Micropatterned सतहों Hyaluronic एसिड कैंसर कोशिकाओं के साथ संबंधों का अध्ययन करने के लिए
Summary
एक उपन्यास दृष्टिकोण है कि exogenous hyaluronic एसिड (हेक्टेयर) के साथ कैंसर कोशिका बातचीत के उच्च संकल्प विश्लेषण की अनुमति देता है वर्णित है. नमूनों सतहों carbodiimide रसायन शास्त्र और microcontact मुद्रण के संयोजन के द्वारा गढ़े हैं.
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 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.
Protocol
Discussion
हा micropatterning विधि प्रस्तुत exogenous हा के साथ सेल बातचीत के अध्ययन की अनुमति देता है. हा कैंसर 1 प्रगति में एक महत्वपूर्ण भूमिका निभा जाना जाता है लेकिन वहाँ सीमित कैंसर कोशिकाओं के दो आयामी हा नमूनों सतहों ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखक जॉन्स हॉपकिन्स, सामग्री अनुसंधान विज्ञान और राष्ट्रीय विज्ञान फाउंडेशन के माध्यम से इंजीनियरिंग सेंटर के हिस्से के रूप में वित्त पोषित सतह विश्लेषण प्रयोगशाला का उपयोग स्वीकार करते हैं. एलईडी एक IGERT प्रशिक्षु और एक राष्ट्रीय विज्ञान फाउंडेशन ग्रेजुएट बंदे है. इस शोध आंशिक रूप से NIH अनुदान U54CA143868 द्वारा समर्थित किया गया.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| SU-2025 photoresist | MicroChem Corp. | Y111069 | ||
| SU-8 developer | MicroChem Corp. | Y020100 | ||
| Sylgard 184 | Dow Corning | |||
| 3-aminopropyltrimethoxysilane (APTMS) | Sigma-Aldrich | 281778 | ||
| 2- [methoxy(polyethyleneoxy) propyl] trimethoxysilane (Peg-silane) | Gelest Inc | SIM6492.7 | ||
| 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide | Thermo Scientific | 22980 | ||
| N-hydroxysuccinimide (NHS) | Thermo Scientific | 24500 | ||
| Fluorescein labeled hyaluronic acid (FL-HA) | Sigma-Aldrick | F1177 | Reconstitute with 10ml of DI water | |
| MDA-MB-231 breast carcinoma cells | ATCC | HTB-26 | ||
| LS174t colon carcinoma cells | ATCC | Cl-188 |
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