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. 2001 Oct 23;98(22):12485-90.
doi: 10.1073/pnas.171460498. Epub 2001 Oct 16.

Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor

J C Rodriguez-Manzaneque  1 , T F Lane, M A Ortega, R O Hynes, J Lawler, M L Iruela-Arispe
Affiliations

Thrombospondin-1 suppresses spontaneous tumor growth and inhibits activation of matrix metalloproteinase-9 and mobilization of vascular endothelial growth factor

J C Rodriguez-Manzaneque et al. Proc Natl Acad Sci U S A. .

Abstract

Growth of tumors and metastasis are processes known to require neovascularization. To ascertain the participation of the endogenous angiogenic inhibitor thrombospondin-1 (TSP1) in tumor progression, we generated mammary tumor-prone mice that either lack, or specifically overexpress, TSP1 in the mammary gland. Tumor burden and vasculature were significantly increased in TSP1-deficient animals, and capillaries within the tumor appeared distended and sinusoidal. In contrast, TSP1 overexpressors showed delayed tumor growth or lacked frank tumor development (20% of animals); tumor capillaries showed reduced diameter and were less frequent. Interestingly, absence of TSP1 resulted in increased association of vascular endothelial growth factor (VEGF) with its receptor VEGFR2 and higher levels of active matrix metalloproteinase-9 (MMP9), a molecule previously shown to facilitate both angiogenesis and tumor invasion. In vitro, enzymatic activation of proMMP9 was suppressed by TSP1. Together these results argue for a protective role of endogenous inhibitors of angiogenesis in tumor growth and implicate TSP1 in the in vivo regulation of metalloproteinase-9 activation and VEGF signaling.

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Figures

Figure 1
Figure 1
Generation and analysis of transgenic animals. (A) Southern blots were probed with a fragment of the simian virus 40 polyA sequence that identifies a 7.5-kb band for the neu (lanes 1, 2, 3, 7, 9, and 11) and a 0.9-kb band for the human TSP1 transgenes (lanes 2, 3, and 5), respectively. A mouse TSP1 probe was used to identify offspring homozygous for the mutated allele (tbsp1−/−) (5.8 kb) (lanes 10 and 11), homozygous for the wild-type allele (4.4 kb) (lanes 6 and 9), or heterozygous with both alleles (lanes 7 and 8). (B) ELISA analysis of total TSP1 protein levels from: (i) wild type (WT), TgN-hTSP1 and tbsp1−/− mammary gland tissue; and (ii) TgN-neu, neu-hTSP1, and neu-tbsp1−/− mammary tumor tissue. Bars indicate standard error. (C) Localization of TSP1 in tumors. In situ hybridization for mouse TSP1 mRNA in TgN-neu (a) and for human TSP1 mRNA in neu-hTSP1 (b) tumors. Immunodetection of TSP1 protein on TgN-neu (c) and neu-hTSP1 (d) tumors. Red arrows indicate TSP1 expression and dashed line (in b) denotes the boundary between tumor and stroma. Yellow arrowheads identify blood vessels. T, tumor; S, Stroma.
Figure 2
Figure 2
Characterization of tumors. (A) Tumor formation in transgenic female carriers of neu alone (TgN-neu) or neu in the absence (neu-tbsp1−/−) or presence of constitutive TSP1 (neu-hTSP1) were followed for 48 weeks. The trend for earlier appearance of tumors across the three groups was statistically significant (P < 0.001). Pair-wise comparisons demonstrated that the differences among the three curves were also statistically significant (P < 0.01). n = 20 in each category. (B) Average size of tumors at 24 weeks. The difference between neu-tbsp1−/− and TgN-neu tumors was statistically significant (P = 0.0039, Student's t test). (C) Kinetics of tumor growth over 30 days. Average values ± SE are presented (n = 5).
Figure 3
Figure 3
Vascular profile of tumors. (A) Evaluation of TgN-neu (a–c), neu-hTSP1 (d–f), and neu-tbsp1−/− (g–i) tumors. a, d, and g, show macroscopic images of tumors. In b, e, and h, vessels are identified with a PECAM antibody. In c, f, and i, vascular structures are visualized after a single intravascular injection of FITC-conjugated lectin followed by confocal microscopy. (B) Quantitation of vascular parameters. Real and computerized images of vessel perimeter (green), lumen area (red), and vessel number are shown alongside quantitation. Bars indicate standard error. Statistical significance (Student's t test) was found between neu-tbsp1−/− and TgN-neu groups for vessel perimeter (P = 0.001) and for lumen area (P = 0.00046). For number of vessels, statistical significance (Student's t test) was found between TgN-neu and neu-hTSP1 tumors (P = 0.006).
Figure 4
Figure 4
Expression of gelatinases in tumors. (A) Gelatin zymography of tumor extracts (5 μg of protein). Percentage of active versus total MMP9 is shown for each lane. The graph represents a summary of the percentage of active MMP9 versus total MMP9 for each group. Differences are statistically significant (Student's t test) compared with TgN-neu control group (for neu-hTSP1, P = 0.01; and for neu-tbsp1−/−, P = 0.002). (B) Immunoblot analysis of tumor extracts with MMP9 antibody. Two images of the blot appear representing different exposure times to show the rarer low-molecular-weight active MMP9 form. (C) The same blot was reprobed with MMP2 antibody.
Figure 5
Figure 5
In vitro inhibition of MMP9 activation by TSP1. (A) Purified proMMP9 preincubated in the presence or absence of purified TSP1 was subsequently treated with the activators MMP3 or p-aminophenyl mercuric acetate. The reaction was analyzed by immunoblot (Top) and gelatin zymography (Bottom). (B) Purified proMMP9 preincubated in the absence (1, 3, and 4) or presence of TSP1 (2 and 5) was left untreated (1 and 2) or treated with MMP3 (–5). After MMP3 activation, TSP1 was added to sample 4. All samples were analyzed for gelatinase activity. Data are presented as percentage of maximum activation with MMP3. Each reaction was also analyzed by zymography (Inset). (C) Purified proMMP9 preincubated in the presence of increasing amounts of TSP1 was treated with MMP3. Half of the reaction was diluted in 200 μl and analyzed for gelatinase activity. Data are presented as percentage of maximum activation with MMP3. Each point represents the average of three independent experiments.
Figure 6
Figure 6
Evaluation of VEGF levels and distribution in tumors. (A) Equal protein amounts were analyzed by immunoblot with a specific antibody for VEGF. (B) Mammary tumor sections from TgN-neu (a–c), neu-hTSP1 (d–f), neu-tbsp1−/− (g–i) mice were evaluated for expression of VEGF by using two antibodies: GV39 M, which recognizes VEGF bound to VEGFR2 (b, e, h) and a polyclonal anti-mouse VEGF, which recognizes the receptor-free form (c, f, i). Staining with PECAM-specific antisera was used for identification of vessels (a, d, g).
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