Background Adjustments in fibronectin (Fn) matrix remodeling contribute to mammary tumor angiogenesis and are related to altered behavior of adipogenic stromal cells; yet the underlying mechanisms remain unclear due in part to a lack of reductionist model systems that allow the inherent complexity of cell-derived extracellular matrices (ECMs) to be deciphered. on the behavior of 3T3-L1 preadipocytes. Changes in cell adhesion and proangiogenic capability were tested via cell counting and by quantification of vascular endothelial growth factor (VEGF) secretion respectively. Integrin-blocking antibodies were utilized to examine varied integrin specificity as a potential mechanism. Results Our findings suggest that tumor-associated partial unfolding of Fn decreases adhesion while enhancing VEGF secretion by breast cancer-associated adipogenic precursor FLJ42958 cells and that altered integrin specificity may underlie these changes. Conclusions and general significance These results not only have important implications for our understanding of tumorigenesis but also enhance knowledge of cell-ECM interactions that may be harnessed for other applications including advanced tissue engineering approaches. tests were used to compare pairs of data sets and a p-value of less than 0.05 was considered statistically significant. Data are represented as average ± standard deviation of at least 3 independent experiments. 3 Results and Discussion 3.1 Tumor stromal cell-derived matrices modulate stromal cell proangiogenic capability We previously reported that tumor-derived soluble factors induce adipogenic precursor cells to (i) self-stimulate their proangiogenic capability in an ECM-dependent manner [3] and (ii) elevate Fn matrix deposition [11]. However it remains unclear whether or not altered Fn matrix assembly directly contributes to the increased proangiogenic potential of tumor-associated adipogenic precursors. To investigate this possible functional link we first evaluated the effect of ECMs deposited by control and tumor-associated adipogenic stromal cells on the behavior of native adipogenic precursors. To this end Hordenine 3 preadipocytes were cultured in TCM and control media followed by detergent-based decellularization as Hordenine previously reported [3]. Immunostaining of these matrices confirmed our ability to generate cell-free tumor-mimicking matrices characterized by increased levels of fibrillar Fn (Fig. 2A) that is partially unfolded [11] relative to the control matrices. Interestingly reseeding and analysis of new 3T3-L1s onto these matrices revealed that tumor-conditioned matrices inhibited adhesion (30%) relative to control ECMs (Fig. 2B) whereas VEGF secretion per cell was enhanced (47%) in the tumor relative to the control condition (Fig. 2C). Different VEGF levels were related to Hordenine altered VEGF synthesis rather than differential VEGF sequestration in the matrices; control and tumor-conditioned matrices contained only negligible amounts of VEGF (approx. 8% of VEGF measured in the media) that were not significantly different between conditions (Fig. 2D) and VEGF mRNA levels of cells cultured on tumor-conditioned matrices were greater (59%) relative to cells on control ECMs. These data suggest that Fn matrix content/conformation and stromal cell VEGF secretion may be correlated. However as decellularized matrices contain various other ECM molecules including collagen I and proteoglycans [27] we next performed experiments to more directly confirm the contribution of Fn to the observed changes. We produced decellularized matrices devoid of Fn fibrils using pUR4 a peptide capable of blocking Fn polymerization [28]. Indeed addition of pUR4 inhibited Fn incorporation into the TCM-treated stromal cell matrices (Fig. 3A) which increased adhesion (Fig. 3B) and decreased VEGF secretion (Fig. 3C) of reseeded 3T3-L1s to levels comparable to control matrices. Hordenine While these experiments confirm that Fn regulates the adhesive and proangiogenic ability of stromal cells observed with decellularized matrices we note that blockade of Fn matrix assembly may interfere with deposition of other ECM components including collagen [29] and it is possible that these changes partially contribute to our results. Figure 2 Decellularized tumor-associated matrices influence adipogenic stromal cell adhesion and proangiogenic factor secretion Figure 3 Fn influences cell behavior in response to control and tumor ECMs To determine the functional relevance of stromal cell secretory changes to endothelial cell behavior we Hordenine collected media from 3T3-L1 cells cultured on control and tumor ECMs and studied their effect on HUVEC migration via a transwell assay. Corresponding with the previously detected increased.