Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
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 ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (25)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Gutierrez, L. S.
Right arrow Articles by Castellino, F. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gutierrez, L. S.
Right arrow Articles by Castellino, F. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Carcinogenesis, Vol. 24, No. 2, 199-207, February 2003
© 2003 Oxford University Press

CANCER BIOLOGY

Thrombospondin 1—a regulator of adenoma growth and carcinoma progression in the APCMin/+ mouse model

Linda S. Gutierrez1, Mark Suckow1, Jack Lawler2, Victoria A. Ploplis1 and Francis J. Castellino1,3

1 Walther Cancer Research Center, W. M. Keck Center for Transgene Research, Department of Chemistry and Biochemistry, Freimann Life Sciences Center University of Notre Dame, Notre Dame, IN 46556, and
2 Department of Pathology, Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA 02215, USA


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thrombospondin 1 (TSP-1) is a multifunctional extracellular matrix protein that is an endogenous regulator of tumor angiogenesis. The effects of TSP-1 on adenoma formation and development into cancerous lesions has been evaluated in the Min/+ (multiple intestinal neoplasia) mouse model. These mice develop multiple adenomas in the small intestine due to a mutation in the homologous APC (adenomatous polyposis coli) gene. As in its human counterpart, these adenomas may progress to carcinomas. Intestines of APCMin/+ mice were dissected and histologic evaluation of adenomas was then conducted. Significant increases in vascularization and proliferation were observed in adenomatous, as compared with normal, mucosa. TSP-1 immunostaining revealed significant decreases in the number and intensity of positive cells in adenomas, as compared with normal mucosa. TSP-1 scores were inversely correlated with vascularity and proliferation rate. Cross breeding of mice homozygous for a deletion of the TSP-1 gene (TSP-1-/-) with mice heterozygous for the APC gene mutation (APCMin/+), resulted in animals that showed a significant increase in adenoma number and diameter. Also, histopathological examination of these adenomas showed accelerated dysplasic changes, carcinoma in situ and early invasion, compared with their APCMin/+ littermates. Moreover, a significant decrease of TUNEL-positive cells was observed in intestinal adenomas of TSP-1-/-/APCMin/+ mice. This study reports the first in vivo impact of TSP-1 during early stages of tumor initiation and development in an intestinal carcinogenesis model and demonstrates that TSP-1 affects both angiogenesis and tumor cell apoptosis.

Abbreviations: APC, adenomatous polyposis coli; H&E, hematoxylin–eosin; Min, multiple intestinal neoplasia; TSP-1, thrombospondin 1; vWF, von Willebrand Factor; WT, wild-type.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Recognition of the role of angiogenic regulators during neoplastic development is important for a more complete understanding of the mechanisms involved in tumor growth and metastasis. One class of such regulators is represented by the thrombospondins. Thrombospondin (TSP)-1 and TSP-2 are large multifunctional structurally similar proteins secreted by activated platelets and other types of cells (1,2). TSP-3, TSP-4 and cartilage oligomeric protein belong to a separate TSP subgroup and possess structural homologies with TSP-1 and TSP-2 in their C-terminal segments.

While structurally homologous, the spatial and temporal expression of the mRNAs for TSP-1 and TSP-2, as well as the types of proteins with which they interact, suggest that both similarities and differences exist in their functions (3–7). Accordingly, TSP-1 and TSP-2 are believed to possess important functions in embryonic development, cell growth and proliferation, tissue remodeling, wound healing, inflammation and fibrinolysis (1,8–12). TSP-1 and TSP-2 are also involved in regulation of angiogenesis, as well as tumor progression and metastasis, perhaps due to the inverse correlation between TSP-1 and TSP-2 mRNA levels and bFGF expression (13); to the up regulation of TSP-1 expression by the product of the wild-type (WT) tumor suppressor gene, p53 (14); to their inhibition of endothelial cell proliferation (15); and/or to their induction of receptor-mediated apoptosis in activated microvascular endothelial cells (16). Despite this, the roles of TSP-1 and TSP-2 in neoplasia remain controversial. On one hand, higher levels of TSP-1 have been detected in plasmas of patients with a variety of cancers (17), including breast cancer (18). Additionally, in a murine metastasis model, TSP has been shown to promote metastasis of fibrosarcoma tumor cells to the lung (19). However, other in vitro and in vivo studies have demonstrated inhibitory functions for TSP-1 and TSP-2 (20–27), as well as the TSP-1 receptor (28), in vessel formation, tumor angiogenesis and metastatic potential; properties that should attenuate progression of the tumor. In addition, it has been found that TSP-1 expression is reduced in carcinomas of the colon (29), breast (30) and bladder (31). Moreover, evidence has been presented indicating that activation of the proangiogenic factor, VEGF (vascular endothelial growth factor), occurs in the pre-malignant phase of colorectal tumor development (25), and the switch to an angiogenic phenotype may in part be the result of down regulation of inhibitors, such as TSP-1 (27).

In the current investigation, the relationships between TSP-1 expression and colorectal tumor progression have been assessed using the APCMin/+ mouse model (a mouse heterozygous for a chain-termination mutation in the 15th exon of the APC gene) that develop multiple intestinal adenomas that clinically mimic those observed in patients with familial adenomatous polyposis (32). These adenomas undergo early transformation into colon carcinomas. The effects of TSP-1 on adenoma growth and progression to carcinomas were also examined by cross breeding mice with a genetic total deficiency of TSP-1 (TSP-1-/-) with APCMin/+ mice. The results are summarized in the current communication.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Mice and tissue preparation
APCMin/+ male mice were purchased from the Jackson Laboratory (Bar Harbor, ME). TSP-1-/- mice were generated as described (33). Mice were maintained on an 11% fat diet, as were their littermate controls. After 90 or 120 days, the animals were killed using CO2 asphyxiation. Immediately thereafter, the whole intestine was exposed and rinsed with a buffer consisting of 10 mM phosphate, 0.15 M NaCl, pH 7.5 (PBS). The tissue was fixed with 6% formalin in PBS for 6 h, then embedded in paraffin. Sections (3 µm) were stained with H&E (hematoxylin–eosin) and also subjected to immunohistochemical analysis (4 µm).

Adenoma scoring
Small intestinal adenomas and colonic adenomas were scored for numbers and diameters at postnatal days 75 or 150 for the cross between APCMin/+ (n = 13) and APCMin/+/TSP-1-/- (n = 7), respectively. The small intestines and colons were dissected free of mesentery and opened along the longitudinal axis. Intestinal contents were cleared with PBS. The intestines were fixed in 6% formalin/PBS for 6 h, followed by 70% ethanol. Using a dissecting microscope (X10-30) and digital calipers, adenoma numbers and diameters were obtained for the entire length of the small intestine and colon. Adenoma analyses were performed without any knowledge of genotype by a pathologist (M.S.) and confirmed independently by another (L.G.)

Histology and immunohistochemistry
Colon samples and small intestine segments were paraffin-embedded and sectioned. Stained sections were viewed without knowledge of genotypes and evaluated by an independent pathologist (Dr Luis Galup, South Bend Medical Foundation, South Bend, IN). All adenomas were checked by H&E and changes such as carcinoma in situ and stromal invasion were evaluated. Adenomas were characterized by expansion of the mucosa layer, reduction in goblet cell number, moderate loss of mucosal architecture by glandular growth and dilated cysts. Only non-lymphoid adenomas were evaluated. Adenomas with >50% of high-grade dysplasia (severe distortion of the glandular architecture and prominent cellular atypia) were considered carcinomas in situ. However, only the lesions showing invasion through the muscularis mucosa were identified as carcinomas. Sections for immunohistochemistry were cleared with xylene and rehydrated by ethanol. Endogenous peroxidase was blocked with peroxyblock (Zymed, San Francisco, CA). Sections designated for TSP-1 and vWF (von Willebrand Factor) immunostaining were treated with 0.2% trypsin/PBS at 37°C for 10 min. Slides for PCNA (proliferant cell nuclear antigen) were boiled in 0.01 M citrate buffer solution, pH 6, for 10 min. The sections were incubated overnight at room temperature with monoclonal antibodies against TSP (Ab4, NeoMarkers, Fremont, CA), PCNA (Biogenex, San Ramon, CA) and VEGF (Oncogene Research Products, Boston, MA). The samples were then incubated with biotinylated rabbit anti-mouse antibody (Vector, Burlingame, CA) for 30 min. After incubation with peroxidase (ABC Kit), the stains were visualized with the chromogen, DAB (3',3-diaminobenzidine; Pierce, Rockford, IL) or AEC (3-amino-9-ethyl carbazole; Vector). For vWF immunohistochemistry, the DAB step was performed immediately after incubation with horseradish peroxidasecomplexed anti-vWF antibody (DAKO EPOS, Carpinteria, CA).

TUNEL
The TUNEL assay was performed according to a published method (34). Tissue sections were incubated with 5 µg/ml of proteinase K for 15 min at room temperature in order to digest proteins. Sections were then covered with a buffer containing 30 mM Tris–HCl, pH 7.2/140 mM sodium cacodylate/1 mM CoCl2. An aliquot of 0.2 µl of terminal deoxynucleotidyl-transferase (Boehringer Mannheim, Gaithersburg, MD) and 10 µM biotinylated dUTP (Boerhringer Mannheim) were added to the sections. The slides were incubated in a humidified chamber at 37°C for 60 min, washed with 50 mM Tris–HCl, pH 7, and finally with PBS. The sections were then incubated for 30 min with the ABC system and DAB was used as the chromogen.

Data analyses
TSP-1 and VEGF stainings were scored in a semiquantitative fashion, incorporating both the intensity and the distribution of specific staining. The evaluations were recorded as the percentage of positive stained cells in each of four categories: 0 (no staining), 1+ (weak), 2+ (distinct) and 3+ (intense). 100 cells were counted in each evaluated field at 200x magnification (16 fields of adenomas, 26 fields of normal areas from APCMin/+ mice and 4 different fields from WT littermates). A HScore value was obtained summing the percentages of cells staining at each category. The PCNA indices were obtained by counting 500 epithelial cells in adenomas and normal mucosa and recording the percentage of positive brown nuclei. MVC (microvessel counts) was obtained by counting adenoma vessels in five different fields and vessels in adjacent normal mucosa separately (5 fields) at 200x magnification. The apoptotic index was obtained by dividing the number of apoptotic cells by the total number of cells counted within a field and then multiplying the results by 1000.

Statistical methods
Non-parametric ANOVA and Fisher‘s tests were used. P values <0.05 were considered significant. The correlations between Hscore of TSP-1, MVC and PCNA indexes were calculated using the Fisher t-test for paired comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Immunohistochemistry of lesions in the APCMin/+ mouse
Normal intestinal mucosa from colon and small intestine revealed strong staining for TSP-1. This stain was diffuse and present mainly in the lamina propria (Figure 1A and BGo). Endothelial cells, fibroblasts and granulocytes showed strong cytoplasmic and membranous immunoreactivity. Staining was also observed in the submucosa and in large vessels of the intestinal wall, as well as in platelets. Positive staining was rarely observed in epithelial cells and, when present, was weak and scattered. This staining pattern also has been described in other types of carcinomas, e.g. squamous cell (30) and breast (35) carcinomas. In general, TSP-1 immunostaining pattern in non-polypoid areas of APCMin/+ intestines (Figure 1A and BGo) and WT intestines was similar. However, segments of APCMin/+ intestine occasionally showed a progressive decrease in staining (Figure 1CGo, arrows), while fully developed adenomas were almost negative for TSP-1. Only scattered epithelial cells were weakly positive (Figure 1DGo). HScores were obtained evaluating the number of positive cells and staining intensity in intestines from WT mice as well as non-polypoid (normal intestine) and adenomas from APCMin/+. No significant differences in TSP-1 expression were observed between intestines from WT mice and non-polypoid areas of intestines from APCMin/+. However, adenomas demonstrated significantly lower Hscore indices compared with WT and normal mucosa from APCMin/+ (Figure 2AGo). The TSP-1 null intestine was employed as a control for the specificity of the antibody (data not shown). In this case, TSP immunohistochemistry showed a complete lack of immunostain in the stroma, endothelial cells and platelets. Only plasmocytes in the lamina propria and plasma in some vessels of the submucosa displayed weak staining. While some mild cross reactivity with TSP-2 has been reported for this antibody by the manufacturer, our immunostaining of intestines of TSP-1-/- mice showed the weak staining pattern outlined above. However, the typical TSP-1 staining in the stroma, macrophages, fibroblasts, platelets and endothelial cells of WT mice was absent in TSP-1-/- mice, suggesting that the antibody was suitable for the purposes of the experiments within this report.



View larger version (102K):
[in this window]
[in a new window]
  		Fig. 1. Immunohistochemical analysis of normal and adenomatous mouse intestine from mice. TSP-1 expression in normal intestine (A and B). Staining for TSP-1 was always present in the lamina propria (LP) and submucosa (SM). Scattered epithelial cells (EC) of the surface were weakly positive. (A) TSP-1 is expressed in some lymphocytic populations in a lymph nodule (LN; 100x original magnification). (B) Fibroblasts and endothelial cells of large vessels in the submucosa were strongly positive, as well as platelets (P) (200x). (C and D) TSP-1 expression in colonic mucosa. A selected area of the APCMin/+ intestine, demonstrated a progressive decrease in TSP-1 immunoreactivity (arrows) (C; 100x) along with a well-developed adenoma (D, 100x) showing very low expression of TSP-1. vWF staining of adenomatous (E; 200x) and normal (F; 200x) intestine show numerous capillaries, mainly at the top (arrows) of the adenomas (E). The vascularity is increased compared with normal intestine (200x). (G) VEGF immunostain of normal mouse intestine (200x) shows that VEGF is expressed mainly in epithelial cells of the crypts (arrows) and becomes weaker through the tips of the villi. (H) VEGF immunostain of an adenoma (200x). Staining is stronger in the adenoma, mainly at the adenomatous areas at the top and luminal surface epithelium (arrows). Staining is localized at the luminal borders of the epithelial cells and adenomatous (H) mouse intestine shows that VEGF levels are significant in both sections. This down regulation of TSP-1 in the mature adenoma (D) coupled with the presence of VEGF favors the neovessel formation observed in (F). In normal tissue, the higher levels of TSP-1 would inhibit the angiogenic tendency of the VEGF observed in (G), resulting in a lack of neovessel formation in (E).

 


View larger version (11K):
[in this window]
[in a new window]
  		Fig. 2. TSP-1 intensity scores (A). Bar chart comparing the TSP-1 HScore in control mucosa of WT, and adenoma and adjacent normal intestinal mucosa of APCMin/+ mice. Adenoma tissues showed significantly lower TSP-1 expression. P = 0.0089 for adenoma versus WT; P = 0.0035 for adenoma versus normal. Comparison of TSP-1 Hscore and vessel counts (B). An inverse correlation was observed between TSP-1 immuno-intensity and vascularity: P < 0.0001. Correlation of TSP-1 HScore on intestinal tissues and PCNA indices (C). TSP-1 HScores correlated inversely with cell proliferation indices. Areas with intense cell proliferation displayed a decrease in TSP-1 expression: P = 0.0015.

 
vWF immunostaining revealed an increased number of vessels in the polyp areas (Figure 1EGo), compared with normal intestine (Figure 1FGo). These vessels were very small and round, with enlarged endothelial cells. Capillaries were observed mainly at the tops of the adenomas. An inverse relationship was clearly observed when vascularity and proliferation were correlated with the TSP-1 score of the intestinal tissues. Here, MVC were significantly higher in areas with decreased TSP-1 immunostains (Figure 2BGo). In addition, the polyps showed higher PCNA indices (Figure 2CGo). Moreover, when TSP-1 scores were compared with PCNA indices, areas of higher proliferation rate showed lower TSP-1 scores. These results demonstrate that TSP-1 is expressed in normal intestinal tissue, and may help to maintain equilibrium in the normal vasculature. The differential expression between normal and adenomatous mucosa may indicate that TSP-1 is an important regulator in early stages of intestinal carcinogenesis. When TSP-1 is down regulated, a more favored environment for intestinal angiogenesis and proliferation is established that may contribute to initiation of the adenoma-carcinoma sequence. In this regard, this work, as well as other studies (20–27), has demonstrated an inverse correlation between TSP-1 expression and tumor vascularity and survival. There is evidence as well of increased vascularity and angiogenic factors, such as VEGF, in human adenomas (36,37).

VEGF immunostaining of normal (Figure 1GGo) and adenomatous (Figure 1HGo) mouse intestine shows that VEGF is clearly present in both samples, but demonstrates a somewhat different expression pattern with variability between regions of the intestine. VEGF is expressed mainly in the proliferative zones of the crypts in normal intestine (Figure 1GGo). Immunoreactivity of the glandular epithelium decreases gradually from the bases of the crypts to the luminal surfaces. In adenomas (Figure 1HGo), the strongest cytoplasmic staining was present in the epithelial cells of the mucosal surface. The increase in VEGF intensity at the tops of the adenomas correlate with higher areas of vascularity.

The data obtained suggest that the down regulation of TSP-1 in the mature adenoma (Figure 1DGo), coupled with the presence of VEGF (Figure 1HGo), shifts the balance toward neovascularization that would be stimulated by VEGF. These observations offer an explanation of the vascularization seen in the adenoma in Figure 1EGo. However, in normal tissue, the presence of TSP-1 (Figure 1A and BGo) would inhibit the angiogenic tendency of the VEGF observed in Figure 1GGo, thus producing the lack of neovessels in Figure 1FGo. Thus, a regulatory switch in TSP-1 levels may play a role in angiogenic changes of normal and adenomatous tissue leading to carcinogenesis of intestinal polyps.

General features of APC Min/+/TSP-/- mice
Because of the relationships suggested above between TSP-1 levels and angiogenesis, TSP-1-deficient mice were crossed with mice to generate APCMin/+/TSP-1-/- mice in order to more directly assess the role of TSP-1 in this genetic model of colonic carcinogenesis. The APCMin/+/TSP-1-/- mice showed the characteristic lordotic spine curvature observed in TSP-1-/- mice (33). They rapidly developed anemia (extremely pale skin and internal organs), bloody feces and weight loss. They expired earlier than their controls (one mouse died from severe anemia before 30 days and one died before 90 days). For this reason, most of the APCMin/+/TSP-1-/- mice were killed before 90 days after birth. No mammary, skin or stomach tumors were observed.

Adenoma counts and diameters in APCMin/+ and APCMin/+/TSP-1-/- mice
Mean adenoma counting revealed a significant increase (P = 0.037) in adenoma number in APCMin/+/TSP-1-/- mice (39 adenomas, n = 7), compared with APCMin/+ control littermates (25 adenomas, n = 13) (Figure 3A–CGo). Whereas the differences in the adenoma diameters were not statistically significant (P = 0.54), adenomas in APCMin/+/TSP-1-/- mice tended to be larger (3.6 mm) compared with their controls (2.1 mm).



View larger version (74K):
[in this window]
[in a new window]
  		Fig. 3. Gross features and adenoma scoring in APCMin/+ and APCMin/+/TSP-1-/- mice. (Top) APCMin/+ mice (A) usually showed fewer adenomas (*) compared with APCMin/+/TSP-1-/- littermates (B). Most of the APCMin/+/TSP-1-/- adenomas were umbilicated, showing central necrosis. The intestinal wall was engrossed and proliferation into the surrounding mucosa was observed. (Bottom) APCMin/+/TSP-1-/- mice showed a significantly higher number of adenomas than APCMin/+ mice. Scoring was performed without knowledge of the genotypes. P = 0.037, n = number of mice per genotype.

 
Histopathological evaluation of colonic adenomas from APCMin/+/TSP-1-/- mice
Each tissue section stained with H&E was evaluated. Features, such as dysplasic changes, carcinoma in situ and stromal invasion were evaluated in each adenoma (Figure 4Go). Intestinal tumors in APCMin/+/TSP-1-/- and APCMin/+ mice shared many similar properties. Both showed adenomas with different grades of inflammation (especially eosinophilic), dysplasia and carcinoma in situ. However, after evaluation of each lesion, histologic features, such as carcinoma in situ and invasion were a more typical pattern in the APCMin/+/TSP-1-/- mice. While most of the lesions were exophytic in the APCMin/+ mice (Figure 4A and DGo), most of the tumors in the APCMin/+/TSP-1-/- mice appeared as complete intramucosal carcinomas, showing central necrosis (Figure 4B and EGo), intense leukocytic infiltrate, and complete loss of glandular and nuclear polarity (Figure 4B, C, E and FGo). Carcinoma in situ was present in almost all the lesions, even those that were smaller (Figure 4CGo). Also, thickening of the walls, glandular invasion of the muscularis and venous embolism were more frequently seen in APCMin/+/TSP-1-/- mice (Figure 4FGo). TSP-/- littermate intestines were also evaluated. Intestinal mucosa of these mice showed some evidence of greater epithelial proliferation, increased mucosal depth growth and villi diameters (data not shown).



View larger version (105K):
[in this window]
[in a new window]
  		Fig. 4. Histological evaluation of adenomas in APCMin/+ and APCMin/+/TSP-1-/- mice. (Top) Each adenoma from the corresponding H&E stain section was evaluated. Most of the adenomas were exophytic lesions, mainly in the adenomas (A, 200x) and carcinomas (D, 100x) that developed in APCMin/+ mice. APCMin/+/TSP-1-/- mice showed more dysplasic areas and carcinoma in situ than their controls (Table 1) even in small lesions (B and C, 200x and 100x, respectively). Central necrosis (arrows in B and E, 100x), stromal invasion, and venous embolisms (arrow in F, 200x and magnified insert, 400x) were more frequently seen in lesions of APCMin/+/TSP-1-/- mice. (Bottom) Summary of features evaluated in H&E stained sections. n = number of animals; A, total number of adenomas evaluated by histology; a, number of adenomas with the indicated feature.

 
Vascularity in adenomas of APCMin/+/TSP-1-/- mice
As TSP-1 is an anti-angiogenic agent, a higher number of vessels in mice lacking TSP-1 was expected. No significant differences were observed between APCMin/+ (vascularity in adenomas, 22.3; vascularity in carcinomas, 29.4) and APCMin/+/TSP-1-/- (vascularity in adenomas, 21.1; vascularity in carcinomas, 27.5) mice. As it has been reported, an increase in angiogenesis was observed in the transition of normal-adenoma to carcinoma in both genotypes, but the vessel counts were similar between both groups (data not shown).

Apoptotic indices
TSP-1 induces apoptosis in endothelial cells and is up regulated by p53 (29). As p53 is a major regulator of apoptosis, we examined in situ apoptosis in APCMin/+/TSP-1-/- mice using the TUNEL assay. Apoptotic indices were significantly lower in adenomas and carcinoma of APCMin/+/TSP-1-/- mice (adenomas, 4.5; carcinomas, 1.0) compared with the lesions of APCMin/+ mice (adenoma, 8.8; carcinomas, 3) (Figure 5Go). Adenomas of the APCMin/+/TSP-1-/- mice showed apoptotic indices very close to those found in the carcinomas of APCMin/+ (Figure 5Go), demonstrating that most of the tumors, even in early lesions in APCMin/+/TSP-1-/- mice are indeed carcinomas.



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5. Apoptosis in APCMin/+ and APCMin/+/TSP-1-/- mice. Apoptotic indices were higher in adenomas of APCMin/+ and lower in tumors of APCMin/+/TSP-1-/- mice. P = 0.0006 for APCMin/+ adenoma versus APCMin/+ carcinoma; P = 0.021 for APCMin/+ adenoma versus APCMin/+/TSP-1-/- adenoma; P < 0.0001 for APCMin/+ adenoma versus APCMin/+/TSP-1-/- carcinoma.

 
PCNA immunohistochemistry
All the tumors from both genotypes showed increase of PCNA labeled cells compared with normal adjacent intestinal mucosa. Most of the adenomas of APCMin/+ mice displayed fewer PCNA-positive cells than carcinomas and lesions of the APCMin/+/TSP-1-/- mice. Positive cells were located mainly in glandular foci and showed fewer positive cells in the upper epithelium (Figure 6AGo). In contrast, carcinomas of APCMin/+ and all lesions of APCMin/+/TSP-1-/- mice, showed higher numbers of positive PCNA cells (Figure 6B–DGo). PCNA-positive cells were found to be more diffuse in the upper epithelium and the entire glandular component. Adenomas in APCMin/+ mice displayed significantly lower PCNA indices as compared with carcinomas and lesions developed in APCMin/+/TSP-1-/- mice (Figure 6EGo). These results indicate that higher proliferation and accelerated carcinogenesis are typical features in the lesions which arose in APCMin/+/TSP-1-/- mice, and even smaller adenomas showed elevated PCNA indices. The latter may be the result of the behavior of malignant cell clones, which eventually will display a more aggressive and invasive phenotype.



View larger version (74K):
[in this window]
[in a new window]
  		Fig. 6. Analysis of cell proliferation in adenomas and carcinomas of APCMin/+/TSP-1-/- and APCMin/+ mice. Diminished PCNA immunohistochemistry was observed in adenomas of APCMin/+ mice (A). No differences in proliferation were observed between the carcinomas that arose in APCMin/+ mice (C) and any tumors that originated in the APCMin/+/TSP-1-/- mice. Adenomas in the APCMin/+/TSP-1-/- mice (B) are highly proliferate tumors and their progression to carcinoma (D) appears to be accelerated. Magnifications, 200x. Graph: scoring of PCNA positive cells showed that all the tumors in the APCMin/+/TSP-1-/- mice had significantly higher PCNA indices when compared with adenomas of APCMin/+ mice. These tumors also showed PCNA indices similar to carcinomas of APCMin/+ mice. P = 0.0001 for APCMin/+ adenomas versus APCMin/+/TSP-1-/- adenomas; P = 0.0006 for APCMin+ adenomas versus APCMin/+ carcinomas; P = 0.0072 for APCMin/+ adenomas versus APCMin/+/TSP-1-/- carcinomas.

 
VEGF immunohistochemistry
VEGF immunostaining was observed in the endothelial cells lining vessels of normal intestine and adenomas. The stain was more intense in the normal colonic mucosa. In contrast to the normal mucosa, adenomas showed a variable intensity, with focal areas of epithelial cells usually strongly positive. The stroma of the lamina propria was positive for VEFG in both groups, and this staining was usually confined to the top area of the adenoma, where vessels and stroma were usually positive. In general, no clear differences in intensity and localization were observed between APCMin/+/TSP-1-/- and APCMin/+ mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The aim of this study was to evaluate the importance of the angiogenic inhibitor TSP-1 in naturally occurring tumors in vivo. As an indicator of the value of this approach, it has been demonstrated that mice with a deficiency of the tumor suppressor gene, p53, showed decreased survival with an additional deficiency of the TSP-1 gene (38). In our case, using an animal model for intestinal adenomatosis, we report the immunolocalization of TSP-1 in normal and pre-malignant intestine. Evidence is presented of a decrease of TSP-1 expression in adenomas and its inverse relationship with more proliferative and vascularized intestine.

On the other hand, no significant differences in vascularization between adenomas and carcinomas in APCMin/+ and all lesions of APCMin/+/TSP-1-/- mice were found. These adenomas, already dysplastic, ultimately become carcinomas, and perhaps no additional vascularization beyond that found in the adenomas is required for the transformation, thus providing a basis for the lack of differences found in adenoma and carcinoma vascularization. These results suggest that TSP-1 may be required in the initial stages of angiogenesis when the vascular supply is low. However, when angiogenesis in the tumor is well established, endogenous TSP-1 has less influence. It is possible that during carcinogenesis, specific clones of cells, less dependent on vessel supply and more resistant to hypoxia, overgrow (39). Therefore, at this stage, vascular supply and angiogenic regulators may play a more secondary role. Also, angiogenesis may be genetically driven (40), and tumor cells at later time points may self-determine their vascular microenvironment. These findings may explain recent reports showing that the efficiency of anti-angiogenic therapy is related to the type, size and location of the tumor (41). Alternatively, TSP-1 is not the only inhibitor of angiogenesis, since other anti-angiogenic factors, e.g. angiostatin and TSP-2, could be produced after the onset of tumor development.

Our results further show TSP-1 to have properties beyond its anti-angiogenic effects. APCMin/+ mice, additionally lacking the TSP-1 gene, were more tumorigenic, indicating that this combination of altered genes leads to a more malignant phenotype. TSP-1 is a complex protein, with the ability to interact with a number of pro-apoptotic and growth factors. Its interactions with these proteins in the tumor microenvironment may regulate apoptosis and proliferation, not only in neovessels, but also in malignant epithelial cells. TSP-1 regulates activation of endogenous proteins, including TGF-ß (40,41), which is involved in multiple pathological events, such as wound healing, proliferation and tumor angiogenesis (42). Also, the loss of tumor suppressor genes may decrease TSP-1 expression and lead to an angiogenic phenotype in tumors (43).

Other studies have indicated that TGF-ß RII expression is diminished in intestinal adenomas of APCMin/+ mice with an associated increase in cyclin D1 and cyclin-dependent kinase 4 (Cdk4) expression, which would facilitate cell proliferation and progression of the disease (44). Additionally, a mutation in the APC gene induces nuclear expression of ß-catenin, which is involved in E-cadherin-mediated cell adhesion and is also a key effector of the prooncogenic Wnt signaling pathway. This latter property has been suggested as a potential cause of development of carcinoma from adenoma in colorectal carcinoma (45). Catenin accumulation induces activation of proliferation-associated genes, cyclin D and c-myc, and the invasion-associated genes, MMP-1 and MMP-7 (46). TSP-1 may be additionally involved in this pathway as it modulates tumor cell adhesion (47) and also induces increased levels of members of the catenin family, e.g. {gamma}-catenin and p120 (Cas), in endothelial cells (48). Of potential importance is the observation that TSP-1 increases the secretion of protease inhibitor PAI-1 (49), which has been reported as a regulator of angiogenesis and tumor growth potentially via its effects on VEGF expression (50). Additionally, TSP-1 is up regulated by the WT product of the P53 gene (51). This induction by p53 may cause tumor cell apoptosis thus directly or indirectly inhibiting tumor angiogenesis.

Numerous reports have shown that high TSP-1 expression in human cancers appears to be a good prognostic factor (52–54). The clinical data, as well as some animal data, emphasize the role of TSP-1 as a protective factor against cancer and a strong modulator of tumorigenesis (38). This has been further reinforced in studies of a spontaneous mammary tumor model (transgenic neu/erbB2 oncogene under control of the mouse mammary tumor virus) with an additional deficiency or overexpression of TSP-1 where it was demonstrated that TSP-1 regulates angiogenesis and tumor burden (55). The current study reports the first in vivo analysis of the impact of TSP-1 in an intestinal carcinogenesis model at early stages of tumor initiation and development. These findings agree with the concept of TSP-1 as a tumor suppressor gene, as its absence enhances tumorigenesis by regulating proliferation and apoptosis, and accelerating the transformation of adenomas to carcinomas. Tumor apoptosis and vascularity are the main outcome predictors in many tumors. These findings are relevant as they highlight the dual properties of TSP-1 as an early anti-angiogenic and pro-apoptotic agent, making it a potential powerful tool against colorectal cancer. Lastly, the presence of early gene mutations, and/or environmental factors, may affect TSP-1 production and secretion. Down regulation of TSP-1 may play an important role in early events of colonic carcinogenesis, and this should be considered in an overall strategy for delaying polyposis in patients with a genetic predisposition to colorectal carcinoma. Thus, this protein may be a valuable marker of malignant transformation.


   Notes
 
3 To whom correspondence should be addressed Email: fcastell{at}nd.edu Back


   Acknowledgments
 
This work was presented in part at the 2nd International Meeting of Thrombospondins and Other Adhesion-Extracellular-Matrix Proteins. Madison, Wisconsin, June 4–8, 2000. The authors thank Ms Stacey Raje and Ms Melanie DeFord for management of veterinary care, Ms Sun Longhua, Valerie Sailes and Mayra Sandoval-Cooper for histologic technical assistance, Ms Juan Fu and Mr Andrew Martin for animal genotyping, and Dr Luis Galup for histologic evaluations. This work was supported in part by NIH grants HL-13423 (to F.J.C.), HL-63682 (to V.A.P.), HL-68003 (to J.L.) and CA-92644 (to J.L.), and also by the Kleiderer-Pezold family endowed professorship (to F.J.C.) and a grant from the W.M. Keck Foundation (to F.J.C.).


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Bornstein,P., Alfi,D., Devarayalu,S., Framson,P. and Li,P. (1990) Characterization of the mouse thrombospondin gene and evaluation of the role of the first intron in human gene expression. J. Biol. Chem., 265, 16691–16698.[Abstract/Free Full Text]
  2. Bornstein,P., O‘Rourke,K., Wikstrom,K., Wolf,F.W., Katz,R., Li,P. and Dixit,V.M. (1991) A second, expressed thrombospondin gene (Thbs2) exists in the mouse genome. J. Biol. Chem.,266, 12821–12824.[Abstract/Free Full Text]
  3. Iruela-Arispe,M.L., Liska,D.J., Sage,E.H. and Bornstein,P. (1993) Differential expression of thrombospondin 1, 2 and 3 during murine development. Dev. Dyn., 197, 40–56.[ISI][Medline]
  4. Chen,H., Sottile,J., O‘Rourke,K.M., Dixit,V.M. and Mosher,D.F. (1994) Properties of recombinant mouse thrombospondin 2 expressed in Spodoptera cells. J. Biol. Chem., 269, 32226–32232.[Abstract/Free Full Text]
  5. Pellerin,S., Lafeuillade,B., Chambaz,E.M. and Feige,J.J. (1994) Distinct effects of thrombospondin-1 and CISP/thrombospondin-2 on adrenocortical cell spreading. Mol. Cell. Endocrinol., 106, 181–186.[CrossRef][ISI][Medline]
  6. Lafeuillade,B., Pellerin,S., Keramidas,M., Danik,M., Chambaz,E.M. and Feige,J.J. (1996) Opposite regulation of thrombospondin-1 and corticotropin-induced secreted protein/thrombospondin-2 expression by adrenocorticotropic hormone in adrenocortical cells. J. Cell. Physiol., 167, 164–172.[CrossRef][ISI][Medline]
  7. Scott-Drew,S. and French-Constant,C. (1997) Expression and function of thrombospondin-1 in myelinating glial cells of the central nervous system. J. Neurosci. Res., 50, 202–214.[CrossRef][ISI][Medline]
  8. Aho,S. and Uitto,J. (1998) Two-hybrid analysis reveals multiple direct interactions for thrombospondin 1. Matrix Biol., 17, 401–412.[CrossRef][ISI][Medline]
  9. Kyriakides,T.R., Zhu,Y.H., Yang,Z. and Bornstein,P. (1998) The distribution of the matricellular protein thrombospondin 2 in tissues of embryonic and adult mice. J. Histochem. Cytochem., 46, 1007–1015.[Abstract/Free Full Text]
  10. Tooney,P.A., Sakai,T., Sakai,K., Aeschlimann,D. and Mosher,D.F. (1998) Restricted localization of thrombospondin-2 protein during mouse embryogenesis: a comparison to thrombospondin-1. Matrix Biol., 17, 131–143.[CrossRef][ISI][Medline]
  11. Kyriakides,T.R., Tam,J.W. and Bornstein,P. (1999) Accelerated wound healing in mice with a disruption of the thrombospondin 2 gene. J. Invest. Dermatol., 113, 782–787.[CrossRef][ISI][Medline]
  12. Vallejo,A.N., Klimiuk,P.A., Weyand,C.M. and Goronzy,J.J. (2000) Central role of thrombospondin-1 in the activation and clonal expansion of cells. J. Immunol., 164, 2947–2954.[Abstract/Free Full Text]
  13. Fontanini,G., Boldrini,L., Calcinai,A., Chine,S., Lucchi,M., Mussi,A., Angeletti,C.A., Basolo,F. and Bevilacqua,G. (1999) Thrombospondins I and II messenger RNA expression in lung carcinoma: relationship with p53 alterations, angiogenic growth factors and vascular density. Clin. Cancer Res., 5, 155–161.[Abstract/Free Full Text]
  14. Dameron,K.M., Volpert,O.V., Tainsky,M.A. and Bouck,N. (1994) Control of angiogenesis in fibroblast by p53 regulation of thrombospondin-1. Science, 265, 1582–1584.[Abstract/Free Full Text]
  15. Panetti,T.S., Chen,H., Misenheimer,T.M., Getzler,S.B. and Mosher,D.F. (1997) Endothelial cell mitogenesis induced by LPA: inhibition by thrombospondin-1 and thrombospondin-2. J. Lab. Clin. Med., 129, 208–216.[CrossRef][ISI][Medline]
  16. Jimenez,B., Volpert,O.V., Crawford,S.E., Febbraio,M., Silverstein,R.L. and Bouck,N. (2000) Signal leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nature Med., 6, 41.[CrossRef][ISI][Medline]
  17. Tuszynski,G.P., Smith,M., Rothman,V.L., Capuzzi,M.D., Joseph,R.R., Katz,J., Besa,E.C., Treat,J. and Switalska,H.I. (1992) Thrombospondin 1 levels in patients with malignancy. Thromb. Haemost., 67, 607–611.[ISI][Medline]
  18. Clezardin,P., Bruno-Bossio,G., Fontana,A., Serre,C.M., Magnetto,S. and Frappart,L. (1999) Thrombospondins, tumor angiogenesis and breast cancer. Pathol. Biol., 47, 368–374.[Medline]
  19. Tuszynski,G.P., Gasic,T.B., Rothman,V.L., Knudsen,K.A. and Gasic,G.J. (1987) Thrombospondin, a potentiator of tumor cell metastasis. Cancer Res., 147, 4130–4133.
  20. Volpert,O.V., Tolsma,S.S., Pellerin,S., Feige,J.J., Chen,H., Mosher,D.F. and Bouck,N. (1995) Inhibition of angiogenesis by thrombospondin-2. Biochem. Biophys. Res. Commun., 217, 326–332.[CrossRef][ISI][Medline]
  21. Tolsma,S., Stack,M.S. and Bouck,N. (1997) Lumen formation and other angiogenic activities of cultured capillary endothelial cells are inhibited by thrombospondin-1. Microvascular Res., 54, 13–26.[CrossRef][ISI][Medline]
  22. Oshika,Y., Masuda,K., Tokunaga,T. et al. (1998) Thrombospondin 2 gene expression is correlated with decreased vascularity in non-small cell lung cancer. Clin. Cancer Res., 4, 1785–1788.[Abstract]
  23. Kyriakides,T.R., Leach,K.J., Hoffman,A.S., Ratner,B.D. and Bornstein,P. (1999) Mice that lack the angiogenesis inhibitor, thrombospondin 2, mount an altered foreign body reaction characterized by increased vascularity. Proc. Natl Acad. Sci. USA, 96, 4449–4454.[Abstract/Free Full Text]
  24. Streit,M., Velasco,P., Brown,L.F., Skobe,M., Richard,L., Riccardi,L., Lawler,J. and Detmar,M. (1999) Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas. Am. J. Pathol., 155, 441–452.[Abstract/Free Full Text]
  25. Streit,M., Riccardi,L., Velasco,P., Brown,L.F., Hawighorst,T., Bornstein,P. and Detmar,M. (1999) Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis. Proc. Natl Acad. Sci. USA, 96, 14888–14893.[Abstract/Free Full Text]
  26. Tokunaga,T., Nakamura,M., Oshika,Y. et al. (1999) Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer. Br. J. Cancer, 79, 354–359.[CrossRef][ISI][Medline]
  27. Maeda,K., Nishiguchi,Y., Yashiro,M., Yamada,S., Onoda,N., Sawada,T., Kang,S. and Hirakawa,K. (2000) Expression of vascular endothelial growth factor and thrombospondin-1 in colorectal carcinoma. Int. J. Mol. Med., 5, 373–378.[ISI][Medline]
  28. Tsuchida,T., Kijima,H., Tokunaga,T. et al. (1999) Expression of the thrombospondin 1 receptor CD36 is correlated with decreased stromal vascularisation in colon cancer. Int. J. Oncol., 14, 47–51.[ISI][Medline]
  29. Tokunaga,T., Nakamura,M., Oshika,Y. et al. (1998) Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 expression in colon cancer cells. Virchows Arch., 433, 415–418.[CrossRef][ISI][Medline]
  30. Bertin,N., Clezardin,P., Kubiak,R. and Frappart,L. (1997) Thrombospondin-1 and -2 messenger RNA expression in normal, benign and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis and fibroblastic desmoplasia. Cancer Res., 57, 396–399.[Abstract/Free Full Text]
  31. Campbell,S.C., Volpert,O.V., Ivanovich,M. and Bouck,N.P. (1998) Molecular mediators of angiogenesis in bladder cancer. Cancer Res.,58, 1298–1304.[Abstract/Free Full Text]
  32. Moser,A.R., Pitot,H.C. and Dove,W.F. (1990) A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science, 247, 322–324.[Abstract/Free Full Text]
  33. Lawler,J., Sunday,M., Thibert,V., Duquette,M., George,E.L., Rayburn,H. and Hynes,R.O. (1998) Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J. Clin. Invest., 101, 982–992.[ISI][Medline]
  34. Gavrieli,Y., Sherman,Y. and Ben-Sasson,S. (1992) Identification of programmed cell death in situ specific labeling of nuclear DNA fragmentation. J. Cell. Biol., 119, 493–501.[Abstract/Free Full Text]
  35. Oshiba,G., Kijima,H., Himeno,S. et al. (1999) Stromal thrombospondin-1 expression is correlated with progression of esophageal squamous cell carcinomas. Anticancer Res., 19, 4375–4378.[ISI][Medline]
  36. Wong,M., Cheung,N., Yuen,S.T., Leung,S.Y. and Vhung,L.P. (1999) Vascular endothelial growth factor is up regulated in the early pre-malignant stage of colorectal tumor progression. Int. J. Cancer, 81, 845–850.[CrossRef][ISI][Medline]
  37. Andre,T., Kotelevets,L., Vaillant,J.C., Coudray,A.M., Weber,L., Prevot,S., Parc,R., Gespach,C. and Chastre,E. (2000) VEGF, VEGF-B, VEGF-C and their receptors KDR, FLT-1 and FLT-4 during the neoplastic progression of human colonic mucosa. Int. J. Cancer, 86, 174–181.[CrossRef][ISI][Medline]
  38. Lawler,J., Miao,W.M., Duquette,M., Bouck,N., Bronson,R.T. and Hynes,R.O. (2001) Thrombospondin-1 gene expression affects survival and tumor spectrum of p53-deficient mice. Am. J. Path., 159, 1949–1956.[Abstract/Free Full Text]
  39. Graeber,T.G., Osmanian,C., Jacks,T., Housman,D.E., Koch,C.J., Lowe,S.W. and Giaccia,A.J. (1996) Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature, 379, 88–92.[CrossRef][Medline]
  40. Murphy-Ullrich,J.E., Schultz-Cherry,S. and Hook,M. (1992) Transforming growth factor-beta complexes with thrombospondin. Mol. Biol. Cell., 3, 181–188.[Abstract]
  41. Schultz-Cherry,S. and Murphy-Ullrich,J.E. (1993) Thrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism. J. Cell. Biol., 122, 923–932.[Abstract/Free Full Text]
  42. Roberts,A.B and Sporn,M.B. (1993) Physiological actions and clinical applications of transforming growth factor-beta (TGF-beta). Growth Fact., 8, 1–9.
  43. Hsu,S.C., Volpert,O.V., Steck,P.A., Mikkelsen,T., Polverini,P.J., Rao,S., Chou,P. and Bouck,N.P. (1996) Inhibition of angiogenesis in human glioblastomas by chromosome 10 induction of thrombospondin-1. Cancer Res., 56, 5684–5691.[Abstract/Free Full Text]
  44. Zhang,T., Nanney,L.B., Peeler,M.O., Williams,C.S., Lamps,L., Heppner,K.J., DuBois,R.N. and Beauchamp,R.D. (1997) Decreased transforming growth factor beta type II receptor expression in intestinal adenomas from Min/+ mice is associated with increased cyclin D1 and cyclin-dependent kinase 4 expression. Cancer Res., 57, 1638–1643.[Abstract/Free Full Text]
  45. Hao,X., Frayling,I.M., Willcocks,T.C., Han,W., Tomlinson,I.P., Pignatelli,M.N., Pretlow,T.P. and Talbot,I.C. (2002) Beta-catenin expression and allelic loss at APC in sporadic colorectal carcinogenesis. Virchows Arch., 440, 362–366.[CrossRef][ISI][Medline]
  46. Brabletz,T., Jung,A., Dag,S., Reu,S. and Kirchner,T. (2000) Beta-catenin induces invasive growth by activating matrix metalloproteinases in colorectal carcinoma. Verh. Dtsch. Ges. Pathol., 84, 175–181.[Medline]
  47. Incardona,F., Lewalle,J.M., Morandi,V., Lambert,S., Legrand,Y., Foidart,J.M. and Legrand,C. (1995) Thrombospondin modulates human breast adenocarcinoma cell adhesion to human vascular endothelial cells. Cancer Res., 55, 166–173.[Abstract/Free Full Text]
  48. Goldblum,S.E., Young,B.A., Wang,P. and Murphy-Ullrich,J.E. (1999) Thrombospondin-1 induces tyrosine phosphorylation of adherens junction proteins and regulates an endothelial paracellular pathway. Mol. Biol. Cell., 10, 1537–1551.[Abstract/Free Full Text]
  49. Arnoletti,J.P., Albo,D., Granick,M.S., Solomon,M.P., Castiglioni,A., Rothman,V.L. and Tuszynski,G.P. (1995) Thrombospondin and transforming growth factor-beta 1 increase expression of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in human MDA-MB-231 breast cancer cells. Cancer, 76, 998–1005.[CrossRef][ISI][Medline]
  50. Gutierrez,L.S., Schulman,A., Brito-Robinson,T., Noria,F., Ploplis,V.A. and Castellino,F.J. (2000) Tumor development is retarded in mice lacking the gene for urokinase-type plasminogen activator or its inhibitor, plasminogen activator inhibitor-1. Cancer Res., 60, 5839–5847.[Abstract/Free Full Text]
  51. Grant,S.W., Kyshtoobayeva,A.S., Kurosaki,T., Jakowatz,J. and Fruehauf,J.P. (1998) Mutant p53 correlates with reduced expression of thrombospondin-1, increased angiogenesis and metastatic progression in melanoma. Cancer Detect. Prev., 22, 185–194.[CrossRef][ISI][Medline]
  52. Alvarez,A.A., Axelrod,J.R., Whitaker,R.S., Isner,P.D., Bentley,R.C., Dodge,R.K. and Rodriguez,G.C. (2001) Thrombospondin-1 expression in epithelial ovarian carcinoma: association with p53 status, tumor angiogenesis and survival in platinum-treated patients. Gynecol. Oncol., 82, 273–278.[CrossRef][ISI][Medline]
  53. Kasper,H.U., Ebert,M., Malfertheiner,P., Roessner,A., Kirkpatrick,C.J. and Wolf,H.K. (2001) Expression of thrombospondin-1 in pancreatic carcinoma: correlation with microvessel density. Virchows Arch., 438, 116–120.[CrossRef][ISI][Medline]
  54. Maeda,K., Nishiguchi,Y., Kang,S.M., Yashiro,M., Onoda,N., Sawada,T., Ishikawa,T. and Hirakawa,K. (2001) Expression of thrombospondin-1 inversely correlated with tumor vascularity and hematogenous metastasis in colon cancer. Oncol. Rep., 8, 763–766.[ISI][Medline]
  55. Rodriguez-Manzaneque,J.C., Lane,T.F., Ortega,M.A., Hynes,R.O., Lawler,J. and Iruela-Arispe,M.L. (2001) Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor. Proc. Natl Acad. Sci. USA, 98, 12485–23490.[Abstract/Free Full Text]
Received August 7, 2002; revised October 4, 2002; accepted October 16, 2002.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
D. Alferez, R. W. Wilkinson, J. Watkins, R. Poulsom, N. Mandir, S. R. Wedge, I. T. Pyrah, N. R. Smith, L. Jackson, A. J. Ryan, et al.
Dual inhibition of VEGFR and EGFR signaling reduces the incidence and size of intestinal adenomas in ApcMin/+ mice
Mol. Cancer Ther., March 1, 2008; 7(3): 590 - 598.
[Abstract] [Full Text] [PDF]

Home page
 Arch SurgHome page
D. M. Iddings, E. A. Koda, S. S. Grewal, R. Parker, S. Saha, and A. Bilchik
Association of Angiogenesis Markers With Lymph Node Metastasis in Early Colorectal Cancer
Arch Surg, August 1, 2007; 142(8): 738 - 745.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Ghoneim, M. Soula-Rothhut, C. Blanchevoye, L. Martiny, F. Antonicelli, and B. Rothhut
Activating Transcription Factor-1-mediated Hepatocyte Growth Factor-induced Down-regulation of Thrombospondin-1 Expression Leads to Thyroid Cancer Cell Invasion
J. Biol. Chem., May 25, 2007; 282(21): 15490 - 15497.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
R.A. Goodlad, A.J. Ryan, S.R. Wedge, I.T. Pyrah, D. Alferez, R. Poulsom, N.R. Smith, N. Mandir, A.J. Watkins, and R.W. Wilkinson
Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer
Carcinogenesis, October 1, 2006; 27(10): 2133 - 2139.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
G. E. Garcia, H.-G. Wisniewski, M. S. Lucia, N. Arevalo, T. J. Slaga, S. L. Kraft, R. Strange, and A. P. Kumar
2-Methoxyestradiol Inhibits Prostate Tumor Development in Transgenic Adenocarcinoma of Mouse Prostate: Role of Tumor Necrosis Factor-{alpha}-Stimulated Gene 6
Clin. Cancer Res., February 1, 2006; 12(3): 980 - 988.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
Y. Moon, F. G. Bottone Jr., M. F. McEntee, and T. E. Eling
Suppression of tumor cell invasion by cyclooxygenase inhibitors is mediated by thrombospondin-1 via the early growth response gene Egr-1
Mol. Cancer Ther., October 1, 2005; 4(10): 1551 - 1558.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y.-W. Zhang, Y. Su, O. V. Volpert, and G. F. V. Woude
Hepatocyte growth factor/scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin 1 regulation
PNAS, October 28, 2003; 100(22): 12718 - 12723.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
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