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

Overview

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).

Representative 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 72 h. Percentage of colonies for every treatment concentration is represented in the included graph. The experiments are triplicates and data represent the mean ± SD. Scale bars = 5 mm. (B) DR/parental relative mRNA fold increase quantified by qRT-PCR of ABCB1, GATA2, CK19, and CDH1 are shown. All qPCR raw data is normalized to Actin (see protocol for details). The experiments are triplicates and data represent the mean ± SD. *p <0.05.

Materials

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