Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical Sciences Oncology Center and Institute for NanoBioTechnology, Johns Hopkins University
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Dickinson, L. E., Gerecht, S. Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells. J. Vis. Exp. (46), e2413, doi:10.3791/2413 (2010).
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 angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. 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 surfaces6-7. 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.
1. Standard Photolithography for Micropatterned Stamp Fabrication
2. HA Micropatterning
3. Cell Culture on HA Micropatterned Surfaces
4. Representative Results:
The carbodiimide chemistry used to covalently immobilize HA to APTMS patterned glass slides is shown in Figure 1A. EDC is a zero-length crosslinking agent that reacts with HA carboxyl groups to form amine-reactive intermediates. As this intermediate is susceptible to hydrolysis, NHS is added to increase carbodiimide reaction efficiency. As HA solution interacts with APTMS micropatterns, a stable amide bond forms between HA intermediate and primary amine of ATPMS. An example of HA patterned surface visualized using a fluorescent microscope and an ImageJ fluorescence intensity analysis demonstrate the patterned immobilization of HA are shown (Figure 1B-C).
The HA micropatterned surfaces enable the study of cancer cell interactions with expgenous HA. Both MDA-MB-231 human breast and LS174t human colon cancer cell lines preferentially adhere to HA micropatterned regions (Figure 2). High resolution imaging using scanning electron microscopy can be used to analyze interactions of cultured cells with HA immobilized surfaces (Figure 3).
Figure 1 HA micropatterned surfaces (A) Schematic describing the development of HA functional surfaces, (B) fluorescence image of fluorescein (green)-labeled HA micropatterned surface. (C) 3D pixel distribution map using ImageJ software demonstrating the discrete HA patterns. Scale bar = 100μm. Modified from 9 with permission from Elsevier.
Figure 2. Cancer cell adhesion on HA micropatterned surfaces. Both colon (A) and breast (B) carcinoma preferentially adhered onto HA patches with 24 hours of culture. Modified from 9 with permission from Elsevier.
Figure 3. Cancer cell interaction with HA. (A) Scanning electron microscopy images of colon cancer cells (A) cultured on HA surfaces shown at low magnification (left) and high magnification (right; of squares on left) and of breast cancer cells (B) cultured on HA surfaces shown at low magnification (left) and high magnification (right; of squares on left).
The HA micropatterning method presented allows the study of cell interactions with exogenous HA. HA is known to play a key role in cancer progression 1 however there have been limited studies investigating the interaction of cancer cells on two dimensional HA patterned surfaces. A controllable study on exogenous HA micropatterns allows high-resolution visualization of cancer cell adhesion, growth, and migration and may elucidate further fundamentals of cancer.
By combining carbodiimide chemistry and microcontact printing, we have developed surfaces with distinct HA patterns to study cancer cell interactions. This method allows consistently patterned surfaces compatible with cell culture and analysis techniques including, but not limited to immunofluorescent staining, scanning electron microscopy, inhibition, migration, etc.
Additionally, photolithographic techniques allow the easy microfabrication of any desired pattern, to control immobilization of HA for specific applications. For example, fabricating PDMS stamps with linear stripes would immobilize HA in a linear array and could be useful in analyzing cell migration along patterned HA.
Although we have primarily utilized this method to elucidate cancer cell interactions with HA, this technique is applicable to study interactions with other cell types. Additionally, this method can be used to immobilize a wide range of HA molecules. The HA we have immobilized is fluorescently labeled with a molecular weight of 800 kDa. However, HA ranges in size from 2000- 25000 repeating disaccharide units, and cellular response has been demonstrated to be dependent on HA molecular weight 10-11. This protocol can therefore be applied to immobilize HA of varying molecular weights to investigate cell response to exogenous HA.
Video schematic figure reproduced with permission; Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Dickinson LE, Kusuma S, Gerecht S. Reconstructing the differentiation niche of embryonic stem cells using biomaterials. Macromol. Biosci. 2010; Oct 21. [Epub ahead of print].
The authors acknowledge the use of the surface analysis laboratory at Johns Hopkins, funded as part of the Materials Research Science and Engineering Center through the National Science Foundation. LED is an IGERT trainee and a National Science Foundation Graduate Fellow. This research was partially supported by NIH grant U54CA143868.
|SU-2025 photoresist||MicroChem Corp.||Y111069|
|SU-8 developer||MicroChem Corp.||Y020100|
|Sylgard 184||Dow Corning|
|2- [methoxy(polyethyleneoxy) propyl] trimethoxysilane (Peg-silane)||Gelest Inc.||SIM6492.7|
|1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide||Thermo Fisher Scientific, Inc.||22980|
|N-hydroxysuccinimide (NHS)||Thermo Fisher Scientific, Inc.||24500|
|Fluorescein labeled hyaluronic acid (FL-HA)||Sigma-Aldrich||F1177||Reconstitute with 10ml of DI water|
|MDA-MB-231 breast carcinoma cells||ATCC||HTB-26|
|LS174t colon carcinoma cells||ATCC||Cl-188|