| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Department of Dermatology,*
Cutaneous Biology
Research Center, Massachusetts General Hospital and Harvard Medical
School, Charlestown, Massachusetts; and the Department of
Pathology,
Beth Israel Deaconess Medical
Center and Harvard Medical School, Boston, Massachusetts
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To address this question, we developed an in vivo xenotransplantation model for the intradermal tumor growth of human SCCs. We chose the human SCC-13 line25 for our studies because of the relatively low endogenous VEGF expression and the high degree of differentiation and circumscribed growth pattern with lack of tumor invasiveness. SCC-13 cells were stably transfected with expression vectors containing murine VEGF (mVEGF)164 in sense (SCC/VEGF+) or antisense (SCC/VEGF-) orientation or with vector alone (SCC/vec), and these cells were studied for up to 8 weeks after intradermal and subcutaneous xenotransplantation. We report here that selective VEGF overexpression in SCC-13 xenotransplants induced tumor invasion and also potently promoted tumor growth and angiogenesis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The human SCC line SCC-13, kindly provided by Dr. James Rheinwald (Harvard Medical School, Boston, MA), was maintained in complete serum-free keratinocyte growth medium (KGM; Life Technologies Inc., Grand Island, NY). A 980-bp full-length mouse VEGF164 complementary DNA (cDNA; GenBank accession number M9520026 ) was cloned into a pCMV-NEO expression vector, which contains a cytomegalovirus enhancer-promoter and a neomycin/geneticin sulfate (G418) selection cassette composed of the Tn5 gene driven by the simian virus 40 promoter. VEGF sense- and antisense-oriented constructs were analyzed by restriction mapping and by direct sequencing using the Sanger dideoxy method.14 DNA transfections were performed by calcium phosphate precipitation as described previously,26 using vector alone and sense and antisense VEGF expression constructs. At 48 hours after transfection, cells were split 1:3 into complete keratinocyte growth medium containing 600 µg/ml G418 to select transfectants. Stably transfected SCC cell clones were expanded, and 10 clones for each construct were analyzed for VEGF messenger RNA (mRNA) expression and protein secretion. Three clones each with high expression of the transfected construct were further expanded and used for the xenotransplantation experiments described below.
RNA Isolation and Northern Blot Analysis
Total cellular RNA was isolated from stable transfectants as described previously.27 Northern blot analyses were performed using BioTrans nylon-supported membranes (ICN, Irvine, CA), according to the manufacturer’s instructions. We used a 980-bp mouse VEGF164 cDNA probe, isolated as described previously.15,26 This cDNA probe recognizes human and mouse VEGF mRNA.14 Endogenous human VEGF165 mRNA yields two bands of 3.7 and 4.2 kb, whereas mVEGF164 mRNAs encoded by the transfection vectors (sense and antisense VEGF) yield bands at 1.0–1.6 kb.14 A cDNA for ß-actin (Clontech, Palo Alto, CA) was used as a probe to control for equal RNA loading, blotting, and hybridization. Hybridization probes were isolated cDNA fragments radiolabeled with a random-primed synthesis kit (MultiPrime; Amersham, Arlington Heights, IL). Blots were washed at high stringency (0.1 x SCC–1% sodium dodecyl sulfate at 65°C) and exposed on Kodak X-OMAT film.
Assays for VEGF Protein in Cell Culture Supernatants
Cultured cells were grown to confluence, the medium was changed, and cells were cultured for an additional 48 hours. Culture supernatants were then collected, cleared by centrifugation, and assayed for mVEGF levels by enzyme-linked immunosorbent assay. We used a commercially available enzyme-linked immunosorbent assay kit to detect mouse VEGF (R&D Systems, Minneapolis, MN), according to the manufacturer’s recommendations. After collection of culture supernatants, cells were trypsinized and counted, using a hemocytometer. Levels of mouse VEGF protein were calculated as nanograms of VEGF/106 cells. In addition, we performed Western blot analyses of secreted mouse and human VEGF. Culture supernatants were applied to a column of heparin-Sepharose CL-6B (Amersham Pharmacia Biotech, Piscataway, NJ) at 4°C, and elution was carried out as previously described.28 Samples were mixed with one-fourth volume of sample buffer (pH 7.5, containing 0.25 mol/L Tris, 5% sodium dodecyl sulfate, 1.25 mol/L sucrose) and heated to 100°C for 1 minute. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on 10% acrylamide gels, using molecular weight calibrations as previously described.28 Proteins were electrophoretically transferred to nitrocellulose paper (Bio-Rad, Hercules, CA) at 50 V for 24 hours, and, after several washes, immunolabeling was performed using either mouse anti-human VEGF monoclonal antibody 263 or a goat anti-mouse VEGF polyclonal antibody that showed a higher sensitivity for transfected mVEGF164 (both obtained from R&D Systems).
Growth of Stably Transfected SCC-13 Cells in Immunodeficient Mice
Three individual clones of stably transfected SCC-13 cells were
used for each construct (vector only, VEGF sense, VEGF antisense). In
addition, the parental cell line was studied. Cells (2 x
106) were injected either intradermally or
subcutaneously into immunodeficient BALB/c(nu/nu) mice (Taconic Farms,
Germantown, NY). Each animal received one such injection in each flank,
and each cell clone was injected into at least five mice. Tumor yields
were determined as the percentage of persisting tumors of at least 1
mm3
tumor volume after 4 weeks. Tumor sizes were
quantitated weekly for 8 weeks by using a digital caliper to measure
the largest and smallest tumor diameters, and tumor sizes were
calculated using the formula: volume = 4/3 x 
x
(1/2 x smaller diameter)2
x 1/2
x larger diameter.29
Significances were calculated using
the one-sided Student’s t-test for unpaired samples.
Animals were euthanized after 8 weeks, the back skin containing tumors
was dissected and photographed, and tumors were harvested and processed
for further analyses as described below. All animal experiments were
approved by the Massachusetts General Hospital Subcommittee on Research
Animal Care.
Histology, in Situ Hybridization, and Immunohistochemistry
For routine histology, tumors were fixed in 4%
paraformaldehyde-phosphate-buffered saline and processed and embedded
in paraffin for hematoxylin and eosin staining as previously
described.14
In situ hybridization of paraffin
and frozen sections was performed as described
earlier,15,27
using pGEM or pBluescript II plasmids
containing mouse VEGF, flt-1 (VEGFR-1), or flk-1 (VEGFR-2) cDNA
fragments. The flt-1 and flk-1 clones were a kind gift from Clive Wood,
Genetics Institute, Cambridge, MA. The flk-1 and flt-1 sequences were
isolated by polymerase chain reaction from a mouse fetal thymus cDNA
library.30
The murine flk-1 transcription template was a
392-bp fragment encompassing amino acids 1 to 130 (nucleotides 268–660
of the flk-1 sequence described previously31
), cloned into
pGEM-T (Promega, Madison, WI). The sequence for murine flt-1 was
obtained by degenerate polymerase chain reaction cloning of kinase
domains, which resulted in a 640-bp cDNA fragment encoding the insert
region from amino acid 832-1045 of a sequence described
previously.30
Transcription reactions were carried out by
using a Riboprobe Gemini II kit (Promega) in the presence of
[
-35S]-UTP. Antisense (and control sense)
probes were evaluated on alternate sections.
For immunohistochemistry, 6-µm cryostat sections were stained with a rat anti-mouse platelet-endothelial cell adhesion molecule-1 (CD31) monoclonal antibody (Pharmingen, San Diego, CA) or with a rabbit anti-mouse collagen type IV polyclonal antibody (Biodesign International, Kennebunk, ME), using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Rat IgG1 (Pharmingen) was used as isotype-matched negative control in monoclonal antibody staining, and normal rabbit serum was used as control for polyclonal rabbit serum. To determine the degree of tumor-induced angiogenesis, cryostat sections of tumor xenografts were stained with an anti-mouse CD31 monoclonal antibody. Representative sections obtained from five tumors from each cell clone were analyzed, using a Nikon E-600 microscope (Nikon, Melville, NY). Images were captured with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI), and morphometric analyses were performed using the IP LAB software program (Scanalytics Inc, Fairfax, VA). Three different fields at x60 magnification were examined on each section, and the number of vessels per square millimeter, the size distribution, and the average and the total areas of tumor blood vessels were determined. The unpaired Student’s t-test was used to analyze differences in the vessel areas.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
SCC-13 cells were chosen for VEGF transfection and subsequent
in vivo transplantation studies because of their high degree
of differentiation and well-circumscribed growth pattern with lack of
tumor cell invasion in vivo.25
Moreover, our
pilot studies had revealed low VEGF expression in SCC-13 cells in
vitro, as compared with SCC-12, SCC-15, HaCaT, or A431 cells (data
not shown). SCC-13 cells were stably transfected with the cDNAs
encoding either sense (SCC/VEGF+) or antisense (SCC/VEGF-)
orientations of the mouse 164-amino-acid VEGF isoform under the control
of a constitutive expression vector. As a control, other SCC-13 cells
were transfected with vector alone without a VEGF insert (SCC/vec). We
characterized VEGF expression in 10 stably transfected clones for
each construct and chose three clones with strong expression of
transfected mVEGF for further in vivo studies. As in the
parental SCC-13 population, transfectants expressed low levels of
endogenous human VEGF mRNA of 4.2 and 3.7 kb, whereas the
vector-directed mVEGF mRNA (both sense and antisense) formed a readily
distinguishable band at 1.9 kb (Figure 1A)
. SCC-13 cells transfected with vector
alone (SCC/vec) expressed low levels of endogenous human VEGF mRNA but
no detectable mVEGF mRNA. Three clones each with high expression of the
transfection constructs were chosen for further analysis of secreted
VEGF protein. Whereas no mVEGF was detectable in SCC/vec and SCC/VEGF-
cell supernatants, between 18 and 23 ng/106
cells
of mouse VEGF were detected in SCC/VEGF+ cell supernatants (data not
shown). Additional Western blot analyses of culture supernatants
confirmed efficient VEGF secretion in SCC/VEGF+ clones, whereas little
or no VEGF was detected in conditioned media harvested from SCC/vec
clones (Figure 1B)
. No VEGF protein was detected in SCC/VEGF- clones.
|
Three clones for each construct (SCC/vec, SCC/VEGF+, SCC/VEGF-),
as well as the parental cell line, were injected in duplicate
intradermally into five mice each. In a separate experiment, three
clones each of sense- versus antisense-transfected cells
were injected subcutaneously into 4–5 nude mice each. Tumor growth was
monitored weekly by measurements of the longest and shortest diameter
of tumors for a total of 8 weeks. After intradermal injection,
VEGF-overexpressing SCC-13 cells were characterized by significantly
increased tumor yields (P < 0.05). Of SCC/VEGF+
transplants, 79% developed into tumors, as compared with 50% for
SCC/vec clones (Figure 2A)
. In contrast,
tumor yields of SCC/VEGF- cells were significantly reduced
(P < 0.01; Figure 2A
). After subcutaneous
injection, however, tumor yields of control-transfected clones were
higher (64%) than after intradermal application and were not
significantly different from SCC/VEGF+ (80%) or SCC/VEGF- (55%)
clones. However, the growth rate of established SCC/VEGF+ tumors was
dramatically higher than that of control tumors in both intradermal and
subcutaneous locations. After intradermal injection, SCC/VEGF+ cells
formed significantly larger tumors (average of 262
mm2
after 8 weeks, compared with 4
mm2
for controls) than SCC/vec or SCC/VEGF-
cells (Figure 2B
and Figure 3, a–c
).
Similar results were found after subcutaneous injection (Figure 2C)
.
Results obtained with the parental cell line were identical to those
obtained with SCC/vec clones (data not shown). SCC/vec cells formed
well-differentiated SCCs with a clearly demarcated border to the
surrounding tumor stroma (Figure 3, d and g)
and without detectable
tumor invasion. In contrast, SCC/VEGF+ tumors were less well
differentiated and were characterized by an irregular infiltrative edge
with small cell groups and single cells invading into the surrounding
stroma (Figure 3, e and h)
. The few SCC/VEGF- transplants that
developed into tumors showed only minimal growth with formation of
well-circumscribed tumor nodules (Figure 3, f and i)
.
|
|
To confirm that VEGF sense or antisense mRNA expression was
maintained in tumor cells after transplantation into the skin of nude
mice, we performed in situ hybridizations after 8 weeks of
tumor growth. The VEGF riboprobe used for these hybridizations
recognizes all isoforms of human and mouse VEGF mRNA.14
These studies confirmed that strong VEGF mRNA expression was maintained
in SCC/VEGF+ tumors (Figure 4, a and b)
whereas only weak VEGF mRNA expression was detected in control SCC/vec
tumors (Figure 4, c and d)
and in SCC/VEGF- tumors (Figure 4, g and h)
. Importantly, SCC/VEGF- tumors maintained high expression of the
VEGF antisense mRNA, as detected by in situ hybridization
with a VEGF sense riboprobe (Figure 4, e and f)
. In accordance with
previously reported findings in a variety of malignant human and murine
tumors, strong expression of flk-1 and flt-1 mRNA was detected in tumor
vessels of SCC/VEGF+ xenotransplants (Figure 5, b, d, f, and h)
, whereas only low
expression levels were detected in SCC/vec (Figure 5, a, c, e, and g)
and SCC/VEGF- transplants.
|
|
Immunohistochemical analysis of tissue sections stained for
collagen IV, a basement membrane component, and for
platelet-endothelial cell adhesion molecule-1 (CD31), an endothelial
junction molecule,32
demonstrated an almost continuous
layer of blood vessels surrounding SCC/vec and SCC/VEGF- tumors
(Figure 3, k, m, n, and p)
. These tumor vessels were focally dilated;
however, ingrowth of vessels into the SCC/vec or SCC/VEGF- tumors was
not observed. In contrast, VEGF-overexpressing SCC/VEGF+ tumors showed
a considerable admixture of blood vessels and tumor cells, most
prominently in areas of tumor invasion (Figure 3, l and o)
and a
significantly increased density of tumor vessels (71.7 ± 13.6
vessels/mm2), as compared with 39.4 ± 4.5
vessels/mm2
in control SCC/vec tumors (Figure 6A)
. In contrast, the vessel density was
significantly reduced to 26.0 ± 5.6
vessels/mm2
in SCC/VEGF- tumors (Figure 6A)
.
Interestingly, the average vessel size in SCC/VEGF+ tumors was greatly
increased to 814 ± 90 µm2, as compared
with 403 ± 52 µm2
in SCC/vec control
tumors and 272 ± 40 µm2
in SCC/VEGF-
tumors (Figure 6B)
. This was mainly due to an almost twofold increase
of the percentage of vessels larger than 500
µm2
in SCC/VEGF+ tumors (18.2%
versus 9.3% in SCC/vec), as well as of grossly dilated
vessels larger than 1000 µm2
(12.6%
versus 6.5%; Figure 6D
). No vessels larger than 500
µm2
were detected in SCC/VEGF- tumors (Figure 6D)
. It is important that, when the parameters vessel density and
average vessel size were combined to calculate total vascular areas, a
much more pronounced increase in tumor angiogenesis was detected in
VEGF-overexpressing tumors. Although vessels accounted for 1.05% ±
0.18% of the total tumor area in SCC/vec tumors (Figure 6C)
, a more
than fivefold increase was observed in SCC/VEGF+ tumors (6.05% ±
0.99%). These data suggest total vascular area measurements as a more
sensitive parameter than vascular density for the evaluation of tumor
angiogenesis.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We chose the established human SCC line SCC-13 for our studies because SCC-13 xenotransplants form well-differentiated, noninvasive tumors after subcutaneous xenotransplantation into immunodeficient mice.25 Therefore, we established stable SCC-13 transfectants with selective overexpression of mVEGF164. To enable an even more direct comparison between high and low VEGF expression by SCC-13 xenotransplants, we also established stable transfectants that overexpressed a VEGF164 antisense construct. Strong expression of the transfected VEGF sense or antisense overexpression constructs was confirmed in stably transfected SCC-13 clones by Northern hybridizations, and Western blot analyses revealed a potent induction of mouse VEGF secretion in SCC/VEGF+ cells. Overexpression of VEGF led to a significantly enhanced rate of SCC tumor formation after intradermal tumor cell application, as compared with control-transfected cells. In contrast, tumor cells overexpressing the VEGF gene in antisense orientation showed significantly reduced tumor yields. These data suggest that VEGF-induced endothelial-cell activation of host vessels plays an important role in establishing the initial environmental milieu for successful growth and survival of the transplanted tumor clones in the dermis. They are in accordance with a previous report demonstrating that overexpression of mVEGF164 by human malignant melanoma cells led to increased metastatic efficiency after intravenous injection of tumor cells into immunodeficient mice, as judged by the number of lung metastases.14 However, after application of tumor cells into the more richly vascularized subcutaneous space, control-transfected SCC clones showed higher tumor yields than after intradermal injection, and no significantly altered tumor yields were detected in clones overexpressing VEGF or a VEGF antisense construct. These findings reveal an important role of the stromal tumor microenvironment for the rate of successful tumor growth and suggest, furthermore, that the initial growth advantage provided by high VEGF secretion may be of less importance in already richly vascularized tissues.
Importantly, VEGF-overexpressing SCC-13 clones showed dramatically enhanced growth of established tumors in both intradermal and subcutaneous locations, as compared with controls transfected with vector without insert only. Results obtained with the parental cell line were identical to those obtained with SCC/vec clones, excluding any negative effects of cell transfection and subsequent cell selection on the tumor growth capacity of stably transfected cells. Eight weeks after transplantation, SCC/VEGF+ clones had formed tumors of more than 60-fold the size of SCC/vec or SCC/VEGF- clones. To the best of our knowledge, this is the first time that an extremely slowly growing tumor with a high degree of differentiation has been investigated for the effects of induced VEGF expression. The VEGF-mediated increase of tumor growth was much more prominent than in previously reported xenotransplant models using highly malignant tumor cell lines.14 It is of interest that SCC/VEGF- xenotransplants maintained tumors of approximately 1 to 2 mm in diameter but were unable to grow beyond this minimal size.
Histological analysis revealed that control SCC/vec xenotransplants
formed well-differentiated, rounded tumors without apparent tumor
invasion. Tumors comprised multilayered epithelium with occasional
mitoses and a clear demarcation of the border between tumor cells and
surrounding tumor stroma. In marked contrast, VEGF-overexpressing
SCC/VEGF+ xenotransplants showed malignant growth behavior with an
irregular tumor stroma border and invasion of small nests of tumor
cells and single tumor cells into the surrounding tissue. These
findings confirm our original hypothesis that selective overexpression
of VEGF enables tumor invasion, and they extend the findings by Skobe
et al that an anti-flk-1 antibody inhibited tumor
invasion.23
The mechanisms by which VEGF mediates the
induction of tumor invasiveness are presently unknown. However, it is
conceivable that VEGF released by tumor cells induces release of matrix
metalloproteinases by endothelial cells that, in turn, might help to
create an environment that is permissive to tumor cell invasion.
Indeed, induction of matrix metalloproteinases by VEGF has been
demonstrated in human endothelial cells.37
Alternatively,
VEGF-stimulated tumor vessels might release activators of matrix
metalloproteinase expression by tumor cells themselves. Recently,
inhibition of the vß3 integrin on angiogenic blood vessels was
shown to inhibit invasiveness of experimental breast
carcinomas.38
Combined with our previously reported
findings that VEGF potently induced vß3 expression in skin dermal
microvascular endothelial cells,39
these results suggest a
potential role of integrins and their receptors in mediating
VEGF-induced tumor invasion. In addition, VEGF-induced vascular
hyperpermeability, leading to extravasation of plasma proteins and
formation of a fibrin-rich stroma,17
may further
facilitate ingrowth of fibroblasts and blood vessels to form a tumor
stroma submissive for tumor invasion.
In accordance with previous reports on the induction of tumor vessels by VEGF,14 we detected a significantly increased density of tumor vessels in SCC/VEGF+ xenotransplants. Whereas tumor vessels in control tumors were found to merely surround the well-differentiated tumors, VEGF-overexpressing SCCs were characterized by an intimate admixture of vessels and tumor cells. It is of interest that measurement of total vascular areas as a percentage of tumor area occupied by vessel provided a much more sensitive parameter (a more than fivefold increase in SCC/VEGF+ tumors) than vessel density or average vessel size to detect VEGF-induced tumor angiogenesis. In conclusion, our results demonstrate that selective overexpression of the angiogenesis factor VEGF in highly differentiated SCCs is sufficient to induce tumor invasiveness, in addition to promotion of tumor growth and angiogenesis. Our studies also provide direct evidence for an active role of the tumor stroma in cancer progression.
|
Footnotes |
|---|
Supported by National Institutes of Health/National Cancer Institute grant CA69184 (to M. D.), by Deutsche Forschungsgemeinschaft Grant De483/3–2 (to M. D.), by American Cancer Society Research project grant 99–23901 (to M. D.), by Deutscher Akademischer Austauschdienst (to M. St.), by the Human Frontier Science Program (to M. Sk.), and by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement (to M. D.).
Accepted for publication September 15, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
v ß 3 blocks human breast cancer growth and angiogenesis in human skin. J Clin Invest 1995, 96:1815-1822
vß3 integrin, osteopontin, and thrombin. Am J Pathol 1996, 149:293-305[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  B. Cat, D. Stuhlmann, H. Steinbrenner, L. Alili, O. Holtkotter, H. Sies, and P. Brenneisen Enhancement of tumor invasion depends on transdifferentiation of skin fibroblasts mediated by reactive oxygen species J. Cell Sci., July 1, 2006; 119(13): 2727 - 2738. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-T. Zhou, Y. Yang, and J.-P. Ge The preventive effect of salvianolic acid B on malignant transformation of DMBA-induced oral premalignant lesion in hamsters Carcinogenesis, April 1, 2006; 27(4): 826 - 832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. O. Yee, M. Streit, T. Hawighorst, M. Detmar, and J. Lawler Expression of the Type-1 Repeats of Thrombospondin-1 Inhibits Tumor Growth Through Activation of Transforming Growth Factor-{beta} Am. J. Pathol., August 1, 2004; 165(2): 541 - 552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Stuhlmann, N. Ale-Agha, R. Reinehr, H. Steinbrenner, M. C. Ramos, H. Sies, and P. Brenneisen Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells Carcinogenesis, November 1, 2003; 24(11): 1737 - 1748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Dadras, T. Paul, J. Bertoncini, L. F. Brown, A. Muzikansky, D. G. Jackson, U. Ellwanger, C. Garbe, M. C. Mihm, and M. Detmar Tumor Lymphangiogenesis: A Novel Prognostic Indicator for Cutaneous Melanoma Metastasis and Survival Am. J. Pathol., June 1, 2003; 162(6): 1951 - 1960. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Furumatsu, Z. N. Shen, A. Kawai, K. Nishida, H. Manabe, T. Oohashi, H. Inoue, and Y. Ninomiya Vascular Endothelial Growth Factor Principally Acts as the Main Angiogenic Factor in the Early Stage of Human Osteoblastogenesis J. Biochem., May 1, 2003; 133(5): 633 - 639. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Barbera-Guillem, J. K. Nyhus, C. C. Wolford, C. R. Friece, and J. W. Sampsel Vascular Endothelial Growth Factor Secretion by Tumor-infiltrating Macrophages Essentially Supports Tumor Angiogenesis, and IgG Immune Complexes Potentiate the Process Cancer Res., December 1, 2002; 62(23): 7042 - 7049. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Rice and C M Quinn Angiogenesis, thrombospondin, and ductal carcinoma in situ of the breast J. Clin. Pathol., August 1, 2002; 55(8): 569 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hawighorst, M. Skobe, M. Streit, Y.-K. Hong, P. Velasco, L. F. Brown, L. Riccardi, B. Lange-Asschenfeldt, and M. Detmar Activation of the Tie2 Receptor by Angiopoietin-1 Enhances Tumor Vessel Maturation and Impairs Squamous Cell Carcinoma Growth Am. J. Pathol., April 1, 2002; 160(4): 1381 - 1392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Senger, C. A. Perruzzi, M. Streit, V. E. Koteliansky, A. R. de Fougerolles, and M. Detmar The {alpha}1{beta}1 and {alpha}2{beta}1 Integrins Provide Critical Support for Vascular Endothelial Growth Factor Signaling, Endothelial Cell Migration, and Tumor Angiogenesis Am. J. Pathol., January 1, 2002; 160(1): 195 - 204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Rodriguez-Manzaneque, T. F. Lane, M. A. Ortega, R. O. Hynes, J. Lawler, and M. L. Iruela-Arispe Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor PNAS, October 12, 2001; (2001) 171460498. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Viloria-Petit, T. Crombet, S. Jothy, D. Hicklin, P. Bohlen, J. M. Schlaeppi, J. Rak, and R. S. Kerbel Acquired Resistance to the Antitumor Effect of Epidermal Growth Factor Receptor-blocking Antibodies in Vivo: A Role for Altered Tumor Angiogenesis Cancer Res., July 1, 2001; 61(13): 5090 - 5101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Straume and L. A. Akslen Expresson of Vascular Endothelial Growth Factor, Its Receptors (FLT-1, KDR) and TSP-1 Related to Microvessel Density and Patient Outcome in Vertical Growth Phase Melanomas Am. J. Pathol., July 1, 2001; 159(1): 223 - 235. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Laconi, P. Pani, S. Pillai, D. Pasciu, D. S. R. Sarma, and E. Laconi A growth-constrained environment drives tumor progression invivo PNAS, June 20, 2001; (2001) 131210498. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Giavazzi, R. Giuliani, D. Coltrini, M. R. Bani, C. Ferri, B. Sennino, M. P. M. Tosatti, A. Stoppacciaro, and M. Presta Modulation of Tumor Angiogenesis by Conditional Expression of Fibroblast Growth Factor-2 Affects Early but not Established Tumors Cancer Res., January 1, 2001; 61(1): 309 - 317. [Abstract] [Full Text]
|
|
|
|
|
|
S. Laconi, P. Pani, S. Pillai, D. Pasciu, D. S. R. Sarma, and E. Laconi A growth-constrained environment drives tumor progression invivo PNAS, July 3, 2001; 98(14): 7806 - 7811. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Rodriguez-Manzaneque, T. F. Lane, M. A. Ortega, R. O. Hynes, J. Lawler, and M. L. Iruela-Arispe Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor PNAS, October 23, 2001; 98(22): 12485 - 12490. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
