Abstract The impact of psychosocial stress on breast cancer pathogenesis has become an area of intense research in the past several years. Sympathetic nervous system (SNS) activation and release of the catecholamine norepinephrine (NE) is a major stress pathway that promotes breast cancer pathogenesis. We have evidence that SNS activation can regulate breast tumor growth and metastasis by altering the tumor extracellular matrix (ECM), specifically fibrillar collagen. Alterations in fibrillar collagen microstructure can be detected using second harmonic generation multiphoton laser scanning microscopy (SHG MPLSM). This non-linear optical technique takes advantage of endogenous light scattering by non-centrosymmetric structures, including collagen. Stress-induced changes to the tumor collagen microstructure can be quantified by measuring the ratio of forward- to backward-scattered light (F/B ratio), as well as the directional coherence of resident collagen fibers. Here, we demonstrate that NE can directly stimulate breast cancer cells and tumor stromal cells to elicit changes in collagen microstructure. In these experiments, cells were pretreated with NE, washed to remove NE, and placed in 3D-type I collagen matrices. Pretreatment of MDA-MB-231 metastatic breast cancer cells with NE significantly lowered the F/B ratio and decreased fiber directional coherence of type I collagen gels. Treatment of MB-231 cells with the β2-adrenergic receptor (β2-AR) selective agonist, terbutaline, mimicked the effects of NE. Furthermore, NE pretreatment of the β-AR-negative metastatic breast cancer line, 4T1, did not elicit alterations in SHG F/B ratio or fiber coherence in collagen gels, compared to control cells. These results suggest that NE acts through tumor cell β2-AR to alter collagen microstructure. Future experiments will elucidate the molecular mediators of this collagen reorganization and determine the impact of such changes on tumor metastatic capacity. These experiments will provide new insights into mechanisms underlying stress-induced tumor pathogenesis that may reveal new therapeutic avenues for the treatment of breast cancer. Citation Format: Ryan P. Dawes, Kathleen A. Burke, Petr Stastka, Edward B. Brown, Kelley S. Madden. The neurotransmitter norepinephrine and β2-AR activation of MB-231 breast cancer cells alters fibrillar collagen structure. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A103.