ताजा ऊतकों से सामान्य और कैंसर से जुड़े fibroblasts की प्रतिदीप्ति सक्रिय सेल छँटाई (FACS) द्वारा अलगाव

Summary

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.

Abstract

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 prognosis^1,2. 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 invasion^5-8. 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 culture^11,12 . 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 CAFs^9,15 . 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.

Protocol

1. चूहों से स्तन या त्वचा के ऊतकों विदारक चूहों से वांछित ऊतक विदारक से पहले, निम्नलिखित आपूर्ति और अभिकर्मकों तैयार: आपूर्ति: शल्य चिकित्सा उपकरण (डिटर्जेंट से धोया और फिर 70% इथेनॉल में…

Representative Results

Fibroblasts के ऊतक अत्यधिक समृद्ध आबादी के अलगाव में fibroblasts परिणामों के लिए एक मार्कर के रूप में PDGFRα का उपयोग. छंटाई के बाद शुद्धता का स्तर 99% थी, के रूप में बाद सॉर्ट विश्लेषण (2A चित्रा) द्वारा मात्रा. सेल विशि…

Discussion

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 ?…

Materials

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