Differentiation of Normal and Cancer Cell Adhesion on Custom Designed Protein Nanopatterns

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...

Modulating Tumor Cell Functions by Tunable Nanopatterned Ligand Presentation

“…Interestingly, invadopodia formation can be initiated by large distances between certain ligands (i.e., low ligand density), as shown by fibronectin-coated gradient nanopatterns created by electron beam lithography [62]. Similar fibronectin-coated gradient nanopatterns demonstrated that breast cancer cells but not normal mammary cells can adapt flexibly to different ligand densities [63]. Marked cellular flexibility on variable ligand densities was also detected in melanoma cells exposed to Au nanopatterns functionalized with N-or E-cadherin.…”

“…The second method that is widely used to create nanopatterns to address biological questions is electron beam lithography (EBL, Figure 2b). This top-down technique uses a focused electron beam to draw nanoscopic patterns on a surface covered with an electron-sensitive layer [62][63][64]. The latter is called a resist.…”

“…In general, biophysical parameters such as ligand density as well as ligand specificity determine important progression-related functions of tumor cells. On the one hand, tumor cells can adapt flexibly to different ligand site densities [63,76]. On the other hand, ligand density variations can induce phenotypic switches resembling EMT [80].…”

Biomimicking Nano‐Micro Binary Polymer Brushes for Smart Cell Orientation and Adhesion Control

“…However, these uniform topographies or micropatterned chemical cues do not mimic well the real ECM. In contrast, ECM in vivo consists of a combination of directional nano‐ and microscale features that are both cell‐adhesive . Very recently, several papers have demonstrated the uniqueness of these multiscale surfaces in regulating cell adhesion, migration, orientation, and differentiation .…”

“…In contrast, ECM in vivo consists of a combination of directional nano‐ and microscale features that are both cell‐adhesive . Very recently, several papers have demonstrated the uniqueness of these multiscale surfaces in regulating cell adhesion, migration, orientation, and differentiation . It is therefore highly desirable that one can mimic such multiscale surfaces for the better understanding of the cell behavior, and for the development of more efficient and smarter substrates for biomedical engineering applications.…”