Functional Chemoresistance Characterization: A Method to Evaluate Drug Resistance in Cancer Cells Using Clonogenic Assay

Abstract

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 describes a method for evaluating chemotherapeutic drug resistance in prostate cancer cells using colony formation assay. This assay can be used as a functional tool to identify novel molecular and cellular mechanisms of chemoresistance in cancer cells.

Protocol

1. Functional Characterization of Acquired Docetaxel Resistance by Colony Formation Assay

NOTE: In this protocol, chemoresistance has been evaluated using colony formation assays. Alternative methods used to evaluate cell viability (i.e., MTS assays) can be used based on investigators' preferences.

1. Plate 2,000 cells (DU145 or 22Rv1, both parental and Docetaxel-resistant) per well in 6-well plates, using 2 mL of media per well.
2. After 24 h, add increasing concentrations of Docetaxel (parental cells: 0.25, 0.5, 1, 2.5 and 5 nM; DR cells: 50, 125, 250, 500 and 1,000 nM). For both DU145 and 22Rv1 cell lines. Add DMSO only to one well as a control at the same volume used for the highest Docetaxel dose.
3. After 72 h, aspirate the drug-containing media and add fresh Docetaxel-free media.
4. Incubate plates for 1-2 weeks until colonies are visible under the microscope.
5. To stain the colonies, wash them gently with 2-3 mL PBS and incubate with 2-3 mL crystal violet solution (0.1% w/v in 10% formalin) for 20 min inside the tissue culture hood or a fume hood.
6. Remove staining solution, wash plates with 2-3 mL of H₂O, remove H₂O and air-dry plates.
7. Analyze the result by visualizing the wells and manually counting colonies with the help of a marker pen (to avoid counting the same colonies twice) and represent the percentage of cell viability in a graph. Take digital images of the plates for figure representation (Figure 1A).

Results

Figure 1: Functional and phenotypic characterization of the Docetaxel-resistant cell models. (A) Representative colony formation assays of parental and Docetaxel-resistant cells treated with the indicated Docetaxel concentrations for 7...

Materials

List of materials used in this article

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 150 sq cm  	 Falcon	355001
6-well tissue culture plates 	 Falcon	353046
RPMI 1640 Medium  	Gibco	11875093	Supplemented with 10% FBS and 1% Penicillin/Streptomycin
Foundation B Fetal Bovine Serum  	Gemini	900208
1X Phosphate-Buffered Saline (PBS) 	Corning	 21-040-CM
0.05%Trypsin-EDTA 	Gibco	 25300-062
Penicillin-Streptomycin  	Gibco	15140122
Crystal Violet  	Sigma-Aldrich	C0775
10% Formalin Solution  	Sigma-Aldrich	HT501128