मानव कोशिका लाइनों से Chemoresistant डिम्बग्रंथि के कैंसर स्टेम सेल के लिए संवर्धन
Summary
कैंसर स्टेम कोशिकाओं (सीएससी) के कारण chemoresistance को ट्यूमर पतन में फंसा रहे हैं. हम चयन गर्भाशय के कैंसर कोशिका लाइनों से सीएससी के विस्तार के लिए एक प्रोटोकॉल अनुकूलित है. Chemotherapeutic सिसप्लैटिन साथ कोशिकाओं के इलाज और हम गैर पक्षपाती सीएससी संस्कृतियों के लिए बेहतर मीडिया को बढ़ावा देने के लिए एक स्टेम सेल में संवर्धन द्वारा.
Abstract
कैंसर स्टेम कोशिकाओं (सीएससी) धीमी साइकिल और अत्यधिक, proliferative आक्रामक उत्पन्न करने के लिए asymmetrically कि विभाजन undifferentiated कोशिकाओं, और chemoresistant ट्यूमर कोशिकाओं के एक सबसेट के रूप में परिभाषित कर रहे हैं. इसलिए, सीएससी therapeutically लक्षित करने के लिए कोशिकाओं का एक आकर्षक आबादी हैं. सीएससी रक्त, मस्तिष्क, फेफड़े, जठरांत्र संबंधी मार्ग, प्रोस्टेट, और अंडाशय में उन सहित कैंसर के प्रकार के एक नंबर के लिए योगदान करने के लिए भविष्यवाणी कर रहे हैं. अलग और सीएससी के लिए एक ट्यूमर सेल आबादी सीएससी के गुण, जेनेटिक्स, और चिकित्सीय प्रतिक्रिया का अध्ययन करने के लिए सक्षम शोधकर्ताओं जाएगा समृद्ध. हम reproducibly डिम्बग्रंथि के कैंसर कोशिका लाइनों (SKOV3 और OVCA429) से डिम्बग्रंथि के कैंसर सीएससी के लिए समृद्ध करती है कि एक प्रोटोकॉल उत्पन्न. सेल लाइनों 3 दिन के लिए 20 माइक्रोन सिसप्लैटिन के साथ व्यवहार कर रहे हैं. जीवित कोशिकाओं साइटोकिन्स और वृद्धि कारकों युक्त सीरम मुक्त स्टेम सेल मीडिया में पृथक और सुसंस्कृत हैं. हम कश्मीर के लिए अलग कक्षों का विश्लेषण करके इन शुद्ध सीएससी की एक संवर्धन का प्रदर्शनNown सेल मार्कर Oct4, Nanog, और Prom1 (CD133) और CD177 और CD133 की कोशिका की सतह अभिव्यक्ति स्टेम. सीएससी प्रदर्शनी chemoresistance वृद्धि हुई. सीएससी के अलगाव के लिए इस विधि chemoresistance और ट्यूमर पतन में सीएससी की भूमिका के अध्ययन के लिए एक उपयोगी उपकरण है.
Introduction
Resistance to chemotherapy is a major impediment to the treatment and cure of cancer. Ovarian cancer is the 5th leading cause of cancer-related deaths among women in the United States (American Cancer Society Facts and Figures 2013). Patients initially respond well to chemotherapy, but most patients relapse1. After relapse patients are treated with a variety of additional chemotherapy agents with very little benefit2. General properties of CSCs include unlimited proliferative capabilities, retention of an undifferentiated state, resistance to drug treatment, efficient DNA repair, and ability to generate malignant tumor cells with different phenotypes3. CSCs frequently exhibit expression of stem cell genes such as Nanog, Oct4, Sox2, Nestin, CD133, and CD117. CSCs often express elevated levels of ABCG2, and ALDH genes that may contribute to drug efflux and metabolism3,4.
The first definitive evidence for CSCs was demonstrated by isolating acute myeloid leukemia-initiating cells that were capable of self-renewal and tumor generation5. These leukemic stem cells expressed surface CD34 and generated leukemia in NOD/SCID (immunocompromised) mice5. Since then CSCs have been identified in many cancer types including leukemias/lymphomas, breast, bladder, colorectal, endometrial, sarcomas, hepatocellular carcinoma, melanomas, gliomas, ovarian, pancreatic, prostate, squamous cell carcinoma, and lung6. Therefore, being able to study this subtype of cancer cell is advantageous.
The goal of this study is to create a protocol for the selection and isolation of chemoresistant CSCs. Several methods have been reported for the isolation of CSCs from ovarian cancer cell lines. Non-adherent spheroids isolated from OVCAR-3, SKOV3, or HO8910 cultures demonstrate stem-like properties7,8. Isolation of CD133+ cells from OVCAR-3 cultures also yields CSCs. CSCs have also been selected in culture by treatment with chemotherapeutic agents. Treating tumor cell lines (OVCA433, Hey, and SKOV3 cells) with cisplatin and paclitaxel allows for the expansion and isolation of CSCs4,9. While culture of some cell types in CSC media leads to isolation of CSCs, SKOV3 cells did not survive culture in serum-free media or form sphere cells4. Therefore, treatment of cells with cisplatin and paclitaxel aided the expansion or isolation of this population4.
Using a modification of the procedure presented by Ma and colleagues4 we developed a method to isolate CSCs from the ovarian cancer cell lines. Our protocol is advantageous as it yields more viable cells while using less toxic chemotherapeutic agents. Cells are treated with cisplatin and subsequently grown in serum-free media supplemented with growth factors (stem cell media). We isolate the resulting non-adherent sphere cells and assay them for their expression of stem cell markers. This model enables the study of CSC properties and response to drug therapy.
Protocol
Representative Results
Discussion
उपचार के लिए प्रतिरोधी रहे हैं कि सीएससी प्राथमिक ट्यूमर के इलाज के बाद पतन के लिए जिम्मेदार हो सकता है. सीएससी की विशेषता डिम्बग्रंथि के कैंसर के लिए सुधार के उपचारों के लिए नेतृत्व कर सकते हैं. ऊपर प्?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Serene Samyesudhas and Dr. Lynn Roy assisted in preparing samples for filming.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| McCoy | Life Technologies | 16600-108 | Warm to 37C prior to use |
| DMEM / F12 serum free | Life Technologies | 11320-033 | Warm to 37C prior to use |
| Minimal Essential Media | Life Technologies | 42360032 | Warm to 37C prior to use |
| Sodium Pyruvate | Life Technologies | 11360070 | |
| Polybrene | Millipore | TR-1003-G | |
| Blasticidin | Life Technologies | R21001 | |
| Fetal Bovine Serum | Atlas Biologicals | F-0500-A | |
| Penicillin-streptomycin | Life Technologies | 15070-063 | |
| Cisplatin | Sigma-Aldrich | T7402-5MG | Caution: Toxic Use precautions |
| pLenti-suCMV-Rsv | Gentarget | LVP023 | BSL2 approval needed |
| Insulin | Sigma-Aldrich | I-1882 | |
| Human Recombinant EGF | Cell Signaling Technology | 8916LC | |
| bFGF | BD biosciences | 354060 | |
| LIF | Santa Cruz | sc-4988A | |
| Bovine Serum Albumin | Roche | 03 116 956 001 | |
| TRIzol | Life Technologies | 15596-018 | |
| High Capacity cDNA Reverse Transcription Kit | Applied Biosystems | 4368813 | |
| IQ Multiplex Powermix | BioRad | 1725849 | |
| Accumax | Millipore | ||
| Primers | Integrated DNA Technology | individually designed and ordered (see protocol for sequnces) | |
| Anti-CD133 PE | Milenyl | 130-098-826 | Primer/probe sets are light sensitive |
| CD117-Biotin | Miltenly | 130-098-570 | |
| AntiBiotin-FITC | Miltenly | 130-098-796 | |
| Paraformaldehyde | Sigma-Aldrich | P6148-1KG | Caution: Toxic always prepare in hood and make fresh. |
| Trypsin | Life Technologies | 25300062 | |
| MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) | Sigma-Aldrich | 25200-072 | |
| EVOS Fl Epifluorescence and Transmitted Light Microscope | Advanced Microscopy Group | ||
| Biorad CFX96 C1000 System | Biorad | ||
| Beckman Coulter FC500 Flow Cytometer | Beckman Coulter | ||
| Spectramax 340PC384 | Molecular Devices |
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