कार्सिनोमा - एसोसिएटेड Fibroblasts के प्रायोगिक मानव स्तन Fibroblasts से जनरेशन (CAFs)
Summary
कार्सिनोमा – जुड़े fibroblasts (CAFs) ट्यूमर stroma भीतर मौजूद myofibroblasts में अमीर, ट्यूमर प्रगति ड्राइविंग में एक प्रमुख भूमिका निभाते हैं. हम प्रयोगात्मक मानव स्तन fibroblasts से CAFs पैदा करने के लिए एक coimplantation ट्यूमर xengraft मॉडल विकसित किया है. प्रोटोकॉल का वर्णन कैसे स्थापित करने के लिए CAF myofibroblasts कि tumourigenesis को बढ़ावा देने की क्षमता हासिल है.
Abstract
Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the ‘stroma’. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of α-smooth muscle actin (α-SMA) expression4,5. These cells are a hallmark of ‘activated fibroblasts’ that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.
Protocol
Discussion
CAF विशेष मार्करों और विविधता के स्तर की कमी मनाया बीच CAFs इस सेल प्रकार अपने आप में एक चुनौती के लक्षण वर्णन सौंपनेवाला. इन विट्रो में अध्ययन CAFs भी अतिरिक्त जटिलता द्वारा निस्र्द्ध गया है कि इन कोशिकाओ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम उदार और इस काम और इस पांडुलिपि के महत्वपूर्ण संपादन के लिए श्री कीरन Mellody (मैनचेस्टर, मैनचेस्टर विश्वविद्यालय) के समर्थन के पर्यवेक्षण के लिए डॉ. रॉबर्ट ए Weinberg (व्हाइटहेड जैव चिकित्सा अनुसंधान, कैम्ब्रिज के लिए संस्थान) धन्यवाद. इस परियोजना अनुसंधान (सीआर ब्रिटेन) अनुदान C147/A6058 संख्या (एओ) ब्रिटेन द्वारा समर्थित किया गया
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
| DMEM | Invitrogen | 61965-026 | ||
| Fetal calf serum | GIBCO | 10270 | ||
| Penicillin-streptomycin | Invitrogen | 15140-122 | ||
| Collagenase type I | Sigma | C0130-1G | ||
| hyaluronidase | Sigma | H4272 | ||
| Vimentin (V9) antibody | Novocastra Laboratories | NCL-L- VIM-V9 |
||
| Tenascin C (BC-8) antibody | a gift from |
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| α-SMA-Cy3 (1A4) antibody | Sigma | C6198 | ||
Prolyl-4-hydroxylase |
Dako | M0877 | ||
| (5B5) antibody | ||||
| Collagen type1 1A antibody | Sigma | HPA011795 | ||
| Pan-cytokeratin antibody | Sigma | C5992 | ||
| Fibronectin antibody | BD Biosciences | 610077 | ||
S100A4/FSP-1 (fibroblast- specific protein-1) antibody |
Dako | A5114 | ||
Fibroblast surface protein (clone 1B10) antibody |
Abcam | ab11333 | ||
| MSCV-IRES-GFP construct | Request to the the authors | |||
| pBabe-puro construct | Purchase from Addgene |
|||
| Puromycin | Sigma | P8833 | ||
| DAPI | Sigma | D9564 | ||
| 15 ml conical tube | Corning | 430766 | ||
| Nude mouse | Taconic | NCRNU-F | Female NCr nude | |
| C3H/10T1/2 cells | ATCC | CCL-226 |
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