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. Author manuscript; available in PMC: 2014 Jan 1.
Published in final edited form as: Nat Cell Biol. 2013 Jun 2;15(7):807–817. doi: 10.1038/ncb2767

Figure 3.

Figure 3

Thrombospondin-1 is an angiocrine tumor suppressor. Lung- and BoMa-like stroma and microvascular niche cultures were decellularized and residual proteins were acid extracted and subjected to LC-MS/MS analysis. (a) Heatmap of ECM proteins (spectral counts) from lung-like microvascular niche (LF + EC) normalized by lung stroma (LF; sorted high to low) and BoMa-like microvascular niche (MSC + EC) normalized by BoMa stroma (MSC). Log2 intensity scale shown at lower left. Localized expression of TSP-1 at the interface between dormant DTCs and lung microvasculature was confirmed in (b) spontaneous and (c) experimental metastasis models (white arrowheads). Scale bars = 10 μm. TSP-1 localization to the vascular BM was also confirmed in non-tumor bearing mice in (d) lung, (e) bone and (f) brain. Scale bars: 10 μm for lung, 20 μm for others. (g) Endothelial source of TSP-1 was confirmed by utilizing a 3D model of capillary morphogenesis where sprouting ECs are separated from inductive LFs by several millimeters. TSP-1 colocalized with type IV collagen in the BM of established microvessel stalks (white arrowheads), but TSP-1 appeared to be downregulated at neovascular tips (white asterisks). Scale bar = 50 μm. This was confirmed by (h) quantification of TSP-1 intensity at stalks vs. tips (n=16 microvessels pooled from 3 different experiments). Error bars denote s.e.m. ***p=0.0005 by two-tailed paired t-test. (i) Add-back of TSP-1 to T4-2 cells plated on lung stroma effectively substituted for the presence of ECs by causing a significant reduction in tumor cell growth (normalized to vehicle condition; n=3 sets of co-cultures analyzed per condition). Error bars denote s.e.m. * p<0.05 when compared to Vehicle by one-way ANOVA and Dunnett’s multiple comparisons test. (j) Treating lung-like microvascular niches with a TSP-1 blocking antibody resulted in significantly enhanced tumor cell growth (vs. IgG control; n=5 sets of co-cultures analyzed per condition). Error bars denote s.e.m. **p=0.0054 by two-tailed unpaired t-test.