उपगोल TGF-β प्रेरित आक्रमण उपाय परख
Summary
मात्रात्मक ग्रोथ फैक्टर (TGF) β प्रेरित 3 आयामी कोलेजन जैल में आक्रमण बदलने को मापने के लिए एक परख वर्णित है. यह परख सेल लाइनों के MCF10A श्रृंखला है, जो स्तन कैंसर के विकास के विभिन्न चरणों का प्रतिनिधित्व करते हैं का लाभ लेता है. इस विधि के अन्य सेल लाइनों के साथ इस्तेमाल किया जा और अन्य संभावित activators या आक्रमण की inhibitors की जांच करने के लिए इस्तेमाल किया जा सकता है अपनाया जा सकता है.
Abstract
TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-β induces invasion are not well understood.
TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-β in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-β-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-β signaling on the invasive properties of breast cells in different stages of malignancy.
Protocol
Discussion
हम एक उपगोल मॉडल में जो MCF10A1 कोशिकाओं और उसके घातक डेरिवेटिव TGF-β निर्भर तरीके में एक मैट्रिक्स कोलेजन में आक्रमण की स्थापना की. सबसे पहले, spheroids methylcellulose की उपस्थिति में 96 कुओं दौर नीचे निलंबन प्लेटों में बनते ?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
हम केन और अभिकर्मकों फ्रेड मिलर (बारबरा Ann Karmanos कैंसर Intitute, डेट्रोइट, संयुक्त राज्य अमेरिका) सेल लाइनों के लिए के लिए Iwata (OSI फार्मास्यूटिकल्स, न्यू यार्क, संयुक्त राज्य अमेरिका) के लिए आभारी हैं.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Methyl cellulose | Sigma-Aldrich | M0512 | |
| PureCol | Advanced Biomatrix | 5005-B | |
| pH indicator strips | Merck | 9533 | |
| 96-well suspension plates |
Greiner Bio-one | 650 185 | |
| 96-well adhesion TC plates |
Greiner Bio-one | 655 180 |
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