Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas

Abstract

The function of the endogenous angiogenesis inhibitor thrombospondin-1 (TSP-1) in epithelial tumor development has remained controversial. We studied the in vitro growth characteristics and the in vivo tumor xenograft growth of the human squamous cell carcinoma cell lines A431 and SCC-13, stably transfected to overexpress human TSP-1. Overexpression of TSP-1 inhibited tumor growth of A431 xenotransplants, and completely abolished tumor formation by SCC-13 cells. TSP-1 overexpressing A431 tumors were characterized by extensive areas of necrosis and by decreased tumor vessel number and size. The effects of TSP-1 on tumor cell growth were indirect since tumor cell proliferation rates in vivo and in vitro, anchorage-dependent and -independent growth in vitro, and susceptibility to induction of apoptosis by serum withdrawal were unchanged in TSP-1 overexpressing tumor cells. However, TSP-1 overexpression up-regulated the TSP-1 receptor CD36, leading to enhanced adhesion of A431 cells to TSP-1. These findings establish TSP-1 as a potent inhibitor of angiogenesis and tumor growth in carcinomas of the skin.

Figure 1.

A: Overexpression of human TSP-1 mRNA in stably transfected A431 and SCC-13 cell clones as compared to vector transfected control clones was confirmed by Northern Blot analysis. mRNA expression of human VEGF remained unchanged in TSP-1 overexpressing and control clones. Hybridization with a human β-actin probe served as control for equal loading. B: Western blot analysis demonstrated increased amounts of TSP-1 protein in cell lysates and conditioned media obtained from TSP-1 transfected A431 cells. TSP-1 migrates at approximately 180 kd. Control transfected SCC-13 cell clones synthesized substantial amounts of TSP-1. Increased TSP-1 secretion was detected in all three TSP-1 transfected cell clones (C1–3). The secretion of VEGF is not modified by TSP-1 overexpression in A431 and SCC-13 cell clones. HDMEC synthesize but do not secrete detectable amounts of TSP-1. C: Mitogenic effect of conditioned media obtained from control-transfected A431 clones (C1–3) as compared to unconditioned medium (M). Conditioned media obtained from TSP-1 transfected A431 clones (T10, T12, T19) significantly inhibited HDMEC proliferation, as determined by BrdU incorporation (P < 0.01). Bars represent mean values ± SD of two independent experiments. D: Co-incubation with a TSP-1 neutralizing antibody (anti-TSP-1) but not with an isotype-specific control antibody (IgG) abolished the inhibitory effect of TSP-1 (25 μg/ml) added to unconditioned medium (open bars) or of conditioned medium harvested from a TSP-1 overexpressing A431 cell clone (T19) (closed bars) on HDMEC proliferation. E: Conditioned media obtained from TSP-1 transfected SCC-13 cell clones (T1–3) but not from control-transfected clones (C1–3) inhibited HDMEC proliferation.

Figure 2.

A: No influence of transfected TSP-1 on the anchorage-dependent growth of A431 cell clones (T10, T12, T19), as compared to control transfectants (C1–3). B: No major differences in anchorage-independent growth of TSP-1 transfected and control-transfected A431 clones, as determined in a soft agar assay. Mean values ± SD of two independent experiments. C, D: Rarefication of large tumor blood vessels supplying TSP-1 overexpressing A431 xenotransplants (D), as compared to control transfected A431 tumors (C). Scale bar = 1 cm. E: Decreased tumor growth of TSP-1 overexpressing A431 cells (T10, T12, T19), as compared to control-transfected clones (C2, C3). Values represent mean values ± SEM for 10 tumors for each clone and time point. F: Complete inhibition of tumor growth of TSP-1 overexpressing SCC-13 clones T2 and T3 (values identical to T2), as compared to control clones C2 and C3. Note different scale of the y axis, as compared to Figure 2E ▶ .

Figure 3.

In situ hybridization demonstrates strong TSP-1 mRNA expression in 3-week-old TSP-1 transfected A431 xenografts (C, D), whereas little or no TSP-1 mRNA expression was detected in control tumors (A, B). In contrast, VEGF mRNA expression remained unchanged in TSP-1 transfected A431 xenotransplants (G, H), as compared to control tumors (E, F). Scale bar = 200 μm.

Figure 4.

Extensive areas of tumor necrosis (*) in TSP-1 overexpressing A431 xenotransplants (B), as compared to control tumors (A); hematoxylin-eosin stain. Scale bar = 200 μm. Immunohistochemical staining with an anti-mouse CD31 monoclonal antibody revealed inhibition of angiogenesis in TSP-1 overexpressing tumors (D), as compared to control tumors (C). E: Moderate reduction of microvascular densities in TSP-1 overexpressing tumors, as measured by the vessel number per mm tumor area. F: Significant decrease of the area covered by blood vessels in TSP-1 overexpressing tumors compared to control tumors (P < 0.001). G, significant reduction of the average blood vessel area (P < 0.001); H, loss of blood vessels of more than 2000 μm in TSP-1 overexpressing tumors, as compared to control tumors. CD31-stained blood vessels were evaluated in three different 10× fields in sections of five different tumors derived from each A431 cell clone. Data are expressed as mean values ± SEM.

Figure 5.

A–D: Immunohistochemical analysis demonstrates strong TSP-1 staining in TSP-1 transfected A431 xenotransplants, predominantly localized in the tumor stroma (B), as compared to the sparse labeling observed in control tumors (A). Little or no detectable CD36 expression in control A431 tumors (C), but strong CD36 expression in TSP-1 transfected xenotransplants (D). Scale bar = 200 μm. E, F: Western blot analysis demonstrates increased CD36 expression in cell culture lysates obtained from TSP-1 transfected A431 clones (T10, T12, and T19) (E) and SCC-13 cell clones (T1-T3) (F), as compared to control clones. G: CD36 expression is induced in cultured A431 cells (C1, ninefold; C2, sixfold; C3, 1.5-fold) after 24 h incubation with conditioned medium obtained from TSP-1 overexpressing clones. H: Significantly increased adhesion of TSP-1 transfected A431 cells to immobilized TSP-1, as compared to control cells (P < 0.001). Incubation with an anti-CD36 blocking antibody significantly (P < 0.001) reduced adhesion of TSP-1 transfected A431 cells to levels observed in control cells. I: No significant difference in adhesion to collagen type I between control and TSP-1 transfected A431 cells. An anti-CD36 blocking antibody did not modify A431 cell adhesion to collagen type I. Scale bars represent mean values ± SD of two independent experiments.