Name: enrichment for chemoresistant ovarian cancer stem cells from human cell lines
Uploaded: 2014-09-10T15:00:00
Description: Watch this Scientific Journal Video about Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines at JoVE.com

Serene Samyesudhas and Dr. Lynn Roy assisted in preparing samples for filming.

1. Hücre Yumurtalık Kanseri Hücre Hatları Kültür ve Floresan Etiketleme <ol><li> SKOV3 ortamı hazırlayın:% 10 fetal sığır serumu (FBS) ile desteklenmiş McCoy ortam, 0.1 mM L-glutamin, 50 U / ml penisilin ve 50 ug / ml streptomisin. % 10 FBS, 1 mM sodyum piruvat, 0.1 mM L-glutamin, 50 U / ml penisilin ile takviye edilmiş minimum gerekli ortamında (MEM) olarak OVCA429 hücreler ve 50 ug / ml streptomisin koruyun. </li><li> % 5 CO2 ile 37 ° C de, nemlendirilmiş bir kuluçka makinesi içinde SK...

<ol>
	<li>Pennington, K., Pulaski, H., Pennington, M., Liu, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+Much+of+a+Good+Thing:+Suicide+Prevention+Promotes+Chemoresistance+in+Ovarian+Carcinoma.">Too Much of a Good Thing: Suicide Prevention Promotes Chemoresistance in Ovarian Carcinoma.</a> Curr Cancer Drug Targets. 10, 575-583 (2010).</li><li>Thigpen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rational+approach+to+the+management+of+recurrent+or+persistent+ovarian+carcinoma.">A rational approach to the management of recurrent or persistent ovarian carcinoma.</a> Clin Obstet Gynecol. 55, 114-130 (2012).</li><li>Burgos-Ojeda, D., Rueda, B. R., Buckanovich, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+stem+cell+markers:+prognostic+and+therapeutic+implications.">Ovarian cancer stem cell markers: prognostic and therapeutic implications.</a> Cancer letters. 322, 1-7 (2012).</li><li>Ma, L., Lai, D., Liu, T., Cheng, W., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem-like+cells+can+be+isolated+with+drug+selection+in+human+ovarian+cancer+cell+line+SKOV3.">Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3.</a> Acta Biochim Biophys Sin (Shanghai). 42, 593-602 (2010).</li><li>Bonnet, D., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+acute+myeloid+leukemia+is+organized+as+a+hierarchy+that+originates+from+a+primitive+hematopoietic+cell.">Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.</a> Nature Medicine. 3, 730-737 (1997).</li><li>Alison, M. R., Lim, S. M., Nicholson, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+problems+for+therapy.">Cancer stem cells: problems for therapy.</a> The Journal of pathology. 223, 147-161 (2011).</li><li>Luo, X., Dong, Z., Chen, Y., Yang, L., Lai, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+ovarian+cancer+stem-like+cells+is+associated+with+epithelial+to+mesenchymal+transition+through+an+miRNA-activated+AKT+pathway.">Enrichment of ovarian cancer stem-like cells is associated with epithelial to mesenchymal transition through an miRNA-activated AKT pathway.</a> Cell proliferation. 46, 436-446 (2013).</li><li>Wang, L., Mezencev, R., Bowen, N. J., Matyunina, L. V., McDonald, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+stem-like+cells+from+a+human+ovarian+cancer+cell+line.">Isolation and characterization of stem-like cells from a human ovarian cancer cell line.</a> Molecular and cellular biochemistry. 363, 257-268 (2012).</li><li>Abubaker, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+single+treatment+of+chemotherapy+results+in+the+enrichment+of+ovarian+cancer+stem+cell-like+cells+leading+to+an+increased+tumor+burden.">Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden.</a> Molecular cancer. 12, 24 (2013).</li><li>Conic, I., Dimov, I., Tasic-Dimov, D., Djordjevic, B., Stefanovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+epithelial+cancer+stem+cells.">Ovarian epithelial cancer stem cells.</a> ScientificWorldJournal. 11, 1243-1269 (2011).</li><li>Liu, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+stem-like+cells+show+induced+translineage-differentiation+capacity+and+are+suppressed+by+alkaline+phosphatase+inhibitor.">Ovarian cancer stem-like cells show induced translineage-differentiation capacity and are suppressed by alkaline phosphatase inhibitor.</a> Oncotarget. 4 (12), 2366-2382 (2013).</li><li>Liu, T., Cheng, W., Lai, D., Huang, Y., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primary+ovarian+cancer+cells+in+different+culture+systems.">Characterization of primary ovarian cancer cells in different culture systems.</a> Oncology reports. 23, 1277-1284 (2010).</li></ol>

Tedavisine dirençli CSCS primer tümörün tedavisi sonrası nüks sorumlu olabilir. CSCS karakterizasyonu yumurtalık kanseri gelişmiş terapi yöntemlerine yol açabilir. Yukarıdaki protokol kullanılarak Kemorezistans CSCS kurulmasında önemli parametreler kemoterapi ile tedavi uzunluğu ve kemoterapi konsantrasyonunu zamanlama. Ma ve arkadaşları protokolünü kullanırken. Bu sisplatin ve paklitaksel ile tedaviden 7 gün sonra canlı hücre kalmamıştır 4 kaldığı tespit edildi. (20 uM ...

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.

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enrichment for chemoresistant ovarian cancer stem cells from human cell lines

Kanser kök hücreler (rulmanları) yavaş bisiklet ve yüksek ölçüde proliferatif invaziv oluşturmak için asimetrik olarak bölmek farklılaşmamış hücreleri ve Kemorezistans tümör hücrelerinin bir alt kümesi olarak tanımlanmaktadır. Bu nedenle, CSCS terapötik hedef hücrelerin çekici bir nüfus vardır. CSCS kan, beyin, akciğer, sindirim yolu, prostat ve yumurtalık da dahil olmak üzere habis türde bir dizi katkı tahmin edilmektedir. Yalıtımlı ve CSCS için bir tümör hücre popülasyonu CSCS özellikleri, genetik ve tedavi yanıtı çalışma için araştırmacılar sağlayacaktır zenginleştirmek. Bu yeniden üretilebilir bir yumurtalık kanseri hücre hatları (SKOV3 ve OVCA429) yumurtalık kanseri için CSCS zenginleştiren bir protokol oluşturulur. Hücre hatları, 3 gün boyunca 20 uM Sisplatin ile tedavi edilir. Canlı kalan hücreler sitokinleri ve büyüme faktörlerini içeren bir serumsuz ortam içinde kök hücre izole edilir ve kültürlenmiştir. Biz k izole hücreler analiz ederek bu saflaştırılmış CSCS bir zenginleştirme göstermeknown hücre işaretçilerini Eki4, Nanog ve Prom1 (CD133) ve CD177 ve CD133 hücre yüzey ifadesine kaynaklanıyor. CSCS sergi kemoterapi direncinin artmıştır. CSCS izolasyonu için bu yöntem, kemoterapi ve tümör relaps CSCS rolü üzerinde çalışılması için yararlı bir araçtır.

Biz sisplatin tedavisi ile epitelyal yumurtalık kanseri hücre çizgilerinden CSCS izole edilmiş olduğunu göstermek için, ilk olarak, tedaviden önce ve sonra seçim hücre çizgilerinin görüntüleri elde. Biz yapışmış (işlenmemiş) SKOV3 ve OVCA429 hücreleri ve SKOV3 ve OVCA429 CSCS (Şekil 1) görüntüleri yakalamak için ışık mikroskobu kullanılmaktadır. CSCS doku kültürü plakalarına bağlı yuvarlak olup görünen (Şekil 1 ve 2). Biz ayrıca ...

Watch this Scientific Journal Video about Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines at JoVE.com

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Kanser kök hücreleri (rulmanları) nedeniyle kemoterapi direncinin tümör nüks sorumlu tutulmaktadır. Biz seçimi ve yumurtalık kanseri hücre hatları CSCS genişlemesi için bir protokol optimize ettik. Kemoterapötik Sisplatin ile hücrelerin tedavisi ve biz yapışmayan CSC kültürler için zenginleştirmek medyayı teşvik kök hücre kültürleme tarafından.

İnsan Hücre Lines Kemorezistans Yumurtalık Kanser Kök Hücreleri Zenginleştirme

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

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Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Çoklu ve tek hücre bazlı küreler tahliller kullanılarak, insan yumurtalık karsinoma hücrelerinde Kök Hücre Özelliklerinin Değerlendirilmesi

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Yumurtalık Kanseri Tümör başlatan Hücrelerinin in vitro Zenginleşmeden içinde

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...