Kræft stamceller (CSCS) er impliceret i tumor tilbagefald på grund kemoterapiresistens. Vi har optimeret en protokol for udvælgelse og udvidelse af CSCS fra ovariekræft cellelinjer. Ved at behandle celler med det kemoterapeutiske cisplatin og dyrkning i en stamcelle fremme medier vi berige for ikke-adhærente CSC kulturer.
Kræft stamceller (CSCS) defineres som en delmængde af langsom cykling og udifferentierede celler, der deler asymmetrisk til at generere meget proliferativ, invasive, og kemoterapi tumorceller. Derfor cscs er en attraktiv population af celler til at målrette terapeutisk. Cscs forventes at bidrage til en række typer af maligniteter inklusive dem i blod, hjerne, lunger, mave-tarmkanalen, prostata og ovarie. Isolere og berige en tumorcellepopulation til CSCS vil give forskerne mulighed for at studere egenskaber, genetik, og terapeutisk respons CSCS. Vi genererede en protokol, der reproducerbart beriger for kræft i æggestokkene CSCS fra æggestokkene cancercellelinjer (SKOV3 og OVCA429). Cellelinjer behandlet med 20 pM cisplatin i 3 dage. Overlevende celler isoleres og dyrkes i et serumfrit stamceller medier indeholdende cytokiner og vækstfaktorer. Vi demonstrerer en berigelse af disse oprensede CSCS ved at analysere de isolerede celler for Known stamcellemarkører Oct4, Nanog, og Prom1 (CD133) og celleoverfladeekspression af CD177 og CD133. Den CSCS udstilling øget kemoterapiresistens. Denne metode til isolering af CSCS er et nyttigt redskab til at studere den rolle, CSCS i kemoterapiresistens og tumor tilbagefald.
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.
CSCS, der er resistente over for behandling, kan være ansvarlig for tilbagefald efter behandling af primær tumor. Karakterisering af CSCS kan føre til bedre behandlinger for kræft i æggestokkene. De kritiske parametre i etableringen af kemoresistent CSCS hjælp af ovenstående protokol er timing længden af behandling med kemoterapi og koncentrationen af kemoterapi. Ved brug af protokollen i Ma et al. det blev konstateret, at efter 7 dages behandling med cisplatin og paclitaxel, forblev i…
The authors have nothing to disclose.
Serene Samyesudhas and Dr. Lynn Roy assisted in preparing samples for filming.
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 |