAR), anti-uPA (American Diagnostica, Stanford, CT); anti-VEGF, anti–basic fibroblast growth factor (bFGF; R&D, Eschborn, Germany); biotinylated, horseradish peroxidase– and fluorescence dye (FITC, APC, rhodamine, Cy2)–labeled secondary antibodies, and FITC-labeled phalloidin (BD PharMingen and Biotrend).

Flow cytometry. Flow cytometry followed routine procedures using 1 to 3 x 105 cells per sample. Trypsinized cells were allowed to recover for 2 hours at 37°C in RPMI 1640 and 10% FCS. For intracellular staining, cells were fixed and permeabilized in advance. Samples were analyzed with FACSCalibur (Becton Dickinson).

Immunohistochemistry. Cryostat sections (5 µm) of snap-frozen tissue were fixed in chloroform/acetone (1:1, 4 minutes). For intracellular staining, sections were fixed in paraformaldehyde (4%) and were permeabilized (0.1% Triton X-100, 4 minutes, 4°C). Tissues were incubated for 1 hour with the first antibody, washed and exposed to the biotinylated secondary antibodies (30 minutes), and alkaline phosphatase–conjugated avidin-biotin complex (Vector Laboratories, Burlingame, CA) solutions (5-20 minutes). Sections were counterstained with Mayer's hematoxylin. The primary antibody was replaced by normal mouse, rat, or rabbit IgG for negative controls.

For immunofluorescence microscopy, cells were seeded on matrigel-coated cover slides. After 8 hours, cells were fixed in 4% paraformaldehyde (w/v in PBS) and, where indicated, were permeabilized (0.1% Triton X-100, 4 minutes). After washing and blocking (PBS/0.2% gelatin/0.5% bovine serum albumin), cells were incubated with the primary antibody (2-10 µg/mL, 60 minutes, 4°C). Slides were rinsed and incubated for 60 minutes at 4°C with a fluorochrome-conjugated secondary antibody. Washed cells were incubated with an excess of unlabeled mouse or rabbit IgG to block free binding sites of the secondary antibody. Unlabeled IgG was also added during incubation with the second, dye-labeled antibody (60 minutes, 4°C). Phalloidin-FITC (0.5 µg/mL) was used for filamentous actin staining (60 minutes, 4°C). After washing thrice in PBS and once in water, slides were mounted in Elvanol. Digitized images were generated using a Leica DMRBE Microscope equipped with a SPOT CCD camera from Diagnostic Instruments, Inc., and Software SPOT2.1.2 (Visitron Systems, Puchheim, Germany).

Preparation of exosomes. AS and AS-D6.1A cells were cultured in RPMI 1640 and 10% exosome-depleted FCS. Exosomes were prepared from supernatant after 2 days of culture by differential centrifugation. Supernatants were centrifuged twice for 10 minutes at 500 x g, once for 20 minutes at 2,000 x g, and once for 30 minutes at 10,000 x g to eliminate cell debris. The purified supernatant was centrifuged for 90 minutes at 100,000 x g using a SW41 rotor. The pellet was resuspended in PBS and centrifuged again for 90 minutes at 100,000 x g.

Western blotting. Cell lysates (1% Triton X-100) were resolved on 12% or 15% SDS-PAGE under nonreducing conditions. Proteins were transferred to Hybond enhanced chemiluminescence at 30 V overnight. After blocking (5% fat-free milk powder), immunoblotting was done with the indicated antibodies, followed by horseradish peroxidase–labeled secondary antibodies. Blots were developed with the enhanced chemiluminescence detection system.

Zymography. Cells (106) were seeded in 24-well plates (35). After overnight culture, cells were washed and starved in serum-free medium. Conditioned medium was collected after 24 hours and was centrifuged (15 minutes, 15,000 x g) to remove cell debris. Aliquots were incubated with Laemmli buffer (15 minutes, 37°C) and separated in a 10% acrylamide gel containing 1 mg/mL of gelatin. The gel was washed for 30 minutes each in 2.5% Triton X-100, 2.5% Triton X-100/50 mmol/L Tris (pH 7.5), and 2.5% Triton X-100/50 mmol/L Tris (pH 7.5), 5 mmol/L CaCl2, 1 µmol/L ZnCl2. Gels were incubated (24-48 hours, 37°C) in 50 mmol/L Tris (pH 7.5), 5 mmol/L CaCl2, 1 µmol/L ZnCl2, and stained with Coomassie blue.

Matrigel assay. Tumor cells (105) were seeded on matrigel-coated 24-well plates. Where indicated, antibodies (10 µg/mL) were added to the culture medium. Cable formation was evaluated after 7 and 48 hours and was documented by light microscopy.

In vitro angiogenesis. The mesentery was collected from 6-week-old rats and 1 cm2 pieces were placed in six-well plates in RPMI 1640 and 10% FCS. Tumor cells (5 x 105), supernatant of confluent tumor cell cultures (1:1 diluted with fresh medium), exosome-depleted supernatant (1:1 diluted with fresh medium), or exosomes (equivalent to supernatant in fresh medium and equilibrated for comparable amounts of CD9, CD81, and CD151) were placed on top of the mesentery. Antibodies (10 µg/mL) were added as indicated. Endothelial cell sprouting was microscopically evaluated.

In vivo angiogenesis and intravital microscopy. BDX rats received an i.p. injection of 5 x 106 EGFP-transfected tumor cells and, where indicated, i.p. antibody injections (200 µg/injection, every 3rd day; ref. 36). At the indicated time points, rats were sacrificed, the mesentery was excised and shock-frozen for immunohistology. Alternatively, rats were anesthetized (xylazine and ketamine) and the abdomen was opened by a midline incision. Rats were fixed on a special plate automatically maintained at 37°C. The mesentery with tumors was immobilized in 50 mL of Ringer's solution at 37°C in an immersion chamber. Microscopic images were taken with a specialized microscope and a 40x magnification objective using two fluorescence filter blocks (I3 and N2.1; Leica GmbH, Wetzlar, Germany). Images were transmitted by a video camera (Kappa GmbH, Gleichen, Germany) to a monitor (PVM-1440M, Sony, Tokyo, Japan), and recorded (sVHS, AG-7350-E, Panasonic, Osaka, Japan) for subsequent off-line analysis. Evaluation of vessel diameter and vessel density was done with special software (CapImage, Zeintl GmbH, Heidelberg, Germany). Relative vessel width was defined as the ratio between vessel area to vessel-tumor contact length. The surface of tumor blood vessels and the relative vessel width was evaluated on 20 randomly chosen fields per rat. In vivo experiments were approved by the local animal care committee (Regierungspräsidium Karlsruhe, Karlsruhe, Germany).

Statistical analysis. Significance of differences was evaluated by the Wilcoxon rank test or the two tailed Student's t test. P values were adjusted for multiple comparisons by the step-down Bonferroni method of Holm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Involvement of D6.1A in cellular morphogenesis. D6.1A-overexpressing tumor cells can induce disseminated intravascular coagulation (13), and i.p.-injected D6.1A-overexpressing tumor cells induce hemorrhagic ascites (15). Because the tetraspanin CD151 has been reported to be involved in cellular morphogenesis (37) with anti-CD151–inhibiting cord-like structure formation of tumor cells and fibroblasts when grown on matrigel, it became interesting to see whether D6.1A would exert similar features.

When D6.1A-overexpressing AS cells were grown on matrigel, AS-D6.1A, but not AS cells, formed cord-like, anastomosing structures. Although both lines express CD9, CD81, and CD151 at a high level, cord formation could only be inhibited by the D6.1A-specific antibody, D6.1, but not by anti-CD9, anti-CD81, or anti-CD151. AS/AS-D6.1A cells express the {alpha}3ß1 and {alpha}6ß1 integrins, where the latter has been described to account for anti-CD151–inhibitable cord formation of NIH3T3 cells (37). Instead, cord formation of AS-D6.1A cells was inhibited by anti-{alpha}3 and anti-ß1, but not by anti-{alpha}6 (Fig. 1A ).


Figure 1
View larger version (61K):
[in this window]
[in a new window]
  		Figure 1. Cord formation of AS-D6.1A cells on matrigel. A, AS and AS-D6.1A cells were cultured for 7 and 48 hours at 37°C on matrigel-coated 24-well plates. Only AS-D6.1A cells formed cord-like structures. To control for the effect of D6.1A on cord formation, AS-D6.1A cells were cultured for 7 hours on matrigel in the presence of D6.1, anti-CD9, anti-CD81, anti-CD151, anti-{alpha}3, anti-{alpha}6, or anti-ß1 (10 µg/mL). Phase contrast microscopy (bar, 100 µm). B, AS and AS-D6.1A cells which were cultured for 7 hours on matrigel-coated cover slides were stained with anti-ß1, anti-{alpha}3, anti-{alpha}6, anti-VEGF, and secondary Cy2-labeled antibody or with Phalloidin-FITC. AS-D6.1A cells were counterstained with rhodamine-labeled D6.1. Single fluorescence and merged overlays (bar, 10 µm).

 
Tetraspanins exert functional activities via their associating molecules, this is in line with the observed inhibition of cord formation by integrin-specific as well as tetraspanin-specific antibodies. To control for this assumption, the localization of {alpha}3, {alpha}6, and ß1, and colocalization with D6.1A was evaluated, respectively, in AS and AS-D6.1A cells that had been grown on matrigel for 7 hours (Fig. 1B). D6.1A colocalized with {alpha}3 and ß1, but hardly with {alpha}6. There has been no pronounced colocalization of D6.1A with actin bundles. Instead, D6.1A colocalized with VEGF.

Thus, D6.1A exhibited morphogenic features, which differed from those described for CD151 inasmuch as cord formation was inhibited by D6.1 and anti-{alpha}3, but not by anti-CD151 and anti-{alpha}6. Although these unexpected differences between the two tetraspanins are interesting and require further elucidation, we were primarily concerned as to whether the morphogenic features of D6.1A might be of functional relevance in vivo. To control the hypothesis, we first evaluated whether D6.1A-expressing tumor cells support angiogenesis in vivo.

D6.1A induces angiogenesis that is inhibited by D6.1. EGFP-transfected AS and AS-D6.1A cells were injected i.p. into syngeneic BDX rats. After 3 and 6 days, rats were anesthetized, the peritoneal cavity was opened and vessel formation was evaluated by intravital microscopy. Vessel density and vessel width were evaluated in at least 20 tumor fields per rat in different parts of the mesentery. In addition, individual fields were surveyed for up to 2 hours to see whether tumor cells would infiltrate preexisting vessels.

There was no evidence for immigration of AS or AS-D6.1A cells into preexisting vessels (data not shown). However, both tumor lines induced angiogenesis. Three days after tumor cell injection, capillary formation was seen in small AS-D6.1A tumor nodules. After 6 days, the relative vessel density was increased by more than five times in AS-D6.1A as compared with AS tumor nodules. Unlike the vessel density, the vessel width, defined by the ratio of vessel area to contact length between vessels and tumor, was only slightly increased in AS-D6.1A as compared with AS tumors. When rats received the D6.1 antibody concomitantly with the tumor cells, angiogenesis was largely abolished in both AS-D6.1A and AS tumor nodules. Anti-CD151, expressed in both tumor lines, served as a control. Vessel density was slightly reduced by anti-CD151 treatment. However, reduction in vessel density did not reach a significant level in either AS or AS-D6.1A tumors. The antiangiogenic effect of D6.1 was not a consequence of tumor growth inhibition. Neither D6.1 nor anti-CD151 inhibited tumor growth in vitro (data not shown) or in vivo (Fig. 2A and B ).


Figure 2
Figure 2
View larger version (87K):
[in this window]
[in a new window]
  		Figure 2. Angiogenesis (in vivo) and endothelial cell growth (in vitro) induction by AS-D6.1A tumor cells. A, AS and AS-D6.1A cells were injected i.p. Where indicated, rats received two i.p. injections (days 0 and 3) of 200 µg control IgG or D6.1 or anti-CD151. After 3 and 6 days, the size of the tumor nodules, vessel density, and vessel width were evaluated by intravital microscopy. The relative size of the tumor area is presented, with the mean tumor area of AS cells taken arbitrarily as 1. Relative vessel width was calculated after 6 days as the ratio of vessel area to vessel-tumor contact length. Columns, mean values for eight rats (except for anti-CD151 treatment with two rats per group) evaluating 20 fields per rat; bars, ±SD. B, tumor nodules were collected 2 weeks after i.p. injection of AS and AS-D6.1A cells. Sections of shock-frozen tissue were stained with control IgG or anti-CD31 (bar, 50 µm). C, the mesenteric sheet of untreated rats was cultured in the absence of tumor cells or was cocultured with AS or AS-D6.1A cells or supernatants. Cultures contained 10 µg/mL control IgG, D6.1, anti-CD9, anti-CD151, or anti-{alpha}3. D, cell lysate, exosomes (100,000 x g pellet of supernatant), cleared supernatant (10,000 x g), and exosome-depleted supernatant (100,000 x g) of AS and AS-D6.1A cultures were separated by SDS-PAGE and blotted with D6.1, anti-CD9, anti-CD81, anti-CD151, and anti-VEGF. Gels were loaded with 2.5 µg of exosome preparation (corresponding to 2 mL of supernatant) and 40 µL of supernatants, Western blot of supernatant preparations being exposed to X-ray films overnight. D6.1A, CD9, CD81, CD151, and VEGF bands (arrow). The mesentery of untreated rats was cultured in the presence of AS cell–derived or AS-D6.1A cell–derived unfractionated or exosome-depleted supernatants or exosomes. AS-D6.1-derived exosomes corresponded to 2 mL of supernatant. Because AS-D6.1A supernatant-derived exosomes contained a higher amount of CD9, CD81, and CD151 than AS supernatant–derived exosomes, the relative protein content was estimated by densitometry. AS supernatant–derived exosomes added to the mesentery were adjusted to contain amounts of the tetraspanins CD9, CD81, and CD151 equivalent to that in AS-D6.1 supernatant–derived exosome preparations. To control for the D6.1A specificity of endothelial cell branching, endothelial cell–exosome cocultures contained, in addition, control IgG, D6.1, anti-CD9, or anti-CD151 (10 µg/mL). Phase contrast microscopy is shown after 72 hours of coculture (bar, 50 µm).

 
To confirm the involvement of D6.1A in angiogenesis, AS-D6.1A cells were seeded in vitro on small pieces of the mesentery. After 72 hours of coculture, the mesentery was filled with uncountable small, branching endothelial strings. Significantly less branching endothelial cells were seen in mesenteries cocultured with AS cells. Morphogenic changes were largely absent in mesenteries cultured in the absence of tumor cells. In line with the in vitro studies on cable formation by the tumor cells and the in vivo analysis of angiogenesis induction, in vitro–induced endothelial cell branching was inhibited by D6.1 and anti-{alpha}3, but not by anti-CD9 and anti-CD151. This accounted for the strong morphogenic alterations induced by AS-D6.1A tumor cells and the weaker changes induced by AS cells. Notably, endothelial cell branching was also induced, albeit very weakly, by AS cell supernatant, but strongly by AS-D6.1A supernatant (Fig. 2C). The latter finding might be explained by the fact that tumor cells shed D6.1A, which was recovered in tumor-derived exosomes. AS and AS-D6.1A supernatant contain VEGF, which was recovered in the soluble fraction following ultracentrifugation. Instead, the tetraspanins, CD9, CD81, CD151 (AS supernatant) plus D6.1A (AS-D6.1A supernatant), are exclusively recovered in the exosome-containing pellet. Notably, although the amount of CD9, CD81, and CD151 is roughly comparable in AS and AS-D6.1A lysates, fewer tetraspanins are recovered in AS-derived than in AS-D6.1A-derived exosomes. To exclude the idea that the amount of tetraspanins is decisive for functional activities, the amount of CD9, CD81, and CD151 in AS- and AS-D6.1A-derived exosome preparations was estimated by densitometry of Western blotting and exosome preparations, which were cocultured with the mesentery and adjusted to contain comparable amounts of the tetraspanins CD9, CD81, and CD151. When mesenteries were cultured with AS-derived or AS-D6.1A-derived exosomes or with exosome-depleted supernatant, branching was exclusively induced by AS-D6.1A-derived exosomes. In line with antibody-mediated inhibition of angiogenesis in vivo, exosome-induced endothelial cell branching was inhibited by D6.1, but not by anti-CD9 and anti-CD151, which instead supported endothelial cell sprouting (Fig. 2D).

Thus, D6.1A-expressing tumor cells as well as D6.1A-containing, tumor-derived exosomes, strongly induced angiogenesis in vivo and endothelial cell branching in vitro. Surprisingly, D6.1 completely inhibited not only AS-D6.1A-induced, but also low-level AS-induced angiogenesis. The latter observation required further exploration.

Angiogenesis is accompanied by D6.1A expression in the capillary endothelium. We first explored how D6.1 could inhibit low-level angiogenesis induced by a D6.1A-negative tumor, and considered it likely that endothelial cells might express D6.1A because low-level D6.1A expression is seen on small capillaries, although not on larger veins and arteries (38). When mesenteries were collected 5 days after i.p. tumor cell application, it became obvious that newly formed capillary endothelium strongly expresses D6.1A, i.e., CD31-positive cells were also stained by D6.1. In addition, the expression of {alpha}3 was up-regulated in endothelial cells and the expression of ß3 integrin was augmented (Fig. 3A ). Notably, capillary D6.1A expression was independent of D6.1A expression by the tumor cell, i.e., after i.p. application of AS cells, newly formed capillaries also expressed D6.1A, but far fewer capillaries were recovered. Furthermore, AS-D6.1A-induced angiogenesis was not restricted to the tumor and the mesentery, but even in the pancreatic gland, angiogenesis was significantly augmented, as shown by CD31 staining. The pancreatic gland was apparently tumor-free. No GFP-expressing tumor cells were detected (data not shown) and only endothelial cells were stained by D6.1 (Fig. 3B).


Figure 3
View larger version (102K):
[in this window]
[in a new window]
  		Figure 3. Capillary endothelium expresses D6.1A. One week after i.p. application of AS or AS-D6.1A tumor cells, the mesentery (A) and the pancreatic gland (B) were excised, tissue was shock-frozen and 5-µm slices were stained with (A) anti-CD9, anti-CD81, anti-CD151, D6.1 (tetraspanins), anti-CD31 (endothelial cells), anti-{alpha}3, anti-{alpha}6, and anti-ß3 (integrins) or (B) control IgG, anti-CD31, D6.1, and anti-{alpha}3. C, shock-frozen (5-µm slices) tissue of a human pancreatic gland, of two sections from chronic pancreatitis, and of four sections from pancreatic adenocarcinoma were stained with a control IgG or anti-CD31 and anti-CO-029 (bar, 50 µm).

 
The finding that sprouting endothelial cells of the rat up-regulated D6.1A expression prompted us to see whether this also accounts for the human homologue CO-029, high CO-029 expression being associated with poor prognosis in gastrointestinal tumors (24, 25). Expression of CO-029 in endothelial cells of the normal pancreatic gland is hardly detectable. However, endothelial cell CO-029 expression is up-regulated in inflamed pancreatic tissue and in pancreatic cancer including the tumor stroma (Fig. 3C, third row). Capillary CO-029 expression in the tumor tissue could be most clearly visualized in a tumor not expressing this tetraspanin (Fig. 3C, fourth row, right).

D6.1A expression in sprouting capillary endothelium could well explain that the D6.1 antibody inhibited tumor-induced angiogenesis irrespective of whether or not the tumor cells express the D6.1A tetraspanin. On the other hand, D6.1A expression in endothelial cells may further sustain angiogenesis.

There remains the question regarding the mechanism whereby AS-D6.1A cells initiate the overshooting of angiogenesis. The findings that few i.p. seeded AS-D6.1A cells suffice for angiogenesis-induction in an adjacent, although tumor-free organ (pancreatic gland), that AS-D6.1A-derived exosomes induce endothelial cell sprouting, and that D6.1A transcription is initiated in sprouting endothelial cells, point towards the activation of cells in the tumor environment by tumor-derived D6.1A, rather than suggesting that AS-D6.1A cells themselves provide all the requirements for angiogenesis induction.

D6.1A expression in AS cells is accompanied by minor up-regulation of VEGF and MMP-13. The integrin expression profile of AS and AS-D6.1A cells has already been partly described (13, 15). AS/AS-D6.1A cells do not express {alpha}1, {alpha}2, ß2, and ß4 (data not shown). They express {alpha}3, {alpha}4, {alpha}5, {alpha}6, and ß1, with up-regulation of {alpha}4 expression on AS-D6.1A as compared with AS cells. Both lines express the tetraspanins CD9, CD81, and CD151, CD151 expression being slightly increased in AS-D6.1A cells. The angiogenic factors VEGF, bFGF, and TNF{alpha} are expressed in both lines at low to intermediate levels, with a moderate up-regulation of VEGF expression in AS-D6.1A cells. Both lines express the antiangiogenic factors IFN{gamma}, TIMP-1, TIMP-2, and IL4 at low to intermediate levels (Fig. 4A-C ). The two lines do not express uPA or uPAR (also evaluated by Western blotting; data not shown). AS cells express MMP-2, MMP-9, and MMP-13 at an intermediate level. MMP-13 is expressed at a higher level, and MMP-9 at a lower level, in AS-D6.1A than in AS cells. Also, MMP-9 secretion is reduced in AS-D6.1A cells (Fig. 4A and D).


Figure 4
View larger version (40K):
[in this window]
[in a new window]
  		Figure 4. VEGF and MMP expression in AS and AS-D6.1A cells. A, AS and AS-D6.1A cells were stained with integrin-, tetraspanin-, angiogenic factor-, antiangiogenic factor-, and matrix-degrading enzyme-specific antibodies. The mean (three to five separate experiments) percentage of stained cells and intensity of staining are shown. *, significant differences in the percentage of stained cells; s, significant differences in the intensity of staining. B, an example of staining with D6.1, anti-bFGF, and anti-VEGF. Single fluorescence overlays of the negative control (nonbinding control antibody; light gray area) and the stained samples (black line). C and D, AS and AS-D6.1A cells were cultured overnight in the absence of FCS. C, lysates of AS and AS-D6.1A cells as well as medium and supernatants (concentrated 20-fold) were separated by SDS-PAGE and blotted with anti-VEGF. Arrowhead, the VEGF band. D, zymography of medium and supernatants of AS and AS-D6.1A cells (concentrated 20-fold) were run on gelatin gels. Arrow, gelatin digest by MMP-2 (72 kDa) and MMP-9 (92 kDa).

 
Although by no means were all proangiogenic and antiangiogenic factors evaluated, with respect to the major components, we only observed a moderate up-regulation of VEGF and MMP-13 expression in AS-D6.1A cells. Therefore, it became likely that D6.1A-expressing tumor cells may, at least in part, induce angiogenesis by transactivation.

D6.1A-expressing tumor cells initiate angiogenesis-promoting activities in the tumor environment. The effect of D6.1A-expressing tumor cells on surrounding tissues was evaluated for peritoneal exudate cells and cells of the mesentery 1 week after i.p. application of AS or AS-D6.1A tumor cells. With few exceptions ({alpha}6, uPA), integrin, tetraspanin, angiogenic factor, and matrix-degrading enzyme expression was up-regulated in peritoneal macrophages from rats receiving AS or AS-D6.1A cells. Major differences between peritoneal macrophages from AS-D6.1A-bearing versus AS-bearing rats were only noted for ß3 and uPAR, that expression was more strongly up-regulated in peritoneal macrophages of AS-D6.1A-bearing rats (Table 1 ).


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of D6.1A-expressing tumor cells on surrounding cells and tissue

 
More striking differences were observed between mesenteric cells collected from AS-D6.1A-bearing versus AS-bearing rats. Notably, VEGF, bFGF, MMP-2, MMP-9, uPA, uPAR, CD31, VEGFR1, and VEGFR2 expression were significantly more up-regulated in mesenteric cells of AS-D6.1A-bearing than AS-bearing rats. This was evaluated quantitatively after digestion of the mesentery by flow cytometry (Table 1) and zymography (data not shown) as well as semiquantitatively by immunohistochemistry (Fig. 5A ). Immunohistochemistry provided evidence that capillary endothelial cells might be the major source of VEGFR1, VEGFR2, uPA/uPAR, and MMPs.


Figure 5
View larger version (43K):
[in this window]
[in a new window]
  		Figure 5. Angiogenic factor induction by AS-D6.1A cells and supernatant. Rats received AS or AS-D6.1A cells, i.p., and were sacrificed after 7 days. A, the mesentery was excised and shock-frozen. Sections (5 µm) were stained with anti-VEGF, anti-bFGF, anti-VEGFR1, anti-VEGFR2, anti-uPA, anti-uPAR, anti-MMP-2, anti-MMP-9, anti-MMP-13, and anti-TIMP-2 (bar, 50 µm). B and C, flow cytometry analysis of the aortic endothelial cell line RAEC (B) and a BDX fibroblast line (C) after 72 hours of coculture with supernatant of AS or AS-D6.1A. Cells were stained with the indicated antibodies. Columns, mean percentage of stained cells from three independently done assays; bars, ±SD. *, significant differences between cells cultured in the absence of AS and AS-D6.1A supernatants; *, significant differences between cells cultured in the presence of AS-D6.1A as compared with AS supernatant.

 
To substantiate this assumption, a syngeneic fibroblast line and a rat aortic endothelial cell line were cultured in the presence of AS and AS-D6.1A supernatant. AS-D6.1A supernatant induced strong up-regulation of TNF{alpha}, uPA, and uPAR in the syngeneic fibroblast line. Increased VEGF, bFGF, and MMP expression were also induced by AS supernatant. Coculture of the aortic endothelial cell line with AS-D6.1A supernatant induced a selective up-regulation of CD31, VEGFR1, MMP-9, and uPAR. D6.1A and VEGFR2 expression became up-regulated, but to a lesser degree (Fig. 5B and C). These in vitro studies with histologically defined tissue lines confirmed that D6.1A influences gene expression (particularly TNF{alpha}) in fibroblasts and (particularly D6.1A, VEGFR, and CD31) in endothelial cells.

Taken together, D6.1A is the strongest angiogenesis inducer and a D6.1A-specific antibody selectively prevents tumor angiogenesis. Tumor cell–bound and tumor cell–released D6.1A obviously initiated an angiogenic loop by inducing D6.1A expression on sprouting endothelial cells. The mode of D6.1A-induced angiogenic gene transcription in endothelial cells and adjacent fibroblasts remains to be elucidated.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
Angiogenesis is a hallmark for tumor growth and progression (2) and, accordingly, interference with the angiogenic switch is considered an important therapeutic tool (39). Here, we reported that the tetraspanin D6.1A strongly induces angiogenesis, which is strikingly inhibited by a D6.1A-specific antibody. There is evidence that tumor cell–associated D6.1A provides only the initial trigger, with the tumor microenvironment accounting for an angiogenic amplification loop.

D6.1A-expressing tumor cells form cable-like structures on matrigel and induce pronounced angiogenesis when injected i.p. The former has also been seen in NIH3T3 cells overexpressing the tetraspanin CD151 (28), the latter has, to our knowledge, not yet been explored for other tetraspanins. Cable formation by {alpha}6ß1 has been found to be due to its association with CD151, where the cytoplasmic tail is essentially required to promote outside-in signaling by {alpha}6ß1 (28). CD82, instead, interferes with cable formation of a prostate tumor line, likely by down-modulating {alpha}6ß1 expression. Anti-CD9, anti-CD81, and anti-CD151 did not interfere with the cable-like growth of this tumor line (40). D6.1A-induced cable formation is inhibited by D6.1 and anti-{alpha}3, but not by anti-{alpha}6, anti-CD9, anti-CD81, and anti-CD151. Thus, the morphogenic features of tetraspanins obviously proceed differently, even if the cells have a similar tetraspanin expression profile. We do not yet know the underlying mechanisms, but we speculate that the cell type–specific integrin profile will be of importance. This assumption is derived from our observation that overexpression of {alpha}6ß4 in AS-D6.1A cells inhibits cable formation and angiogenesis induction in vivo, but neither cable formation nor angiogenesis can be rescued by an anti-{alpha}6ß4 antibody.4

AS-D6.1A-promoted angiogenesis was independent of cell-cell contact, i.e., supernatant from AS-D6.1A cells sufficed for "angiogenesis" induction, and addition of the D6.1 antibody inhibited supernatant-induced angiogenesis. This points towards D6.1A contained in the supernatant as being the responsible element. Tetraspanins are known to be released in vesicles, called exosomes (41, 42). Indeed, D6.1A shed by AS-D6.1A cells is exclusively recovered in exosomes, whereas VEGF is recovered in the soluble fraction. The components of AS-D6.1A-derived exosomes have not yet been fully resolved; however, we know that aside from tetraspanins, they also contain integrins.5 Importantly, D6.1A enriched exosomes, which do not contain VEGF and induce endothelial cell branching. The enrichment of D6.1A in exosomes also provides an explanation for the observation that the D6.1 antibody inhibited AS-D6.1A supernatant–induced and exosome-induced endothelial cell branching. D6.1A-initiated angiogenesis independent of cell-cell contact was also evidenced by strong angiogenesis in an adjacent organ, the pancreatic gland, a few days after i.p. tumor cell application. Thus, transactivation via D6.1A became a likely feature.

In fact, AS-D6.1A cells/supernatant do not provide the full requirement for angiogenesis induction, but rather stimulate adjacent cells. Peritoneal exudate cells are obviously not a major target of D6.1A, only uPA and uPAR expression became selectively up-regulated in AS-D6.1A-bearing compared with AS-bearing rats. This was different for the mesentery. The expression of several angiogenesis-related genes, such as bFGF, VEGF, MMP-2, and MMP-9 became up-regulated in the presence of AS as well as AS-D6.1A supernatant. However, the expression of some genes (CD31, D6.1A, and VEGFR) was forced by AS-D6.1A cells/supernatants. Up-regulated expression of these endothelial cell markers was confirmed in cocultures of a rat aortic endothelial cell line with AS and AS-D6.1A supernatant. In fact, up-regulated expression of D6.1A/CO-029 is a general feature of newly formed capillaries in rats and humans, which is independent of whether tumor cells express D6.1A/CO-029, albeit in the absence of tumor-derived D6.1A, only weak angiogenesis was observed. Therefore, we suggest that the D6.1A-mediated and up-regulated expression of these and additional genes is a secondary phenomenon, i.e., D6.1A does not directly promote multiple gene transcription, but initiates the angiogenic switch by an as yet undefined mechanism. The finding that AS-D6.1A supernatant selectively induced changes in angiogenesis-related molecule expression was less pronounced in tissue culture lines than on the mesentery provides additional support for our interpretation and indicates the requirement for crosstalk not only between tumor cells/tumor-derived exosomes and endothelial cells, but also with the surrounding stroma. Nonetheless, strong D6.1A expression on newly formed capillaries supports the creation of an angiogenic loop.

Notably, this D6.1A-initiated angiogenic switch is hypoxia-independent. The assumption is derived from the observations of "angiogenesis" induction in vitro and in vivo at a very early stage of tumor growth as well as in a tumor-free organ.

We have not yet explored the biochemical events of D6.1A-initiated angiogenesis. Because strong D6.1A expression was noted in sprouting endothelial cells and because D6.1 inhibited angiogenesis, even if the tumor cells did not express D6.1A, we suggest that D6.1A expression on endothelial cells is an early event in tumor-derived D6.1A-initiated angiogenesis, where D6.1A-expressing endothelial cells subsequently support the maintenance of angiogenesis. Tetraspanins, including D6.1A, are located in glycolipid-enriched membrane microdomains (37), which serve as a scaffold for signal-transducing molecules (10, 16–18). Some tetraspanins associate with G protein–coupled receptors (GPCR) and mediate signal transduction via associated heterotrimeric G proteins (43). Although a GPCR association is not yet known for D6.1A, D6.1A associates with prostaglandin F2{alpha} receptor regulatory protein (14), which regulates the binding of ligands to GPCRs (44). D6.1A also associates with PKC and a type II phosphoinositide-4-kinase, which initiates signal transduction via PIP2 phosphorylation (14). Finally, tetraspanins could induce the activation of associated integrins (10, 16, 18). D6.1A associates with {alpha}3 (13, 14), which, similar to {alpha}4 expression, becomes strongly up-regulated in endothelial cells during D6.1A-initiated angiogenesis. Both {alpha}3 and {alpha}4 have been implicated in MMP transcription (45, 46). Thus, there are several pathways whereby D6.1A can contribute to transcription/activation of angiogenesis-promoting molecules.

There remains the question regarding the primary interaction of D6.1A with endothelial cells. We hypothesize that such an interaction is mediated either by cell membrane D6.1A-associated molecules and/or by molecules enriched in D6.1A-containing exosomes. Tetraspanin-mediated protein sorting as described for the immunologic and the so-called infectious synapse (10, 11, 17, 47) could well facilitate such a mechanism of transactivation. Alternatively, one could speculate that D6.1A-containing microdomains or exosomes may be fusogenic, a feature well known, e.g., CD9, that is essential for egg-sperm and myoblast fusion (48, 49). We will explore these hypotheses by analyzing the content of AS-D6.1A-derived compared with AS-derived exosomes and by evaluating the mode of exosome-mediated intercellular communication.


   Conclusions
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
The tumor growth–promoting activity of the tetraspanin, D6.1A/CO-029, is due to its capacity to induce the angiogenic switch. Notably, membrane-bound as well as exosomal D6.1A supports angiogenic factor transcription in targeted cells. As newly formed capillaries also strongly express D6.1A, an angiogenic loop is created that further sustains capillary formation. Supporting the central role of the tetraspanin, the process can be inhibited by a D6.1A-specific antibody. Thus, antibody blockade of this tetraspanin could well become a new and supposedly highly selective and efficient drug.


   Acknowledgments
 
Grant support: Deutsche Forschungsgemeinschaft (Zo40-8/3, M. Zöller) and the Tumorzentrum Heidelberg Mannheim (M. Zöller and E. Ryschich).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
4 Unpublished observation. Back

5 Unpublished finding. Back

Received 1/31/06. Revised 4/23/06. Accepted 5/23/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

  1. Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med 2003;3:643–51.[CrossRef][Medline]
  2. Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol 2002;29:15–8.[Medline]
  3. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003;3:401–10.[CrossRef][Medline]
  4. Giordano FJ, Johnson RS. Angiogenesis: the role of the microenvironment in flipping the switch. Curr Opin Genet Dev 2001;11:35–40.[CrossRef][Medline]
  5. Ribatti D. The crucial role of vascular permeability factor/vascular endothelial growth factor in angiogenesis: a historical review. Br J Haematol 2005;128:303–9.[CrossRef][Medline]
  6. Kraling BM, Wiederschain DG, Boehm T, et al. The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro. J Cell Sci 1999;112:1599–609.[Abstract]
  7. Oh J, Takahashi R, Kondo S, et al. The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis. Cell 2001;107:789–800.[CrossRef][Medline]
  8. Sato Y, Abe M, Tanaka K, et al. Signal transduction and transcriptional regulation of angiogenesis. Adv Exp Med Biol 2000;476:109–15.[Medline]
  9. Hemler ME. Specific tetraspanin functions. J Cell Biol 2001;155:1103–7.[Abstract/Free Full Text]
  10. Levy S, Shoham T. The tetraspanin web modulates immune-signalling complexes. Nat Rev Immunol 2005;5:136–48.[CrossRef][Medline]
  11. Yunta M, Lazo PA. Tetraspanin proteins as organisers of membrane microdomains and signalling complexes. Cell Signal 2003;15:559–64.[CrossRef][Medline]
  12. Berditchevski F. Complexes of tetraspanins with integrins: more than meets the eye. J Cell Sci 2001;114:4143–51.[Abstract/Free Full Text]
  13. Claas C, Seiter S, Claas A, Savelyeva L, Schwab M, Zöller M. Association between the rat homologue of CO-029, a metastasis-associated tetraspanin molecule and consumption coagulopathy. J Cell Biol 1998;141:267–80.[Abstract/Free Full Text]
  14. Claas C, Wahl J, Orlicky DJ, et al. The tetraspanin D6.1A and its molecular partners on rat carcinoma cells. Biochem J 2005;389:99–110.[CrossRef][Medline]
  15. Herlevsen M, Schmidt DS, Miyazaki K, Zöller M. The association of the tetraspanin D6.1A with the {alpha}6ß4 integrin supports cell motility and liver metastasis formation. J Cell Sci 2003;116:4373–90.[Abstract/Free Full Text]
  16. Hemler ME. Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu Rev Cell Dev Biol 2003;19:397–422.[CrossRef][Medline]
  17. Tarrant JM, Robb L, van Spriel AB, Wright MD. Tetraspanins: molecular organisers of the leukocyte surface. Trends Immunol 2003;24:610–7.[CrossRef][Medline]
  18. Wright MD, Moseley GW, van Spriel AB. Tetraspanin microdomains in immune cell signalling and malignant disease. Tissue Antigens 2004;64:533–42.[CrossRef][Medline]
  19. Boucheix C, Rubinstein E. Tetraspanins. Cell Mol Life Sci 2001;58:1189–205.[CrossRef][Medline]
  20. Funakoshi T, Tachibana I, Hoshida Y, et al. Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer. Oncogene 2003;22:674–87.[CrossRef][Medline]
  21. Furuya M, Kato H, Nishimura N, et al. Down-regulation of CD9 in human ovarian carcinoma cell might contribute to peritoneal dissemination: morphologic alteration and reduced expression of ß1 integrin subsets. Cancer Res 2005;65:2617–25.[Abstract/Free Full Text]
  22. Zhou B, Liu L, Reddivari M, Zhang XA. The palmitoylation of metastasis suppressor KAI1/CD82 is important for its motility- and invasiveness-inhibitory activity. Cancer Res 2004;64:7455–63.[Abstract/Free Full Text]
  23. Gesierich S, Paret C, Hildebrand D, et al. Colocalization of the tetraspanins, CO-029 and CD151, with integrins in human pancreatic adenocarcinoma: impact on cell motility. Clin Cancer Res 2005;11:2840–52.[Abstract/Free Full Text]
  24. Kanetaka K, Sakamoto M, Yamamoto M, Takamura M, Kanematsu T, Hirohashi S. Possible involvement of tetraspanin CO-029 in hematogenous intrahepatic metastasis of liver cancer cells. J Gastroenterol Hepatol 2003;18:1309–14.[CrossRef][Medline]
  25. Sela BA, Steplewski Z, Koprowski H. Colon carcinoma-associated glycoproteins recognized by monoclonal antibodies CO-029 and GA22-2. Hybridoma 1989;8:481–91.[Medline]
  26. Tokuhara T, Hasegawa H, Hattori N, et al. Clinical significance of CD151 gene expression in non-small cell lung cancer. Clin Cancer Res 2001;7:4109–14.[Abstract/Free Full Text]
  27. Yunta M, Lazo PA. Apoptosis protection and survival signal by the CD53 tetraspanin antigen. Oncogene 2003;22:1219–24.[CrossRef][Medline]
  28. Zhang XA, Kazarov AR, Yang X, Bontrager AL, Stipp CS, Hemler ME. Function of the tetraspanin CD151-6ß1 integrin complex during cellular morphogenesis. Mol Biol Cell 2002;13:1–11.[Abstract/Free Full Text]
  29. Longo N, Yanez-Mo M, Mittelbrunn M, et al. Regulatory role of tetraspanin CD9 in tumor-endothelial cell interaction during transendothelial invasion of melanoma cells. Blood 2001;98:3717–26.[Abstract/Free Full Text]
  30. De Cicco M. The prothrombotic state in cancer: pathogenic mechanisms. Crit Rev Oncol Hematol 2004;50:187–96.[Medline]
  31. Moseley GW. Tetraspanin-Fc receptor interactions. Platelets 2005;16:3–12.[CrossRef][Medline]
  32. Lau LM, Wee JL, Wright MD, et al. The tetraspanin superfamily member CD151 regulates outside-in integrin {alpha}IIbß3 signaling and platelet function. Blood 2004;104:2368–75.[Abstract/Free Full Text]
  33. Matzku S, Komitowski D, Mildenberger M, Zöller M. Characterization of BSp73, a spontaneous rat tumor and its in vivo selected variants showing different metastasizing capacities. Invasion Metastasis 1983;3:109–30.[Medline]
  34. Matzku S, Wenzel A, Liu S, Zöller M. Antigenic differences between metastatic and nonmetastatic BSp73 rat tumor variants characterized by monoclonal antibodies. Cancer Res 1989;49:1294–9.[Abstract/Free Full Text]
  35. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94:2493–503.[Medline]
  36. Ryschich E, Schmidt J, Loeffler T, et al. Different radiogenic effects on microcirculation in healthy pancreas and in pancreatic carcinoma of the rat. Ann Surg 2003;237:515–21.[CrossRef][Medline]
  37. Zhang XA, Bontrager AL, Hemler ME. Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific ß(1) integrins. J Biol Chem 2001;276:25005–13.[Abstract/Free Full Text]
  38. Claas C, Herrmann K, Matzku S, Möller P, Zöller M. Developmentally regulated expression of metastasis-associated antigens in the rat. Cell Growth Differ 1996;7:663–78.[Abstract]
  39. Folkman J. Endogenous angiogenesis inhibitors. APMIS 2004;12:496–507.
  40. He B, Liu L, Cook GA, Grgurevich S, Jennings LK, Zhang XA. Tetraspanin CD82 attenuates cellular morphogenesis through down-regulating integrin {alpha}6-mediated cell adhesion. J Biol Chem 2005;280:3346–54.[Abstract/Free Full Text]
  41. Bartosch B, Vitelli A, Granier C, et al. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem 2003;278:41624–30.[Abstract/Free Full Text]
  42. Mantegazza AR, Barrio MM, Moutel S, et al. CD63 tetraspanin slows down cell migration and translocates to the endosomal-lysosomal-MIICs route after extracellular stimuli in human immature dendritic cells. Blood 2004;104:1183–90.[Abstract/Free Full Text]
  43. Little KD, Hemler ME, Stipp CS. Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells: central role of CD81 in facilitating GPR56-G{alpha} q/11 association. Mol Biol Cell 2004;15:2375–87.[Abstract/Free Full Text]
  44. Orlicky DJ, Nordeen SK. Cloning, sequencing and proposed structure for a prostaglandin F2 {alpha} receptor regulatory protein. Prostaglandins Leukot Essent Fatty Acids 1996;55:261–8.[CrossRef][Medline]
  45. Clayton A, Turkes A, Dewitt S, Steadman R, Mason MD, Hallett MB. Adhesion and signaling by B cell-derived exosomes: the role of integrins. FASEB J 2004;18:977–9.[Abstract/Free Full Text]
  46. Ito H, Seyama Y, Kubota S. Calreticulin is directly involved in anti-{alpha}3 integrin antibody-mediated secretion and activation of matrix metalloprotease-2. Biochem Biophys Res Commun 2001;283:297–302.[CrossRef][Medline]
  47. Garcia E, Pion M, Pelchen-Matthews A, et al. HIV-1 trafficking to the dendritic cell-T-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse. Traffic 2005;6:488–501.[CrossRef][Medline]
  48. Schwander M, Leu M, Stumm M, et al. ß1 integrins regulate myoblast fusion and sarcomere assembly. Dev Cell 2003;4:673–85.[CrossRef][Medline]
  49. Zhu GZ, Miller BJ, Boucheix C, et al. Residues SFQ (173-175) in the large extracellular loop of CD9 are required for gamete fusion. Development 2002;129:1995–2002.[Medline]



This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
J. L. Johnson, N. Winterwood, K. A. DeMali, and C. S. Stipp
Tetraspanin CD151 regulates RhoA activation and the dynamic stability of carcinoma cell-cell contacts
J. Cell Sci., July 1, 2009; 122(13): 2263 - 2273.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
R. Sadej, H. Romanska, G. Baldwin, K. Gkirtzimanaki, V. Novitskaya, A. D. Filer, Z. Krcova, R. Kusinska, J. Ehrmann, C. D. Buckley, et al.
CD151 Regulates Tumorigenesis by Modulating the Communication between Tumor Cells and Endothelium
Mol. Cancer Res., June 1, 2009; 7(6): 787 - 798.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K. Al-Nedawi, B. Meehan, R. S. Kerbel, A. C. Allison, and J. Rak
Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR
PNAS, March 10, 2009; 106(10): 3794 - 3799.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gesierich, S.
Right arrow Articles by Zöller, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gesierich, S.
Right arrow Articles by Zöller, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online