Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Abstract

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis 1 . Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread 2-3 . HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization [4][5] . Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces [6][7] . In these studies cell adhesion preferentially occurs on non-HA patterned regions.To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8 . Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA. Use forceps to handle wafer and prevent surface defects during the entire process. 2. Transfer wafer to spin coater and cover wafer surface with SU-2025 negative photoresist. Cover at least 80% of wafer with photoresist. Spin coat for 10 seconds at 600rpm, followed by 30 seconds at 3000rpm. Due to the photosensitivity of the photoresist, turn off room lights from this point forward. 3. Transfer photoresist coated silicon wafer to hot plate for "soft-bake" at 95°C for 3 minutes. Remove from hot plate and allow 1 minute to cool. 4. Place a pre-fabricated mask with the desired pattern on top of the photoresist-covered silicon wafer...

Hypoxia and free radicals: Role in tumor progression and the use of engineering-based platforms to address these relationships

“…matrix stiffness) and composition of the ECM, features which are reported to be abnormal in the tumor environment [90], direct cell behavior. In our lab, we have utilized this technique to address vascular network assembly from endothelial progenitor cells on micropatterned fibronectin and fibrin gel strips [91] and the adhesion of different breast cancer cell lines on micropatterned hyaluronan [92], discovering that substrate geometry and topography impact cellular responses. This approach also allows the investigation of single-cell interactions between ECs and cancer cells at different stages of tumorigenesis [92].…”

“…In our lab, we have utilized this technique to address vascular network assembly from endothelial progenitor cells on micropatterned fibronectin and fibrin gel strips [91] and the adhesion of different breast cancer cell lines on micropatterned hyaluronan [92], discovering that substrate geometry and topography impact cellular responses. This approach also allows the investigation of single-cell interactions between ECs and cancer cells at different stages of tumorigenesis [92]. Others have utilized micropatterning to investigate the role of matrix geometry and stiffness on mammary epithelial cell transformation [93] and tumor spheroid generation [94].…”

Hyaluronan Regulation of Endothelial Barrier Function in Cancer

“…We have reported that HMW-HA enhances EC barrier function (Singleton et al, 2006, 2010). The HA in the stroma of several cancers has been implicated in numerous processes including inhibition of apoptosis, stimulation of tumor cell proliferation, migration and invasion, epithelial mesenchymal transition, protection from immune cells, and regulation of intercellular space (Auvinen et al, 2013; Bollyky et al, 2009; Cho et al, 2012; Dickinson & Gerecht, 2010; Sironen et al, 2011; Tammi et al, 2008). Whether this stroma-associated HA also promotes resealing of new capillaries remains to be determined.…”

Engineering Approaches for Investigating Tumor Angiogenesis: Exploiting the Role of the Extracellular Matrix

“…As a proof of principle, Chiu et al (38) micropatterned tumor cells and ECs together in spatially and geometrically defined regions of fibrinogen, methodology the authors believe will not only allow for studies of tumor cell-directed EC migration and capillary formation, but will permit patterning of numerous cell types on the same substrate. Work in our lab has demonstrated the specific adhesion of breast cancer cells on micropatterned HA surfaces (39) and endothelial progenitor cell elongation and unidirectional network assembly on micropatterned fibronectin and fibrin gel stripes (32), suggesting the role of spatial presentation of specific ECM molecules in regulating tumor and vascular cell behaviors. Overall, through control over the geometry and spacing of ECM molecules, investigators will be better equipped to address the complex interactions taking place between cancer and vascular cells during angiogenesis in the tumor environment.…”