Dickinson Laura E, Gerecht Sharon
Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical Sciences Oncology Center and Institute for NanoBioTechnology, Johns Hopkins University, USA.
J Vis Exp. 2010 Dec 22(46):2413. doi: 10.3791/2413.
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.
癌症侵袭和进展涉及一种运动性细胞表型，该表型受到肿瘤微环境中生长因子/细胞因子和细胞外基质（ECM）成分的复杂调控。透明质酸（HA）是一种基质ECM成分，已知其通过增强侵袭、生长和血管生成来促进肿瘤进展（1）。HA与其细胞表面受体CD44的相互作用诱导信号事件，促进肿瘤细胞生长、存活和迁移，从而增加转移扩散（2 - 3）。HA是一种阴离子、非硫酸化的糖胺聚糖，由D - 葡萄糖醛酸和D - N - 乙酰葡糖胺的重复单元组成。由于重复二糖单元上存在羧基和羟基，天然HA在很大程度上是亲水性的，并且适合进行化学修饰以引入硫酸基团用于光反应固定（4 - 5）。先前涉及将HA固定到表面的研究利用了HA及其硫酸化衍生物的生物抗性行为来控制细胞在表面的黏附（6 - 7）。在这些研究中，细胞黏附优先发生在非HA图案化区域。为了分析细胞与外源性HA的相互作用，我们开发了图案化功能化表面，能够对癌细胞与HA的相互作用进行可控研究和高分辨率可视化。我们利用微接触印刷（μCP）在玻璃表面定义HA的离散图案化区域。使用了一种“ tethering”方法，该方法应用碳二亚胺连接化学来固定HA（8）。用氨基硅烷对玻璃表面进行微接触印刷，并与具有优化比例的EDC和NHS的HA溶液反应，以使HA能够固定在图案化阵列中。将碳二亚胺化学与μCP相结合能够将HA固定到特定区域，从而创建适合体外应用的表面。结肠癌细胞和乳腺癌细胞都与HA微图案化表面发生了隐性相互作用。癌细胞在24小时内发生黏附，48小时内发生增殖。使用HA微图案化表面，我们证明癌细胞黏附是通过HA受体CD44发生的。此外，HA图案化表面与扫描电子显微镜（SEM）兼容，并允许对癌细胞黏附突起和在HA图案上的铺展进行高分辨率成像，以分析癌细胞在外源性HA上的运动性。
