Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing in a stem cell promoting media we enrich for non-adherent CSC cultures.

The overall goal of this procedure is to select and isolate chemo resistant ovarian cancer stem cells or CSCs. This is accomplished by first fluorescently labeling ovarian cancer cell lines with red fluorescent protein or RFP and selecting for RFP positive cells using fluorescence activated cell sorting or facts. The second step of the procedure is to treat ovarian cancer cell lines with chemotherapeutic cisplatin for 72 hours and then culture the surviving cells in CSC media.

After two days, the non-adherence steroids will form. Next CSCs are characterized using gene expression analysis for stem cell markers and CSCs are analyzed for cell surface markers using flow cytometry. Ultimately, analysis of CSCs for therapeutic response is achieved by treating cells with cisplatin or paclitaxel and performing an MTT assay to measure viability.

The main advantage of this technique over existing methods like treating cancer cells with high doses of paclitaxel and cisplatin is that our method yields more viable cells while using less toxic amounts of chemotherapeutic agents. The implications of this technique extend towards improving therapy of ovarian cancer because CSCs represent a therapeutically resistant population that will need new treatment modalities. This method can also be applied to other systems such as pancreatic cancer, breast cancer, and other tumor types with therapeutically resistant cells.

Along with Jennifer Demonstrating this technique will be Dr.Leroy, a postdoc from my laboratory. To begin propagate Skov three and O cough 4 2 9 cell lines in a humidified incubator at 37 degrees Celsius with 5%CO2 and grow cells to between 40 and 50%co fluency. To generate SCV three cells expressing red fluorescent protein or RFP add lentiviral particles expressing RFP and 10 micrograms per milliliter of poly brain to SCV three media in the absence of penicillin and streptomycin for 72 hours.

After 72 hours, select for RFP positive cells by fluorescence activated cell sorting or facts by first trypsin RFP and non RFP expressing cells then centrifuge at 125 times G for five minutes following centrifugation resuspend the cells at 10 million cells per milliliter in PBS containing 0.1%BSA on a cell sorter. Use non RFP cells to determine the sorting parameters. Then collect cells that express high RFP using a 561 nanometer laser to enrich for cancer stem cells.

Treat SCV three, SCV three RFP or O cough 4 2 9 cell lines with 20 micromolar of chemotherapeutic cisplatin for 72 hours. Next trypsin is the cells and culture Surviving cells in CSC media. Change the media every two days by centrifuging cells at 125 times G for five minutes and Resus suspending the pellet in fresh CSC media check cells for viability every two days by counting cells and performing trian blue exclusion.

Collect CSCs for further experiments by centrifuging as before and resus suspending the pellet in the appropriate solution. Monitors CSC cell morphology using a light microscope at 20 x and 40 x magnifications. To characterize the CSCs first, collect three preparations of CSCs by centrifuging media containing cells at 125 times G for five minutes.

Then resuspend the pellet in a solution of phenol containing gu indium thiocyanate. For RNA extraction, then synthesize complementary DNA or CD NA from 0.1 to one microgram of RNA using a commercially available CDNA reverse transcription kit. Next, perform quantitative reverse transcribed polymerase chain reaction, abbreviated Q-R-T-P-C-R as detailed in the text protocol.

Perform all Q-R-T-P-C-R in duplicate for each sample using a real-time PCR thermocycler capable of detecting multiple fluorophores. As a final step, normalize stem cell gene expression to gap DH expression for each sample. Using the delta delta CT method.

Harvest CSCs by collecting media containing cells and centrifuging at 125 times G for five minutes. Add 500 microliters of a non proteolytic cell detachment solution to break up the cells before centrifuging again to remove the cell detachment solution. Then wash the cells twice with cold PBS.

Incubate the cells in normal growth media for one hour prior to labeling the cells after spinning the cells. As before, resuspend the cells in PBS with 0.1%BSA containing anti CD 1 33 PE and anti CD one 17 FE.Then fix the cells overnight in 1%Para formaldehyde perform flow cytometry to analyze CD one 17 and CD 1 33 cell surface expression. Collect data on cells in the FL one and FL three channels and generate gates for cells expressing both fits E and pe.

Generate a dot plot with four quadrants and determine the number of cells in each quadrant plate. 5, 000 cells per well for each cell variant on 96 well plates overnight. Then treat the cells with various concentrations of cisplatin or paclitaxel for 96 hours.

After 96 hours, add 10 microliters of 10 milligrams per milliliter. MTT incubate at 37 degrees Celsius for two to four hours until precipitate forms to solubilize.MTT. Add the MTT solvent a solution containing four millimolar hydrochloric acid and 0.1%NP 40 in Isopropanol.

Following incubation for 15 minutes in the dark, read the plates at 570 nanometers on a plate reader to demonstrate that CSCs were isolated from epithelial ovarian cancer cell lines using cisplatin treatment. Images of the cell lines were acquired prior to treatment and after selection, light microscopy captured images of adherence. SCV three and OCA 4 2 9 cells, as well as SCV three CSCs and OCA 4 2 9 CSCs CSCs appear round and not attach to the tissue culture plates.

SCV three cells transduced with RFP retain their fluorescence after isolation of the CSCs demonstrating that the cells are viable.Viable. CSC cultures were assessed for their response to drug therapy. CSCs demonstrated increased resistance to cisplatin and paclitaxel expression of stem cell genes.

CD 1 33 NEIN nano and T four was examined. In initial experiments. T four nano and nein were elevated in S SCV three CSC cultures compared to untreated, but the results were not statistically significant.

CD 1 33 was significantly induced in SCV three CSC cultures compared to untreated cells. Upon repeating these experiments, T four and nano increased in S SCV three CSCs as compared to parental control cells. SCV three, CSCs exhibit increased cell surface expression of the CSC marker CD one 17 as revealed by fax analysis While attempting this procedure, it is important to remember that the major limitation of this protocol is the length of time that the CSC remain viable.

Using this protocol, the CSC remain viable for less than a week. Therefore, the types of experiments and analysis for the CSCs need to be carefully timed. Don't forget that working with cisplatin can be extremely hazardous and precautions such as the use of protective clothing and avoiding contact with skin and mucus membranes should always be taken Following this procedure.

Other methods like triam blue exclusion can be performed in order to answer additional questions regarding the cell's viability, as well as limiting dilution assays to demonstrate the tumor promoting characteristics of the CSCs After its development. This technique paved the way for researchers in the cancer field to explore pathways that lead to chemo resistance.