1. Generation of Docetaxel-resistant Prostate Cancer Cells
NOTE: Perform cell treatments using Docetaxel solution stock used for the determination of IC50 drug concentration (prepare enough stock in advance for experimental use). Parental cells should be grown in parallel in a small flask throughout the whole procedure and exposed to vehicle (DMSO) in corresponding volumes mimicking the amounts used in the Docetaxel treated flasks.
<ol>
	<li>Plate DU145 or 22Rv1 cells in 150 cm2 flasks containing 20 mL of media (3.5 x 106 cells per flask for DU145 and 6.5 x 106 cells per flask for 22Rv1). Use of 10 to 20 flasks of cells is recommended to have enough cells at the end of the protocol.</li>
	<li>After 24 h, when cells are at about 70-80% confluence (Figure 1B, before Docetaxel), add Docetaxel at the IC50 concentration (5 nM for the cells described in this protocol).</li>
	<li>After 72 h, aspirate drug-containing media and add fresh, Docetaxel-free media (Figure 1B, 72 h after Docetaxel). Change the media every 3-4 days. Within approximately 1-2 weeks, resistant clones will appear evident under the microscope (Figure 1B, 7 and 14 days after Docetaxel).</li>
	<li>Aspirate media, carefully wash cells with 15 mL PBS and incubate with 4 mL 0.05% Trypsin-EDTA for 3-5 min at 37 &#176;C to detach cells from the flask surface.</li>
	<li>Resuspend trypsinized cells from the flask using 8 mL of fresh media. Pool cells from all treated flasks. Pellet cells by centrifugation (300 x g, 3 min, RT).</li>
	<li>Remove the supernatant, resuspend the cell pellet in 20 mL of fresh media and plate the cells in 150 cm2 flasks (3.5 x 106 cells per flask for DU145 and 6.5 x 106 cells per flask for 22Rv1).</li>
	<li>After 24 h, when cells are at about 70-80% confluence, add 5 nM Docetaxel (initial IC50 concentration) again.</li>
	<li>Repeat steps 1.3 to 1.6 on day 3.</li>
	<li>After 24 h, when cells are at about 70-80% confluence, treat them with 2X IC50 Docetaxel concentration (10 nM for the cells described in this protocol).</li>
	<li>Repeat steps 1.3 to 1.8, following a dose-escalation of Docetaxel concentrations (25 nM, 50 nM, 100 nM, and 250 nM), to generate a stable population of cells growing in the flasks under the final highest concentration. For higher concentrations, the dose-escalation protocol can be implemented for up to 6 months until the 1,000 nM concentration is reached (Figure 2A).</li>
</ol>

<table><tbody><tr><td>DU145 cells</td><td>ATCC</td><td>HTB-81</td><td></td></tr><tr><td>22Rv1 cells</td><td>ATCC</td><td>CRL-2505</td><td></td></tr><tr><td>Docetaxel</td><td>Selleck Chemicals</td><td>S1148</td><td>in DMSO (10mM)</td></tr><tr><td>Tissue culture flask 150cm^{2}</td><td>Falcon</td><td>355001</td><td></td></tr><tr><td>RPMI 1640 Medium</td><td>Gibco</td><td>11875093</td><td>Supplemented with 10% FBS and&#60;br/&#62; 1% Penicillin/Streptomycin</td></tr><tr><td>Foundation B Fetal Bovine Serum</td><td>Gemini</td><td>900208</td><td></td></tr><tr><td>1X Phosphate-Buffered Saline&#60;br/&#62; (PBS)</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>0.05%Trypsin-EDTA</td><td>Gibco</td><td>25300-062</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr></tbody></table>

generating chemoresistant prostate cancer cells: a procedure for obtaining drug-resistant cancer cells in vitro

Generating Chemoresistant Prostate Cancer Cells: A Procedure for Obtaining Drug-resistant Cancer Cells In Vitro

Source: Mohr, L. et al. <a href='https://www.jove.com/video/56327' target='_blank'>Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel.</a> J. Vis. Exp. (2017)
This video describes a protocol for generating taxane-resistant cell models of prostate cancer. These models can help study the pathways involved in progression to Docetaxel resistance in prostate cancer patients.

<img alt="Cell viability assay, IC50 determination; graphs, micrographs; chemo resistance analysis after docetaxel." src="/files/ftp_upload/20399/20399_Figure1.jpg" data-altupdatedat="1753371239000" data-alt="Figure 1" /> 
Figure 1: Generation of Docetaxel-resistant prostate cancer cell model systems. (A) Diagram depicting the determination of the Docetaxel IC50 in the parental prostate cancer cell lines DU145 and 22Rv1 (Step 1 of the protocol). Expe...

Source: Mohr, L. et al. Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel. J. Vis. Exp. (2017)
This video ...

To generate chemoresistant cells, begin by taking human prostate cancer cell suspension in a culture flask. Incubate the flask to facilitate cell adherence and their division to form a monolayer.
Supplement the cells with an anti-cancer drug solution at an optimum concentration and incubate. The drug molecules enter the cells and induce apoptosis in sensitive cells.
Some cells express special drug efflux transporters or pumps that periodically remove the drugs. This mechanism prevents drug accumulation, enabling these cells to withstand the effect of anti-cancer drugs and become chemoresistant.
Aspirate the drug- and debris-containing media from the culture. Supplement with fresh drug-free growth media to support the growth of chemoresistant cells. Discard the spent media. Trypsinize the cells to detach them from the culture flask and transfer them to a tube.
Centrifuge the tube to pelletize the cells. Remove the trypsin-containing supernatant. Resuspend the cells in fresh growth media. Grow the cells in a new culture flask.
Subsequently, repeat the drug treatment and cell culture steps to expose the chemoresistant cells to escalating doses of the same anti-cancer drug. This technique screens the culture by eliminating all the sensitive cells to generate a pool of highly chemoresistant surviving cells.

To begin this procedure, plate DU145 or 22Rv1 cells in the 150-centimeter squared flasks containing 20 milliliters of media. After 24 hours, when the cells are at about 70% to 80% confluence, add docetaxel at 5 nanomolar.
After 72 hours, aspirate the drug-containing media and add fresh docetaxel-free media. Change the media every 3 to 4 days, and wait for 1 to 2 weeks until clones appear in the flask. Aspirate the media. Carefully wash the cells with 15 milliliters of PBS and incubate them with 4 milliliters of 0.05% trypsin-EDTA for 3 to 5 minutes at 37 degrees Celsius to detach the cells from the flask surface. Then, resuspend the trypsinized cells from the flask using 8 milliliters of fresh media.
Pool the cells from all treated flasks and pellet them by centrifugation. Following that, remove the supernatant. Resuspend the cell pellet in 20 milliliters of fresh media and plate the cells in 150-centimeter squared flasks.
After 24 hours, when the cells are at about 70% to 80% confluence, add 5 nanomolar docetaxel again. Repeat the procedures as shown previously until the clones appear in the flask. Then, plate the cells in 150-centimeter squared flasks again.
After 24 hours, when the cells are at about 70% to 80% confluence, treat them with 10 nanomolar docetaxel. Repeat the same steps in a docetaxel dose-escalating manner and keep pooling the surviving clones after every concentration treatment. At the end of the process, you will obtain a pool of resistant cells ready for experimental use.

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3.0K Views. To generate chemoresistant cells, begin by taking human prostate cancer cell suspension in a culture flask. Incubate the flask to facilitate cell adherence and their division to form a monolayer. Supplement the cells with an anti-cancer drug solution at an optimum concentration and incubate. The drug molecules enter the cells and induce apoptosis in sensitive cells. Some cells express special drug efflux transporters or pumps that periodically remove the drugs. This mechanism pr...

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Protocol

<img title="figure-results-1" alt="Cell viability assay, IC50 determination; graphs, micrographs; chemo resistance analysis after docetaxel." src="/files/ftp_upload/20399/20399_Figure1.jpg" data-altupdatedat="1753371239000" data-alt="Figure 1" /> 
Figure 1: Generation of Docetaxel-resistant prostate cancer cell model systems. (A) Diagram depicting the determination of the Docetaxel IC50 in the parental prostate cancer cell lines DU145 and 22Rv1 (Step 1 of the protocol). Expe...

Generating Chemoresistant Prostate Cancer Cells: A Procedure for Obtaining Drug-resistant Cancer Cells In Vitro

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Name	Company	Catalog Number	Comments
DU145 cells	ATCC	HTB-81
22Rv1 cells	ATCC	CRL-2505
Docetaxel	Selleck Chemicals	S1148	in DMSO (10mM)
Tissue culture flask 150cm^{2}	Falcon	355001
RPMI 1640 Medium	Gibco	11875093	Supplemented with 10% FBS and<br/> 1% Penicillin/Streptomycin
Foundation B Fetal Bovine Serum	Gemini	900208
1X Phosphate-Buffered Saline<br/> (PBS)	Corning	21-040-CM
0.05%Trypsin-EDTA	Gibco	25300-062
Penicillin-Streptomycin	Gibco	15140122