मॉडलिंग और 3 - आयामी इमेजिंग कलेक्टिव सेल आक्रमण
Summary
तीन आयामी मैट्रिक्स कोशिकी में ट्यूमर सेल आक्रमण के मॉडल बेहतर प्रतिबिंबित<em> Vivo में</em> दो आयामी गतिशीलता assays की तुलना में स्थिति है. मैट्रिक्स आक्रमण fluorescently लेबल कोशिकाओं आक्रमण मोड और प्रमुख बनाम निम्नलिखित कोशिकाओं के विशिष्ट योगदान पर विस्तृत जानकारी के confocal इमेजिंग के साथ संयुक्त assays का उपयोग प्राप्त किया जा सकता है.
Abstract
A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90% of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade “upwards” through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.
Protocol
Discussion
Matrigel आक्रमण assays के परंपरागत chemoattractant प्रेरित गतिशीलता के साथ बाह्य मैट्रिक्स प्रोटीन की एक दिशा में परत पर रखा और नीचे में एक फिल्टर के माध्यम से कोशिकाओं के साथ किया गया है स्थापित. Invasiveness कितने कोशिकाओं को फ…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस शोध के लिए अनुदान कैंसर रिसर्च यूके से है.
Materials
| Name of the reagent | Company | Catalogue number |
|---|---|---|
| DMEM (Dulbecco’s Modified Eagle’s Medium) | GIBCO | 21969 |
| Fetal Bovine Serum | PAA | A15-101 |
| Penicillin Streptomycin | GIBCO | 15140 |
| 200 mM L-Glutamine (100x) | GIBCO | 25050-032 |
| Puromycin | Sigma-Aldrich | P8833 |
| 0.05% Trypsin EDTA | GIBCO | 25300 |
| Polybrene | Sigma-Aldrich | AL-118 |
| Lipofectamine 2000 Reagent | Invitrogen | 11668019 |
| 6.5mm Transwells, 8.0 µm pore size | Corning | 3422 |
| Complete Matrigel | BD Biosciences | 354234 |
| Calcein AM | Invitrogen | C1430 |
| RNase | Qiagen | 19101 |
| Propidium Iodide | Sigma-Aldrich | P4864 |
| Confocal microcope | Leica | SP2MP |
References
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