Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells

Abstract

Background Changes 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. In particular, breast cancer-associated adipogenic stromal cells not only enhance the composition, quantity, and rigidity of deposited Fn, but also partially unfold these matrices. However, the specific effect of Fn conformation on tumor angiogenesis is undefined. Methods Decellularized matrices and a conducting polymer device consisting of poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) were used to examine the effect of Fn conformation 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 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.

3D conducting polymer platforms for electrical control of protein conformation and cellular functions

“…39 Moreover, this tumor-associated partial unfolding of Fn has been implicated in the enhanced secretion of pro-angiogenic factors by adipogenic precursor cells, suggesting a link to tumor angiogenesis. 10 Since our experiments showed that we could alter Fn conformation by manipulating the redox state of the PEDOT:PSS scaffolds, we sought to assess whether this could affect the pro-angiogenic capability of 3T3-L1 cells cultured in oxidized and reduced scaffolds. We cultured 3T3-L1s in oxidized and reduced scaffolds, as described above, and after 24 hours, we collected the media from the wells in which the scaffolds were cultured.…”

“…To quantify pro-angiogenic potential, we measured the concentration of vascular endothelial growth factor (VEGF) present in the media using ELISA (normalized to the number of cells, as measured by DNA assay), and found that the cells cultured in reduced scaffolds secreted 4 times more VEGF than those cultured in oxidized scaffolds (Figure 5b). Previous studies using both Fn molecular films 10 and thin Fn fibrillar networks 40 have shown that altered Fn conformation controls adhesion and pro-angiogenic behaviors via an integrin-switching mechanism (in particular, the preferential engagement of α v β 3 rather than α 5 β 1 integrins in the presence of unfolded Fn); however, further studies are required to determine if the voltage-induced alterations of Fn conformation are similarly responsible for the observed cell behavior in our PEDOT:PSS scaffolds. Nonetheless, these results suggest that the reduced Fn-coated scaffolds mimic to some degree the conformation of tumor-associated Fn fibers (as measured by FRET), which enhance the pro-angiogenic capability of adipogenic precursors.…”

“…Interestingly, we note that the difference in VEGF secretion we observed between the two redox states in the 3D PEDOT:PSS scaffolds is significantly enhanced when compared to previous results obtained with a 2D thin film system. 10 This difference points toward the importance of physiologically-relevant 3D cell culture systems, which can perhaps more fully capture the in vivo magnitude of cell responses that might otherwise be muted in 2D cell culture systems.…”

In vitro models of tumor vessels and matrix: Engineering approaches to investigate transport limitations and drug delivery in cancer

“…Applying differential voltages to poly (3,4-ethylenedioxythiophene) doped with p -toluenesulfonate (PEDOT:TOS) permits selective manipulation of the conformation of adsorbed fibronectin (Figure 4D) [151]. Such systems have been used to elucidate that fibronectin conformational changes influence cell adhesion and secretory patterns for both stromal and tumor cells [176, 177]. Yet, fibronectin does not occur in isolation, but is part of complex ECM scaffolds, which can be partially mimicked with fibroblast-derived 3D ECMs.…”

Utilizing Fibronectin Integrin-Binding Specificity to Control Cellular Responses

“…Unfolded Fn favors avb3 engagement over a5b1 which, in turn, enhances the secretion of VEGF, a proangiogenic growth factor, in tumor stromal cells. 61 Stem cell differentiation and renewal. The binding of a5b1 to Fn was shown to be critical for the proliferation and differentiation of progenitor cells into both osteoblasts and myoblasts.…”