Verrijking voor chemoresistent eierstokkanker stamcellen uit menselijke cellijnen
Summary
Kanker stamcellen (CSC) betrokken zijn bij tumor recidief vanwege chemoresistentie. Wij hebben een protocol voor selectie en uitbreiding van CSC van eierstokkanker cellijnen geoptimaliseerd. Door het behandelen van cellen met het chemotherapeutische cisplatine en kweken in een stamcel bevorderen media we verrijken voor niet-hechtende CSC kweken.
Abstract
Kanker stamcellen (CSC) worden gedefinieerd als een deelverzameling van trage fietsen en ongedifferentieerde cellen die asymmetrisch verdelen om zeer proliferatieve, invasieve genereren, en chemoresistent tumorcellen. Daarom CSC een aantrekkelijk populatie van cellen voor het therapeutisch. CSC wordt voorspeld bijdragen aan een aantal typen maligniteiten waaronder die in het bloed, hersenen, longen, maagdarmkanaal, prostaat en ovarium. Isoleren en verrijken van een tumorcel populatie voor CSC zal onderzoekers in staat om de eigenschappen, genetica, en de therapeutische respons van CSC te bestuderen. We genereerden een protocol dat reproduceerbaar verrijkt voor eierstokkanker CSC van eierstokkanker cellijnen (SKOV3 en OVCA429). Cellijnen behandeld met 20 uM cisplatine voor 3 dagen. Overlevende cellen worden geïsoleerd en gekweekt in een serumvrij medium dat stamcellen cytokines en groeifactoren. We demonstreren een verrijking van deze gezuiverde CSC door het analyseren van de geïsoleerde cellen voor known stamcel markers Oct4, NANOG en Prom1 (CD133) en celoppervlak expressie van CD177 en CD133. De CSC vertonen verhoogde chemoresistance. Deze methode voor het isoleren van CSC is een nuttig hulpmiddel voor het bestuderen van de rol van CSCs in chemoresistance en tumor recidief.
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
KSC die resistent zijn tegen behandeling zijn mogelijk verantwoordelijk voor het recidief na behandeling van de primaire tumor. Karakterisering van CSC kan leiden tot verbeterde therapieën voor kanker van de eierstokken. De kritische parameters in de oprichting van chemoresistent CSC's met behulp van het bovenstaande protocol worden de timing van de duur van de behandeling met chemotherapie en de concentratie van de chemotherapie. Bij het gebruik van het protocol in Ma et al. bleek dat na 7 dagen beh…
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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