कैंसर एसोसिएटेड Fibroblasts (cafs) संकेत है कि प्रसार, angiogenesis, और सूजन को बढ़ावा देता है के माध्यम से ट्यूमर दीक्षा, विकास और प्रगति की सुविधा. यहाँ हम एक ताजा माउस और मानव ऊतकों से सेल छँटाई, एक सतह मार्कर के रूप PDGFRα का उपयोग सामान्य fibroblasts और cafs का शुद्ध आबादी को अलग विधि का वर्णन करता है.
Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker α-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFRα as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFRα is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to α-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.
जबकि टिशू कल्चर में प्रदर्शन प्रयोगों जानकारीपूर्ण और कार्यात्मक सिद्धांतों कि vivo में सत्यापित किया जा सकता है सुझाव कर सकते हैं, यह है कि बड़े परिवर्तन होते 11,12 संस्कृति में कोशिकाओं के जीन की ?…
The authors have nothing to disclose.
हम FACS छँटाई के साथ उनकी मदद के लिए डॉ. Yitzchak Oschry और डा. Sagi आसिफ Orit धन्यवाद. इस शोध अनुदान समझौते के एन के तहत यूरोपीय संघ के सातवें फ्रेमवर्क कार्यक्रम (FP7/2007-2013) से पूर्वोत्तर के लिए अनुदान द्वारा समर्थित किया गया ° [276890], इसराइल कैंसर एसोसिएशन (20,110,078 #), और इसराइल कैंसर रिसर्च (फंड रिसर्च से कैरियर विकास) पुरस्कार से सम्मानित किया गया.
Name of the reagent | Company | Catalogue number | Comments (optional) |
DMEM | Gibco | 41965 | |
PBS | Biological Industries | 02-023-1A | |
Collagenase II | Worthington | LS4176 | |
Collagenase IV | Worthington | LS4188 | |
Deoxyribonuclease | Worthington | LS2007 | |
PharmLyse | BD | 555899 | |
Cell strainer 70 μm | SPL | 93070 | |
Purified anti-mouse CD16/CD32 | BD Pharmingen | 553142 | |
Via probe (7AAD) | e-Bioscience | 00-6993-50 | |
Anti-mouse CD140a-PE (PDGFRa) | e-Bioscience | 12-1401-81 | |
Anti-mouse F4/80- FITC | Cederlane | CL8940F | |
DMEM w/o Phenol Red | Gibco | 31053 | |
Collagen Type I | BD Biosciences | 354236 |