ヒト細胞株から化学療法抵抗性の卵巣がん幹細胞の濃縮
Summary
癌幹細胞（CSCは）による化学療法耐性に対する腫瘍の再発に関与している。私たちは、選択および卵巣癌細胞株からのCSCの拡張のためのプロトコールを最適化した。化学療法剤シスプラチンで細胞を処理し、私たちは非接着CSC培養液を濃縮するメディアを促進幹細胞で培養することによる。
Abstract
癌幹細胞（CSCが）をゆっくりサイクリング、高度に増殖性の侵襲性生成するために非対称的に分裂未分化細胞、および化学療法抵抗性の腫瘍細胞のサブセットとして定義される。そのため、CSCは、治療標的とする細胞の魅力的な集団である。 CSCは、血液、脳、肺、胃腸管、前立腺、および卵巣のものを含む悪性腫瘍の種類の数に貢献すると予想される。単離とのCSCのための腫瘍細胞集団を濃縮すると、プロパティ、遺伝学、およびのCSC治療反応を研究する研究者ができるようになります。私たちは、再現可能に卵巣癌細胞株（SKOV3及びOVCA429）から卵巣癌のCSCが富化プロトコルを生成しました。細胞株は、3日間20μMのシスプラチンで処理される。生存細胞は、サイトカインおよび成長因子を含む血清を含まない幹細胞培地中で単離され、培養される。私たちは、kの単離した細胞を分析することによって、これらの精製のCSCの濃縮を実証ノウン細胞マーカーのOct4、Nanogの、およびPROM1（CD133）およびCD177およびCD133の細胞表面発現ステム。 CSCは展示は化学療法抵抗性を増加させた。 CSCを単離するためのこの方法は、化学療法抵抗性および腫瘍の再発でのCSCの役割を研究するための有用なツールである。
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
治療に抵抗性であるCSCは、原発腫瘍の治療後の再発に責任があります。 CSCはの特徴付けは、卵巣癌のための改善された治療法につながる可能性があります。上記のプロトコルを使用して、化学療法抵抗性のCSCの確立において重要なパラメータは、化学療法による治療の長さおよび化学療法の濃度チャートである。 Ma らにプロトコルを使用する場合。それは、シスプラチン及びパクリ?…
Disclosures
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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