Fibroblasts are among the most abundant stromal cells in the tumor microenvironment (TME), progressively differentiating into activated, motile, myofibroblast‐like, protumorigenic cells referred to as Cancer‐Associated Fibroblasts (CAFs). To investigate the mechanisms by which epithelial cells direct this transition, the early stages of tumorigenesis were exemplified by indirect cocultures of WI‐38 or human primary breast cancer fibroblasts with human mammary epithelial cells expressing an inducible c‐Myc oncogene (MCF10A‐MycER). After c‐Myc activation, the conditioned medium (CM) of MCF10A‐MycER cells significantly enhanced fibroblast activation and mobilization. As this was accompanied by decreased insulin‐like growth factor binding protein‐6 (IGFBP‐6) and increased insulin‐like growth factor‐1 and IGF‐II (IGF‐I, IGF‐II) in the CM, IGFs were investigated as key chemotactic factors. Silencing IGFBP‐6 or IGF‐I or IGF‐II expression in epithelial cells or blocking Insulin‐like growth factor 1 receptor (IGF‐1R) activity on fibroblasts significantly altered fibroblast mobilization. Exposure of WI‐38 fibroblasts to CM from induced MCF10A‐MycER cells or to IGF‐II upregulated FAK phosphorylation on Tyr³⁹⁷, as well as the expression of α‐smooth muscle actin (α‐SMA), features associated with CAF phenotype and increased cell migratory/invasive behavior. In three‐dimensional (3D)‐organotypic assays, WI‐38 or human primary fibroblasts, preactivated with either CM from MCF10A‐MycER cells or IGFs, resulted in a permissive TME that enabled nontransformed MCF10A matrix invasion. This effect was abolished by inhibiting IGF‐1R activity. Thus, breast epithelial cell oncogenic activation and stromal fibroblast transition to CAFs are linked through the IGFs/IGF‐1R axis, which directly promotes TME remodeling and increases tumor invasion.

Among peritumoral cells, cancer-associated fibroblasts (CAFs) are major facilitators of tumor progression. This study describes the effects of two urokinase-derived, novel decapeptides, denoted as Pep 1 and its cyclic derivative Pep 2. In a mouse model of tumor dissemination, using HT1080 fibrosarcoma cells, Pep 2 reduced the number and size of lung metastases. Specific binding of fluoresceinated Pep 2 to HT1080 and telomerase immortalised fibroblasts (TIF) cell surfaces was enhanced by αv overexpression or abolished by excess vitronectin, anti-αv antibodies or silencing of ITGAV αv gene, identifying αv-integrin as the Pep 2 molecular target. In 3D-organotypic assays, peptide-exposed TIFs and primary CAFs from breast carcinoma patients both exhibited a markedly reduced pro-invasive ability of either HT1080 fibrosarcoma or MDA-MB-231 mammary carcinoma cells, respectively. Furthermore, TIFs, either exposed to Pep 2, or silenced for αv integrin, were impaired in their ability to chemoattract cancer cells and to contract collagen matrices, exhibiting reduced α-smooth muscle actin (α-SMA) levels. Finally, peptide exposure of αv-expressing primary CAFs led to the downregulation of α-SMA protein and to a dramatic reduction of their pro-invasive capability. In conclusion, the ability of the novel decapeptides to interfere with tumor cell invasion directly and through the down-modulation of CAF phenotype suggests their use as lead compounds for co-targeting anti-cancer strategies.