丰富的人力细胞系化疗耐药性卵巢癌干细胞
Summary
肿瘤干细胞（肿瘤干细胞）有牵连的肿瘤复发，由于化疗。我们已经进行了优化的协议选择和扩展，从卵巢癌细胞系的CSCs的。通过处理细胞与化疗顺铂和在干细胞培养促进我们丰富的非粘附CSC培养介质。
Abstract
癌症干细胞（CSCs的）被定义为慢骑自行车和未分化细胞的不对称分裂，产生高度增殖，浸润和化疗耐药的肿瘤细胞的一个子集。因此，肿瘤干细胞是细胞的一个有吸引力的人群为目标的治疗。 CSCs的被预测为向多种类型的恶性肿瘤，包括那些在血液，脑，肺，胃肠道，前列腺和卵巢的。分离和富集肿瘤细胞群的肿瘤干细胞将使研究人员能够研究性质，遗传学和肿瘤干细胞治疗的反应。我们产生的一种协议，它可再现丰富的卵巢癌细胞株（SKOV3和OVCA429）卵巢癌肿瘤干细胞。细胞系是用20μM的顺铂处理3天。存活细胞的分离及培养在含细胞因子和生长因子的无血清干细胞培养基。我们通过分析分离出的细胞对于k证明这些肿瘤干细胞纯化的浓缩nown干细胞标记物的Oct4，Nanog和PROM1（CD133）和CD177和CD133的细胞表面表达。在社区体育会显示增加的耐药。这种方法对于肿瘤干细胞的分离，是研究耐药和肿瘤复发的肿瘤干细胞的作用，一个有用的工具。
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
肿瘤干细胞有抗药性的治疗可能是治疗原发肿瘤后交代复发。肿瘤干细胞的特性可能导致更好的治疗卵巢癌。在建立使用上述协议化疗耐药的CSCs的临界参数的时序治疗与化疗的长度和化疗的浓度。当使用在Ma 等人的协议。人们发现，在7天后用顺铂和紫杉醇治疗的，无活细胞保持4。通过还原处理，以3天（以20μM的顺铂而不是40μM的顺铂和10μM紫杉醇），可行化疗耐药细胞和肿瘤干细?…
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 |
Referências
- Pennington, K., Pulaski, H., Pennington, M., Liu, J. R. Too Much of a Good Thing: Suicide Prevention Promotes Chemoresistance in Ovarian Carcinoma. Curr Cancer Drug Targets. 10, 575-583 (2010).
- Thigpen, T. A rational approach to the management of recurrent or persistent ovarian carcinoma. Clin Obstet Gynecol. 55, 114-130 (2012).
- Burgos-Ojeda, D., Rueda, B. R., Buckanovich, R. J. Ovarian cancer stem cell markers: prognostic and therapeutic implications. Cancer letters. 322, 1-7 (2012).
- Ma, L., Lai, D., Liu, T., Cheng, W., Guo, L. Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3. Acta Biochim Biophys Sin (Shanghai). 42, 593-602 (2010).
- Bonnet, D., Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nature Medicine. 3, 730-737 (1997).
- Alison, M. R., Lim, S. M., Nicholson, L. J. Cancer stem cells: problems for therapy. The Journal of pathology. 223, 147-161 (2011).
- Luo, X., Dong, Z., Chen, Y., Yang, L., Lai, D. Enrichment of ovarian cancer stem-like cells is associated with epithelial to mesenchymal transition through an miRNA-activated AKT pathway. Cell proliferation. 46, 436-446 (2013).
- Wang, L., Mezencev, R., Bowen, N. J., Matyunina, L. V., McDonald, J. F. Isolation and characterization of stem-like cells from a human ovarian cancer cell line. Molecular and cellular biochemistry. 363, 257-268 (2012).
- Abubaker, K., et al. Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden. Molecular cancer. 12, 24 (2013).
- Conic, I., Dimov, I., Tasic-Dimov, D., Djordjevic, B., Stefanovic, V. Ovarian epithelial cancer stem cells. ScientificWorldJournal. 11, 1243-1269 (2011).
- Liu, K. C., et al. Ovarian cancer stem-like cells show induced translineage-differentiation capacity and are suppressed by alkaline phosphatase inhibitor. Oncotarget. 4 (12), 2366-2382 (2013).
- Liu, T., Cheng, W., Lai, D., Huang, Y., Guo, L. Characterization of primary ovarian cancer cells in different culture systems. Oncology reports. 23, 1277-1284 (2010).
