Berigelse for kemoterapi kræft i æggestokkene stamceller fra humane cellelinier

Summary

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.

Abstract

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.

Introduction

Resistance to chemotherapy is a major impediment to the treatment and cure of cancer. Ovarian cancer is the 5^th 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 metabolism^3,4.

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 properties^7,8. 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 CSCs^4,9. 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

1. Celle Kultur og fluorescensmærkning af æggestokkene cancercellelinjer Forbered SKOV3 medier: McCoy medier suppleret med 10% føtalt bovint serum (FBS), 0,1 mM L-glutamin, 50 U / ml penicillin og 50 ug / ml streptomycin. Oprethold OVCA429 celler i minimale essentielle medier (MEM) suppleret med 10% FBS, 1 mM natriumpyruvat, 0,1 mM L-glutamin, 50 U / ml penicillin og 50 ug / ml streptomycin. Propagate SKOV3 og OVCA429 cellelinier i en befugtet inkubator ved 37 ° C med 5% CO2. Grow cel…

Representative Results

For at demonstrere, at vi isolerede CSCS fra epitelovariecancer cellelinjer hjælp cisplatinbehandlingen, vi først købte billeder af cellelinier før behandling og efter valget. Vi brugte lysmikroskopi at fange billeder af vedhængende (ubehandlet) SKOV3 og OVCA429 celler og SKOV3 og OVCA429 CSCS (figur 1). Cscs vises rundt og ikke bundet til vævskulturpladerne (figur 1 og 2). Vi viser desuden, at SKOV3-celler transduceret med RFP bevarer deres fluorescens efter isol…

Discussion

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…

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