Graphical abstract

Cell adhesion to the extracellular matrix is deregulated in metastasis. However, traditional surfaces used to study cell adhesion do not faithfully mimic the in vivo microenvironment. Electron beam lithography (EBL) is able to generate customized protein nanopatterns. Here, we used an EBL-based green lithography approach to fabricate homogeneous and gradient, single (fibronectin, K-casein) and double (fibronectin, laminin) active component protein nanopatterns with micrometer scale spacing to investigate differences in adhesion of breast cancer cells (BCC) and normal mammary epithelial cells (NMEC). Our results showed that as expected, in contrast to NMEC, BCC were plastic: they tolerated nonadhesion promoting regions, adapted to flow and exploited gradients better. In addition, the number of focal adhesions but not their area appeared to be the dominant parameter for regulation of cell adhesion. Our findings also demonstrated that custom designed protein nanopatterns, which can properly mimic the in vivo microenvironment, enable realistic distinction of normal and cancerous cell adhesion. KEYWORDS: Focal adhesion, fibronectin, laminin, nanopattern, cancer, electron beam lithography T he microenvironment of cells plays key roles in metastasis, angiogenesis, tumorigenesis, embryonic development, tissue homeostasis, atherosclerosis, and wound healing. Studies on cell adhesion to extracellular matrix routinely use uniformly coated surfaces that do not truly represent the extracellular matrix in vivo that is comprised of nanometer scale adhesive patches separated by micrometer scale spacing. 1−5 Focal adhesions (FAs) are protein compositions involved in the biomechanical and biochemical interactions of cells with the extracellular matrix. The extracellular and intracellular parts of a FA are extracellular matrix proteins (e.g., fibronectin, laminin) and cytoskeletal proteins (e.g., actin, vinculin), respectively. 5−10 Although there are significant cell adhesion studies that use surfaces fabricated to mimic the extracellular matrix at the nanoand micrometer scales, 11−24 the regulation of cell morphology and FA features by the spatial arrangement of the extracellular matrix remains incompletely understood. 15,21 Furthermore, there are no studies comparing adhesion of normal and cancer cells on nanopatterns. Cell adhesion to the extracellular matrix is one of the major processes that is disturbed in cancer cells. 25 For instance, expression levels of molecules that mediate cell-tomatrix adhesion such as integrins are known to be altered in cancer cells. 26 Altered adhesion appears to favor metastasis and therefore a deeper understanding of the differences in cell adhesion between normal and cancer cells is desired. 27,28 Among known patterning techniques, electron beam lithography offers considerable practical and technical advantages that provide well-controlled nanofabricated biomimetic surfaces with defined pattern geometries. 29−32 Previous work showed that vinculin and cytoskeletal organization are mo...

Cell adhesion to extracellular matrix is an important process for both health and disease states. Surface protein patterns that are topographically flat, and do not introduce other chemical, topographical or rigidity related functionality and, more importantly, that mimic the organization of the in vivo extracellular matrix are desired. Previous work showed that vinculin and cytoskeletal organization are modulated by size and shape of surface nanopatterns. However, quantitative analysis on cell morphology and focal adhesions as a function of micrometer scale spacings of FN nanopatterns was absent. Here, electron beam lithography was used to pattern fibronectin nanodots with micrometer scale spacings on a Kcasein background on indium tin oxide coated glass which, unlike silicon, is transparent and thus suitable for many light microscopy techniques. Exposure times were significantly reduced using the line exposure mode with micrometer scale step sizes. Micrometer scale spacings of 2, 4 and 8 μm between fibronectin nanodots proved to modulate cell adhesion through modification of cell area, focal adhesion number, size and circularity. Overall, cell behavior was shown to shift at the apparent threshold of 4 μm spacing. The findings presented here offer exciting new opportunities for cell biology research.

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