¹Cutaneous Biology Research Center and Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, MA 02129, USA

²Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, MA 02215, USA

³Department of Genetics, St. Jude Children's Research Hospital, Memphis, Tennessee, TN 38105, USA

⁴IMCR Human Immunology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK

Correspondence to: M Detmar, CBRC/Department of Dermatology, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129, USA; E-mail: michael.detmar@cbrc2.mgh.harvard.edu

The roles played by the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in the early stages of multi-step carcinogenesis and in the control of hematogenous versus lymphatic metastasis are unknown. To investigate these issues we compared tumor development in normal mice and in transgenic mice with targeted overexpression of TSP-1 in the epidermis following a standard two-step chemical skin carcinogenesis regimen. Overexpression of TSP-1 resulted in delayed and reduced development of premalignant epithelial hyperplasias, but did not inhibit the malignant conversion to squamous cell carcinomas. TSP-1 overexpression also suppressed tumor angiogenesis and distant organ metastasis, but failed to inhibit tumor-associated lymphangiogenesis or lymphatic tumor spread to regional lymph nodes. Concomitant with these results, we found that the endothelial TSP-1 receptor CD36 was mostly absent from cutaneous lymphatic vessels. Our findings indicate the potential use of TSP-1 for the prevention of premalignant stages of tumorigenesis and are likely to have implications for the further development of anti-angiogenic cancer therapies.

Oncogene (2002) 21, 7945-7956. doi:10.1038/sj.onc.1205956

TSP-1; VEGF; Prox1; LYVE-1; chemoprevention

Introduction

Thrombospondin-1 (TSP-1) is a 450 kD homotrimeric matricellular glycoprotein (for review, see Bornstein, 2001; Lawler, 2000) that inhibits proliferation and migration of vascular endothelial cells in vitro and inhibits neovascularization in vivo, contributing to the normal quiescence of the vasculature (Jimenez et al., 2000; Tolsma et al., 1993). Correlative expression data in human malignant melanomas (Zabrenetzky et al., 1994) and in cancers of the breast (Volpert et al., 1995), the cervix (Kodama et al., 2001) and the ovary (Alvarez et al., 2001), together with the results of xenotransplant tumor studies in mice (Bleuel et al., 1999; Streit et al., 2000; Weinstat et al., 1994) indicate that increased TSP-1 expression is associated with decreased malignant tumor growth, invasion and organ metastasis. Conversely, antisense inhibition of TSP-1 expression in squamous cell carcinomas (SCC) was reported to suppress tumor growth in vivo (Castle et al., 1991). Thus, TSP-1 has been described as a tumor suppressor (Volpert et al., 1995) and as a tumor promoter (Tuszynski and Nicosia, 1996). Previous experimental investigations into the importance of TSP-1 for tumor growth and angiogenesis have exclusively studied established malignant tumors (Bleuel et al., 1999; Castle et al., 1991; Streit et al., 2000; Weinstat et al., 1994). However, autochthonous human tumors usually develop through a series of preneoplastic and benign neoplastic stages before converting into malignant cancer, and the impact of TSP-1 on the early, premalignant, angiogenic stages of multi-step carcinogenesis is unknown. Based upon our recent findings that TSP-1 mRNA expression was already potently downregulated in the early premalignant stages of multi-step, chemically-induced skin carcinogenesis (Hawighorst et al., 2001), we hypothesized that TSP-1 might play an important role in the control of early carcinogenic events.

Chemically-induced multistage epithelial carcinogenesis is a well established model for skin cancer in mice (Ito et al., 1995) that allows a detailed evaluation of distinct premalignant and malignant stages of carcinogenesis (Marks and Furstenberger, 2000), and that has also provided a more in-depth understanding of human epithelial cancer development (Balmain and Harris, 2000; Digiovanni, 1992; Yuspa, 1994). For many tumors, the initial dissemination occurs via lymphatic vessels to regional lymph nodes, following routes of natural tissue drainage (Sleeman, 2000). However, the biological role of TSP-1 and of other endogenous inhibitors of angiogenesis in tumor lymphangiogenesis and lymphatic metastasis has remained unclear, in part due to the lack of specific lymphatic markers that distinguish lymphatic from blood vascular endothelium.

In this present study, we have investigated the distinct biological role of TSP-1 in multi-step epithelial carcinogenesis and metastasis. We subjected mice with targeted overexpression of TSP-1 in the epidermis (Streit et al., 2000) to a standard two-step chemical skin carcinogenesis regimen, using 7,12-dimethylbenz()anthracene (DMBA) for tumor initiation and phorbol 12-myristate 13-acetate (PMA) to promote tumor growth (Digiovanni, 1992). Here we report that overexpression of TSP-1 in epidermal keratinocytes delayed and diminished the formation of pre-malignant epithelial hyperplasias but did not reduce the rate of malignant conversion to squamous cell carcinomas. TSP-1 overexpression was also associated with decreased tumor angiogenesis and increased tumor cell apoptosis, but failed to inhibit tumor lymphangiogenesis and lymphatic tumor spread to regional lymph nodes. Concomitantly, the major vascular endothelial TSP-1 receptor CD36 was only scarcely expressed or was absent from cutaneous lymphatic vessels. These results reveal a predominant inhibitory effect of TSP-1 on the early stages of multi-step carcinogenesis, with specific inhibition of blood vascular angiogenesis but not of tumor lymphangiogenesis and lymphatic metastasis, suggesting that TSP-1 might most efficiently be used to target early premalignant stages of tumorigenesis.

Results

Delayed and diminished skin carcinogenesis in TSP-1 transgenic mice

We subjected wild-type and TSP-1 transgenic mice to a standard two-step skin carcinogenesis protocol by using topical application of a single subcarcinogenic dose of DMBA for initiation, followed by 20 weekly topical applications of PMA for tumor promotion. TSP-1 transgenic mice showed delayed formation of pre-malignant skin papillomas, with an average latency period of 14 weeks after the first PMA application, as compared with 10 weeks in wild-type mice (Figure 1a). At 17 weeks, all of the wild-type mice, but only 79% of TSP-1 transgenic mice, had developed papillomas. After 20 weeks of PMA promotion, TSP-1 transgenic mice had developed less than six papillomas per mouse, as compared with more than 11 papillomas in wild-type mice (Figure 1b). Malignant conversion of papillomas to SCC was first detected at 16 weeks after TPA promotion in wild-type mice and after 20 weeks in TSP-1 transgenic mice (Figure 1c). After 33 weeks, 85% of wild-type mice but only 50% of TSP-1 transgenic mice had developed SCC. The average number of SCC per mouse was significantly decreased in TSP-1 transgenic mice (0.7±0.15 at week 33; P<0.01), as compared with wild-type mice (1.5±0.19; Figure 1d). However, the ratio of malignant conversion of papillomas to SCC was comparable in wild-type (12.5%) and TSP-1 transgenic mice (12.1%; Figure 1e). No tumors were observed in wild-type or TSP-1 transgenic mice treated with DMBA or with PMA alone (data not shown). In situ hybridization studies and immunohistochemistry confirmed that transgenic expression of TSP-1 mRNA and protein was maintained throughout the successive steps of epithelial carcinogenesis (Figure 2). Immunofluorescence stains for the early and late epidermal terminal differentiation markers keratin 10 and loricrin showed comparable differentiation of normal skin and of epithelial tumors in both genotypes (data not shown).

Decreased tumor angiogenesis and increased apoptosis in TSP-1 transgenic mice

The 'angiogenic switch' from vascular quiescence to upregulation of angiogenesis was already detected at the early stages of epithelial tumorigenesis in wild-type mice, as shown by comparative analysis of CD31-positive vessels in normal skin (Figure 3a), early-stage papillomas (Figure 3c) and SCC (Figure 3e). In contrast, the extent of tumor angiogenesis was less pronounced in TSP-1 transgenic mice (Figure 3b,d,f) which were characterized by reduced numbers of large angiogenic vessels (3d,f). Computer-assisted morphometric image analysis revealed that the relative area occupied by CD31-positive vessels was significantly lower (P<0.001) in TSP-1 transgenic mice throughout the successive stages of skin carcinogenesis (Figure 3g). In agreement with our previous results (Streit et al., 2000), the vascularization of normal skin was comparable in TSP-1 transgenic mice and wild-type mice (Figure 3a,b,g), suggesting that the reduced angiogenic response in early TSP-1 transgenic tumors was not due to already decreased vascularity in untreated skin. The number of apoptotic tumor cells was more than 1.8-fold (P<0.05) increased in TSP-1 transgenic papillomas, as compared with wild-type tumors (Figure 3i), whereas no significant differences of tumor cell proliferation were detected (P=0.84; Figure 3h).

TSP-1 does not modulate VEGF expression, receptor binding or receptor activation during skin carcinogenesis

We next investigated whether the TSP-1 mediated inhibition of vascularization was due to direct or indirect effects of TSP-1 on VEGF expression or bioavailability. In situ hybridization (Figure 4a-d) revealed only minor differences in VEGF mRNA expression between wild-type (Figure 4a,c) and TSP-1 transgenic SCCs (Figure 4b,d), with little or no expression in the tumor stroma of both genotypes. Moreover, Western blot analyses of SCC lysates showed comparable VEGF protein levels in both genotypes (Figure 4e), and immunoprecipitation studies demonstrated comparable phosphorylation levels of VEGF receptor-2 (VEGFR-2, flk-1) in wild-type and TSP-1 transgenic tumors (Figure 4f). Finally, immunostains with the antibody GV39M that recognized VEGF bound to VEGFR-2 (Brekken et al., 1998), revealed similar amounts of VEGF/VEGFR-2 ligand-receptor complexes in tumor-associated vessels of wild-type (Figure 4g) and TSP-1 transgenic mice (Figure 4h). Taken together, these results suggested that the inhibition of skin carcinogenesis and angiogenesis in TSP-1 transgenic mice was not due to reduced VEGF expression, receptor binding or receptor activation.

TSP-1 inhibits organ metastasis but does not affect tumor lymphangiogenesis and lymph node metastasis

After 33 weeks, 25% of SCC-bearing wild-type mice had developed lung metastases, as compared with only 4% of TSP-1 transgenic mice. In contrast, the incidence of regional lymph node metastases was comparable in TSP-1 transgenic (50%) and wild-type mice (58.3%). To investigate whether the inability of TSP-1 to suppress lymphatic metastasis was due to a reduced inhibitory activity of TSP-1 on tumor lymphangiogenesis, as compared with blood vascular angiogenesis, we next investigated tumor-associated lymphatic vessels, using antibodies to two recently identified lymphatic-specific markers, the hyaluronan receptor LYVE-1 (Banerji et al., 1999; Jackson et al., 2001; Prevo et al., 2001) and the transcription factor Prox1 (Wigle et al., 2002; Wigle and Oliver, 1999). Differential immunofluorescence stains of wild-type SCCs for LYVE-1, Prox1 and the panendeothelial junction molecule CD31 (PECAM-1) demonstrated that LYVE-1 was expressed by a subfraction of CD31 positive vessels that were distinguished by their weaker staining for CD31 (Figure 5a-c). Similarly, Prox1 expression was detected in a fraction of tumor-associated CD31-positive vessels, demonstrating the presence of tumor-associated lymphatic vessels in SCCs (Figure 5d-f). Double stains for LYVE-1 and Prox1 revealed that all LYVE-1 positive tumor-associated vessels also expressed the lymphatic-specific marker Prox1 (Figure 5g-i), confirming the specificity of LYVE-1 for tumor-associated lymphatic vessels also in chemically-induced cutaneous SCCs.

We next used LYVE-1 stains for a detailed comparison of tumor lymphangiogenesis in wild-type and TSP-1 transgenic mice. Double staining for CD31 and LYVE-1 revealed similar numbers of peritumoral (data not shown) and intratumoral lymphatic vessels in SCCs of both genotypes whereas the number of blood vessels differed strikingly (Figure 6a,c). No significant differences of lymphatic vascular density or average lymphatic vessel size were found between both genotypes by quantitative image analysis (Figure 6e,f). Double immunofluorescence stains for LYVE-1 and for the proliferation marker BrdU revealed comparable numbers of proliferating lymphatic endothelial cells both surrounding and within wild-type (Figure 6b) and TSP-1 transgenic tumors (Figure 6d).

CD36 is only sparsely expressed in lymphatic vessels

The CD36 receptor on vascular endothelial cells has been reported to play a major role in mediating the anti-angiogenic effects of TSP-1 (Jimenez et al., 2000). Immunofluorescence double stains of wild-type SCCs for CD36 and CD31 revealed that only a fraction of all peritumoral CD31-positive vessels also expressed CD36 (Figure 7a-c), whereas CD36 expression was not detectable in intratumoral vessels (data not shown). Combined immunofluorescence stains for CD36 and LYVE-1 demonstrated little or no expression of CD36 in LYVE-1 positive lymphatic vessels, whereas CD36 was strongly epxressed in LYVE-1 negative blood vessels (Figure 7d-f). Because of the potential clinical implications of these findings, we next evaluated the vascular expression of CD36 in normal human tissue. Double immunofluorescence stains of human skin for CD36 and CD34, a vascular endothelial marker that is absent from LYVE-1 positive lymphatic vessels (Banerji et al., 1999; Jackson et al., 2001) revealed strong expression of CD36 in most CD34 positive dermal blood vessels (Figure 7g-i) but little or no CD36 expression in LYVE-1 positive lymphatic vessels (Figure 7j-l).

Discussion

Our results identify a pronounced inhibitory effect of genetic overexpression of TSP-1 on the early, premalignant stages of tumor formation in an established orthotopic model of multi-step epithelial carcinogenesis (Digiovanni, 1992). However, the ratio of malignant conversion of benign papillomas to SCC was identical in TSP-1 transgenic and in wild-type mice, revealing that TSP-1 expression was unable to prevent the genetic events involved in malignant tumor progression. This conclusion was further supported by the comparable latency period between papilloma and SCC formation in both genotypes. Taken together, our findings demonstrate, for the first time, that overexpression of an endogenous angiogenesis inhibitor predominantly conveys an early growth disadvantage of initiated/promoted premalignant epithelial cells but does not inhibit their subsequent capacity to form malignant tumors.

Pronounced angiogenesis occurred already at the early stages of premalignant epithelial tumor development in FVB wild-type mice, in agreement with our previously reported findings in the 129 genetic background (Hawighorst et al., 2001), indicating that the distinction between benign epithelial hyperplasias and malignant tumors can not be based upon the extent of tumor vascularization (Eberhard et al., 2000). Transgenic overexpression of TSP-1 potently suppressed tumor vascularization in early lesions, with a predominant decrease of large angiogenic blood vessels. These results are in accordance with the inhibitory effects of TSP-1 overexpression on the vascularity of skin wounds (Streit et al., 2000), of malignant mammary gland tumors (Rodriguez-Manzaneque et al., 2001) and of cutaneous SCC xenotransplants (Bleuel et al., 1999; Streit et al., 1999). The strong correlation between inhibition of angiogenesis and early-stage papilloma formation reveals that TSP-1 mediated angiogenesis inhibition functions as an early rate-limiting step during the promotion of premalignant epithelial tumors. The significant increase of tumor cell apoptosis in TSP-1 transgenic mice is in accordance with the previously reported induction of tumor cell apoptosis by therapeutic inhibition of angiogenesis (Bergers et al., 1999; Holmgren et al., 1995).

It has been reported that TSP-1 affected the bioavailability of the major tumor angiogenesis factor, vascular endothelial growth factor (VEGF) in murine mammary tumors (Rodriguez-Manzaneque et al., 2001). Moreover, enhanced secretion of pro-angiogenic factors was found in subpopulations of fibrosarcoma and glioblastoma cell lines that were resistant to the anti-angiogenic effects of TSP-1 (Filleur et al., 2001). We did not detect any modulation of VEGF mRNA or protein expression in TSP-1 overexpressing tumors, in accordance with our previous results in tumor xenotransplants (Streit et al., 1999). Moreover, we found that the phosphorylation levels of VEGFR-2 in tumor lysates and the levels of vascular staining with the antibody GV39M, that recognizes VEGF bound to VEGFR-2 (Brekken et al., 1998), were comparable in SCCs induced in wild-type and TSP-1 transgenic mice. These results suggest that the inhibition of tumor vascularization during multi-step epithelial carcinogenesis by transgenic TSP-1 was not mediated by inhibitory effects on VEGF expression, bioactivity, or bioavailability.

The metastatic spread of tumor cells is responsible for the majority of cancer-related deaths. Continuous transgenic overexpression of TSP-1 during multi-step epithelial carcinogenesis resulted in a reduced incidence of SCC metastasis to the lungs, associated with reduced vascularity of the primary tumors. These results are in agreement with the previously reported inhibition of spontaneous pulmonary metastases by stable overexpression of TSP-1 in MDA-MB-435 breast cancer xenotransplants (Weinstat et al., 1994), and they suggest that the rate of hematogenous metastasis was reduced because of the pronounced inhibitory effect of TSP-1 on blood vascular tumor angiogenesis.

It has remained unclear whether angiogenesis inhibitors currently developed for clinical therapy might also affect lymphangiogenesis, the growth of lymphatic vessels. Lymphatic tumor spread to regional lymph nodes represents the most common pathway of initial tumor dissemination, and the detection of lymphatic metastasis serves as an important prognostic indicator of tumor aggressiveness and frequently determines the choice of anti-tumoral therapy. In contrast to the reduced incidence of lung metastases, we did not detect any difference in the incidence of lymphatic SCC metastasis to regional lymph nodes between TSP-1 transgenic and wild-type mice. Because recent experimental studies have revealed a correlation between tumor lymphangiogenesis and lymphatic metastasis (Karpanen et al., 2001; Mandriota et al., 2001; Skobe et al., 2001; Stacker et al., 2001), we next investigated the density and size of tumor-associated lymphatic vessels by using an antibody against the murine hyaluronan receptor LYVE-1 (Jackson et al., 2001) that specifically stains lymphatic vessels, but not blood vessels, in experimental tumor xenotransplants in mice (Skobe et al., 2001; Wigle et al., 2002) and in human tumors (Beasley et al., 2002). The specificity of the LYVE-1 antibody for tumor-associated lymphatic vessels has recently been questioned (Padera et al., 2002). However, our findings that all LYVE-1-positive, tumor-associated vessels also expressed Prox1, a transcription factor specifically expressed by normal and tumor-associated lymphatic endothelium (Oliver and Detmar, 2002; Wigle et al., 2002), confirm the specificity of LYVE-1 for tumor-associated lymphatic vessels also in chemically-induced murine SCC.

Using combined LYVE-1/CD31 double stains to quantify tumor lymphangiogenesis, we found that both intratumoral and peritumoral lymphatic vessels were present at comparable levels in both genotypes. Moreover, tumor-associated lymphatic endothelial cell proliferation was found at similar levels in wild-type and TSP-1 transgenic mice, revealing that active tumor lymphangiogenesis is not restricted to rapidly growing tumor xenotransplants but also occurs in slowly progressing tumors during orthotopic, multistep carcinogenesis. To the best of our knowledge, this is the first study to investigate the effects of an angiogenesis inhibitor on tumor lymphangiogenesis and demonstrate that TSP-1, despite its potent inhibition of angiogenesis and organ metastasis, did not inhibit lymphangiogenesis and lymphatic tumor spread. These findings have potential implications for the development of anti-angiogenic tumor therapy, because the prevention and/or treatment of cancer metastases, rather than the treatment of primary tumors, will likely represent its major therapeutic target. It remains to be investigated whether specific targeting of tumor lymphangiogenesis, either alone or in combination with anti-angiogenic therapy, might provide additional benefits for the prevention or treatment of advanced human cancers.

TSP-1 is thought to inhibit angiogenesis by a number of different mechanisms whose relative importance for different types of angiogenesis in benign or malignant conditions remain unclear (reviewed in Bornstein, 2001; Lawler, 2000). However, a recent study in CD36-deficient mice has provided strong evidence for an important role of the CD36 receptor on vascular endothelial cells for mediating the in vivo anti-angiogenic effects of TSP-1 (Jimenez et al., 2000). Using differential immunostains of murine SCC and of human skin for CD36, the lymphatic LYVE-1 and the blood vascular marker CD34 (Oliver and Detmar, 2002; Prevo et al., 2001), our study revealed little or no expression of CD36 in lymphatic vessels as compared with its strong expression in blood vessels, suggesting that different CD36 expression levels contribute to the differential effects of TSP-1 on angiogenesis versus lymphangiogenesis.

In summary, our studies provide evidence that TSP-1 mediated angiogenesis inhibition during multistep carcinogenesis predominantly conveys an early growth disadvantage of initiated/promoted premalignant epithelial cells but does not inhibit the subsequent development of malignant tumors, indicating that TSP-1 may prove efficacious in the chemoprevention of the early premalignant stages of epithelial tumorigenesis. Despite its potent suppression of blood vascular angiogenesis and distant organ metastasis, however, TSP-1 was unable to inhibit tumor lymphangiogenesis and lymphatic metastasis, with potential implications for the development of clinical anti-angiogenic therapies.

Materials and methods

Chemical skin carcinogenesis protocol

The construction of the keratin 14 (K14) promoter/TSP-1 transgene vector and the generation of transgenic FVB mice with targeted overexpression of TSP-1 in the skin have been previously described (Streit et al., 2000). Chemically-induced skin carcinogenesis was performed in heterozygous TSP-1 transgenic mice (n=30) and their wild-type littermates (n=29) as previously described (Hawighorst et al., 2001). The ratio of malignant conversion was calculated as the total number of carcinomas divided by the total number of papillomas, expressed as a percentage. At autopsy, all axillary and inguinal lymph nodes as well as the lungs of SCC-bearing mice were examined for the presence of metastases. The two-sided unpaired Student's t-test was used to analyse differences in the number of tumors per mouse between the two genotypes. In vivo metastasis data were evaluated using the Mann-Whiney test and the Alternate Welch test. All animal studies were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.

Histology, immunohistochemistry and in situ hybridization

Hematoxylin and eosin-stained sections were prepared from lymph nodes and from lungs at three random depths. The slides were evaluated using a Nikon E-600 microscope (Nikon, Melville, NY, USA), and mice were designated positive for the presence of metastasis when at least one colony of tumor cells was detected in the draining lymph nodes or lungs as previously described (Leone et al., 1993). Immunohistochemical staining was performed on 6 m paraffin sections or cryostat sections as previously described (Hawighorst et al., 2001), using a rat monoclonal antibody against CD31 (Pharmingen, San Diego, CA, USA) and the antibody GV39M (kindly provided by P Thorpe, Dallas, TX, USA and R Brekken, Seattle, WA, USA) that recognizes VEGF bound to VEGFR-2. For immunofluorescence single and double stains, 6 m frozen sections were stained with antibodies to human TSP-1 (kindly provided by R Swerlick, Atlanta, GA, USA), murine and human LYVE-1 (Prevo et al., 2001), Prox1 (Wigle et al., 2002), mouse keratin 10 and loricrin (Babco, Richmond, CA, USA), mouse CD31 (Pharmingen), human CD34 (Pharmingen), human CD36 (Immunotech, Marseille, France), and mouse CD36 (kindly provided by R Silverstein, New York NY, USA), and with corresponding secondary antibodies labeled with Alexa Fluor488 or 594 (Molecular Probes, Eugene, Oregon, OR, USA). In situ hybridization for human TSP-1 and murine VEGF was performed as described (Detmar et al., 1998; Streit et al., 2000). The VEGF riboprobe used recognizes all isoforms of mouse VEGF mRNA.

Computer-assisted morphometric vessel analysis

Representative CD31-stained frozen sections, obtained from biopsies of normal untreated skin (n=5), small papillomas (1-3 mm in diameter; n=6), large papillomas (>3 mm in diameter; n=10), and squamous cell carcinomas (SCC; n=5) were analysed as described (Hawighorst et al., 2001). For evaluation of lymphatic vessels, frozen sections of five different representative SCCs per group were double-stained with antibodies to LYVE-1 and CD31. Lymphatic vessels were quantified in tumor areas with the highest density of lymphatic vessels, using the IP lab software (Scanalytics). Statistical analysis was performed using the two-sided unpaired student's t-test.

Western blot analyses

Six different wild-type and TSP-1 transgenic SCCs were analysed, and three tumors each were pooled and homogenized in lysis buffer (2% SDS, 50 mM Tris pH 7.4, 20 mM phenylmethylsulfonyl fluoride, 50 g/ml leupeptin and 50 g/ml aproptinin). Samples were analysed by denaturing SDS-PAGE and were immunoblotted with an antibody against mouse VEGF (R&D Systems Inc., Minneapolis, MN, USA). Immunoreactive proteins were visualized using a chemiluminescense detection system (ECL; Amersham Arlington Heights, IL, USA). Levels of VEGF protein expression were quantified by densitometry.

Receptor phosphorylation assays

In vivo receptor phosphorylation assays for flk-1 were performed as recently described (Hawighorst et al., 2002). Briefly, six different wild-type and TSP-1 transgenic SCCs were homogenized in RIPA lysis buffer containing protease inhibitors, followed by immunoprecipitation with polyclonal antibody against mouse VEGFR-2 (flk-1; Santa Cruz). Immunocomplexes were recovered on protein-G-sepharose separated by 7.5% SDS-PAGE, and blotted onto a nitrocellulose membrane (Bio-Rad). Flk-1 phosphorylation was analysed by probing the membranes with a monoclonal anti-phosphotyrosine antibody (PY-20; ICN Biomedicals, Inc., Aurora, OH, USA). The same membranes were then stripped using TBST (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 2.4) and were probed for VEGFR-2 (flk-1; Santa Cruz). Immunoreactive proteins were visualized using the ECL Chemiluminescence detection system. Levels of VEGFR-2 phosphorylation were quantified by densitometry and were normalized to the expression levels of VEGFR-2.

Cell proliferation and apoptosis assays

Large papillomas of similar size were obtained from TSP-1 transgenic (n=3) and wild-type (n=3) mice. Proliferating tumor cells were visualized by double immunofluorescence stains with an FITC-conjugated anti-BrdU antibody (Pharmingen) and a rabbit antibody to mouse keratin K14, and were counted in an area overlying a total length of 21.3 mm of basement membrane in TSP-1 transgenic mice and of 25.4 mm in wild-type mice as described (Hawighorst et al., 2001). Apoptotic cells were identified by TUNEL staining using the Fluorescein-FragEL DNA fragmentation kit (Oncogene, Cambridge, MA, USA) according to the manufacturer's instructions. Nuclei were stained with propidium iodide in PBS (2 g/ml). Labeled tumor cells were counted in an area overlying a total length of 43.2 mm of basement membrane in TSP-1 transgenic mice and of 39.4 mm in wild-type mice. Results are expressed as the mean±s.e.m. of BrdU- or TUNEL-positive nuclei per mm basement membrane. The two-sided unpaired Student's t-test was used to analyse differences of proliferation and apoptosis rates.

Acknowledgements

The authors thank R Silverstein, P Thorpe, R Brekken and R Swerlick for the generous gift of antibodies, M Mihm and S Dadras for advice on mouse skin pathology, J Nagy for statistical advice and M Constant for technical assistance. This work was supported by NIH/NCI grants CA69184, CA86410 and CA91861 (M Detmar), by NIH grants GM58462 and EY12162 (G Oliver), by American Cancer Society Program Project Grant 99-23901 (M Detmar), by the Deutsche Forschungsgemeinschaft (T Hawighorst), by Deutscher Akademischer Austauschdienst (M Streit), by the Dermatology Foundation (M Streit), by the American Lebanese Syrian Associated Charities (G Oliver) and by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd., Agreement (M Detmar).

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Figure 1 Delayed and decreased skin carcinogenesis in TSP-1 transgenic mice. (a) Delayed and diminished incidence of papillomas in TSP-1 transgenic mice (n=30; filled circles), as compared with wild-type mice (n=29; open squares). Incidence is expressed as percentage of mice with detectable tumor formation (>1 mm). (b) Significantly decreased frequency of papilloma formation, expressed as the average number of papillomas per mouse, in TSP-1 transgenic mice. P<0.05 from weeks 7 to 9, P<0.01 from weeks 10 to 16, and P<0.001 after 17 weeks of PMA treatment. (c) Delayed and decreased incidence of squamous cell carcinoma (SCC) in TSP-1 transgenic mice. (d) Decreased average number of SCCs per mouse in TSP-1 transgenic mice. (P<0.05 from weeks 19 to 24, P<0.01 after 25 weeks). (e) Comparable ratio of malignant conversion of papillomas to SCC in TSP-1 transgenic (TG) and wild-type (WT) mice

Figure 2 In situ hybridization confirmed that strong TSP-1 mRNA expression was maintained in tumor cells of TSP-1 transgenic SCCs (b, d), whereas little or no TSP-1 mRNA expression was detected in wild-type SCCs (a, c). The dotted lines delineate the border between SCC and peritumoral mesenchymal stroma. Bright-field (a, b) and dark-field (c, d) micrographs. Immunofluorescence stains for TSP-1 (green) demonstrate deposition of TSP-1 protein in the stroma of TSP-1 transgenic SCC (f) as compared to little TSP-1 protein detection in wild-type SCCs (e). Propidium iodide (red) was used as a nuclear counterstain. (Bars=100 m)

Figure 3 Diminished tumor angiogenesis in TSP-1 transgenic mice. CD31 immunostains of untreated skin (a, b), early papillomas (c, d) and SCCs (e, f) of wild-type and TSP-1 transgenic mice demonstrate increased vascularization of papillomas and SCCs in wild-type mice, as compared with untreated skin. Tumor angiogenesis was less pronounced in TSP-1 transgenic mice, with a prominent reduction of enlarged angiogenic vessels. Bars=200 m. (g) Quantitative image analysis of CD31 stained vessels revealed a significantly decreased average vascular area in TSP-1 transgenic mice (filled bars) already at the early stage of papilloma formation and throughout the progressive stages of skin carcinogenesis, as compared with wild-type mice (open bars). Skin: untreated skin (n=5); SP: small papillomas (3 mm; n=6); LP: large papillomas (>3 mm; n=10); SCC: squamous cell carcinoma (n=5). Data are expressed as mean values+s.e.m. n.s.=not significant; ***P<0.001. (h, i) Significant increase in the number of apoptotic epithelial cells per mm of basement membrane in TSP-1 transgenic papillomas, as compared with wild-type tumors. No significant differences (n.s.; P=0.84) were found in the number of BrdU-labeled proliferating tumor cells. Data are expressed as mean values±s.e.m. (n=3). *P<0.05

Figure 4 Comparable VEGF expression and bioactivity in wild-type and TSP-1 transgenic SCCs. In situ hybridization demonstrates comparable VEGF mRNA expression by tumor cells in SCCs of wild-type (a, c) and TSP-1 transgenic mice (b, d). Bright-field (a, b) and dark-field (c, d) micrographs. Bars=100 m. (e) Western blot analysis revealed comparable VEGF protein expression in SCCs of both genotypes. (f) VEGFR-2 (flk-1) phosphorylation assays revealed no major differences between wild-type and TSP-1 transgenic SCCs. (g, h) Immunostains with the antibody GV39M, which recognizes VEGF bound to VEGFR-2, demonstrates comparable association of VEGF with tumor blood vessels in SCCs of both genotypes. Bars=100 m

Figure 5 LYVE-1 is a specific marker for tumor-associated lymphatic vessels in multistage skin carcinogenesis. (a-c) Double immunofluorescence stains of chemically-induced SCCs for the endothelial junction molecule CD31 (red) and LYVE-1 (green) demonstrated that only a fraction of all CD31-positive vessels, characterized by low-level expression of CD31, expressed LYVE-1 (arrows). (d-f) Differential stains of SCCs for the lymphatic-specific transcription factor Prox1 (green) and CD31 (red) revealed Prox1 positive tumor-associated lymphatic vessels (arrows), characterized by low-level CD31 expression. (g-i) Double immunofluorescence stains of SCCs for LYVE-1 (red) and Prox1 (green) revealed that all LYVE-1 positive lymphatic vessels also expressed Prox1 (arrows)

Figure 6 Transgenic overexpression of TSP-1 does not inhibit tumor lymphangiogenesis. (a, c) Immunofluorescence stains of SCCs for CD31 (red) and LYVE-1 (green) revealed that the density and the size of tumor-associated LYVE-1 positive lymphatic vessels was comparable in TSP-1 transgenic (TG) and wild-type (WT) tumors, whereas LYVE-1 negative/CD31 positive blood vessels were reduced in TSP-1 transgenic tumors. The presence of intratumoral lymphatics is revealed by nuclear counterstain with Hoechst (blue). (b, d) Immunostains for LYVE-1 (green) and BrdU (red) demonstrate comparable numbers of proliferating lymphatic endothelial cells (arrows) in wild-type and TSP-1 transgenic SCC. Bars=100 m. (e, f) Quantitative image analysis of LYVE-1 and CD31 double-stained sections demonstrated no major differences in the area covered by tumor-associated lymphatic vessels (e) or in the density of tumor-associated lymphatic vessels (f) between SCCs induced in wild-type (open bars) or TSP-1 transgenic (filled bars) mice

Figure 7 Sparse expression of the TSP-1 receptor CD36 on mouse and human lymphatic vessels. (a-c) Double immunofluorescence stains of murine SCCs for CD31 (red) and CD36 (green) distinguishes subfractions of CD31 positive vessels with strong (arrows) and sparse (arrowheads) expression of CD36. (d-f) Differential stains of murine SCCs for LYVE-1 (d; red) and CD36 (e; green) revealed little or no expression of CD36 in LYVE-1 positive lymphatic vessels (arrowheads), as compared with strong CD36 expression in LYVE-1 negative blood vessels (arrows). Bars=50 m. (g-i) Double immunofluorescence stains of normal human skin for CD34 (green) and CD36 (red) indicates strong CD36 expression in all CD34 positive blood vessels (arrows) while CD36 is only sparsely expressed in CD34 negative vessels (arrowheads). (j-k) Differential stains of human stains of human skin for LYVE-1 (red) and CD36 (green) revealed absent or low-level CD36 expression pattern in LYVE-1 positive lymphatic vessels (arrowheads) whereas LYVE-1 negative blood vessels strongly express CD36 (arrows). Bars=100 m