Abstract 5083: A comprehensive workflow for screening and real-time analysis of cell invasion
| Autor: | Timothy K. Nadler, Amedeo Cappione |
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| Rok vydání: | 2016 |
| Předmět: | |
| Zdroj: | Cancer Research. 76:5083-5083 |
| ISSN: | 1538-7445 0008-5472 |
| Popis: | Cell migration and invasion are crucial steps in many normal, as well as, aberrant physiological processes, in particular, the highly lethal metastatic progression of cancer. Once the normally restrictive basement membrane boundaries of the tumor have been breached, invasive cells can gain access to the vasculature and lymphatic system permitting systemic spread of the disease. Invasion is a complex multistep process involving cell adhesion, directed migration, and proteolytic degradation of surrounding extracellular matrix (ECM) barriers. Multiple factors present in the local microenvironment, including soluble growth factors or chemokines and ECM-anchored integrins and proteases, act coordinately to regulate the invasive potential. Understanding the cellular and molecular mechanisms underlying invasion is essential to the development of therapeutics that target tumor metastasis. Therefore, techniques for the quantification and dynamic visualization of penetrating cells have become central to oncology research. The most widely employed method for analyzing cell invasion in vitro is a modified Boyden chamber assay using Matrigel, a basement membrane matrix preparation. While amenable to semi-quantitative “screening” assays, these systems suffer from the inability to establish continuous gradients. As a result, this method, such gradients cannot be stably maintained under static culturing conditions, precluding longer-term real-time cell analysis. Moreover, these platforms are not strictly designed for microscope-based visualization and thus cannot be employed for dynamic mechanistic studies through imaging analysis at the inter- and intra-cellular levels. Here we present a two-tiered platform for the analysis of cell invasion. Cell culture insert plates (24 and 96-well capacity), provide the ideal tool for initial compound screening for functional response (inhibit invasion) and toxicity. For selected compounds, mechanistic studies were then performed using a microfluidic-based system. The platform consists of a microfluidic culture plate and environmental control system; the latter regulates matrix loading, media perfusion, the establishment of stable continuous concentration gradients within the plate, as well as temperature and gas control. Most significantly, the plate can be paired with an inverted microscope enabling dynamic analysis of cell movement in real-time. Fluorescent visualization further permits selective discrimination of unique cell types in heterogeneous samples as well as changes in expression patterns of labeled proteins. In summary, the combined workflow platform presented here provides a framework for studying the effects of signaling molecules and growth factors on the migratory and invasive propensities of tumors cells. Citation Format: Amedeo J. Cappione, Timothy Nadler. A comprehensive workflow for screening and real-time analysis of cell invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5083. |
| Databáze: | OpenAIRE |
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