The antiangiogenic extracellular matrix protein thrombospondin-1 (TSP-1) inhibits tumor growth and metastasis in animals. However, the clinical relevance of such findings are equivocal as increased stromal TSP-1 expression has been associated with either good or poor prognosis. In an effort to obtain a more integrated understanding of the role of TSP-1 in breast cancer, we first used a breast tumorigenesis model in which tumor-associated stromal fibroblasts were engineered to produce high levels of TSP-1. We demonstrate here that stromal TSP-1 delayed human MDA-MB-231/B02 breast tumor growth. However, this delay in MDA-MB-231/B02 tumor growth upon exposure to TSP-1 was associated with an increased vascular endothelial growth factor (VEGF) expression in tumor cells themselves, leading to a tumor growth rate comparable to that of tumors whose fibroblasts did not overproduce TSP-1. Clinical evidence also suggested that primary breast carcinomas have adapted to escape the effects of stromal TSP-1. TSP-1 was found to be expressed in the stroma of human breast carcinomas where, although its level correlated with decreased vascularization, it was unexpectedly associated with a reduction of relapse-free survival. In metastatic axillary lymph nodes, tumor cells expressed high levels of VEGF and TSP-1 expression were no longer associated with a decreased vascularization. Overall, these results suggest that a resistance may develop early in human breast cancers as a result of high in situ exposure to stromal TSP-1, leading to disease progression. © 2005 Wiley-Liss, Inc.