Development of Drug-resistant Cell Lines for Experimental Procedures

Summary

Drug-resistant cell lines enable understanding of treatment resistance mechanisms and nonclinical evaluation of new compounds, repurposed drugs, and combination therapies. This paper introduces a protocol for creating resistant cell lines by exposing parent cell lines to stepwise increases in the concentration of the target drug.

Abstract

The development of drug-resistant cell lines is essential for understanding the mechanisms of drug resistance and identifying strategies to overcome treatment failure in cancer therapy. Resistance models enable preclinical evaluation of novel compounds, repurposed drugs, and combination therapies. To generate resistant cells, parental cancer cell lines are repeatedly exposed to incrementally increasing concentrations of the target drug over several weeks. Cells that survive and proliferate at each stage are selected, expanded, and exposed to higher drug doses. The development of resistance is confirmed by quantifying and comparing the half-maximal inhibitory concentration (IC₅₀) values between parental and resistant cells using cell viability assays and nonlinear regression analysis. Significantly increased IC₅₀ values indicate successful adaptation to drug pressure and the development of resistance. These drug-resistant cell lines are available for comprehensive analysis, such as microarray and single-cell sequencing, as well as various in vitro or in vivo experiments. These models provide valuable tools for investigating potential therapeutic strategies to overcome drug resistance.

Introduction

Chemotherapy is the most frequently used treatment option for local cancers not amenable to surgery or radiation therapy or metastatic cancers. Molecularly targeted drugs and chemotherapy are used as first-line treatments for many malignancies, and their efficacy is widely recognized. Some cancers show resistance to certain drugs from the start of treatment, making treatment ineffective. However, in more common cases, treatment that was initially effective becomes ineffective due to the development of drug resistance, leading to the progression of cancer.

Drug resistance is caused by both genetic mutations or epigenetic response in cancer c....

Protocol

1. Cell culture and cell viability assays

NOTE: Paclitaxel is an anticancer drug and is cytotoxic. When handling it, wear a gown, goggles, and gloves, and work inside a safety cabinet to prevent inhalation into the body. DMSO is not highly cytotoxic, but when it comes into contact with skin or mucous membranes, the permeability of dissolved drugs increases, so wear a gown, goggles, and gloves when handling it.

1. Culture DU-145 cells in a 10 cm cell culture dish using complete medium (RPMI-1640 + 1% penicillin-streptomycin [pen-strep] + 10% fetal bovine serum [FBS]) in a 37 °C incubator at 5% CO₂

Results

Using this method, we previously spent 9 months successfully establishing paclitaxel-resistant DU145 cells (DU145-TxR). The results of the cell viability assay showed that the IC₅₀ value of paclitaxel for the DU145 parent cell line was 1.1 nM, whereas the IC₅₀ value for DU145-TxR was 149.6x higher than that of the parent cell line (IC₅₀: 164.6 nM) (Table 1 and Figure 2). This result indicates that DU145-TxR has acquired very high drug resista.......

Discussion

The development of drug-resistant cancer cell lines is an established method for investigating drug toxicity and resistance mechanisms, but the first challenge researchers face is selecting an appropriate model for expressing resistance. A common method for creating drug-resistant cell lines in vitro is to expose the cell line to the target drug, but it is necessary to consider how to set the exposure concentration (stepwise or all at once) and whether to set a drug-free period (pulsed or continuous).

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	Gibco	25300-054
10 mL Disposable serological pipette	Fisher scientific	13-678-11E
100 mm dish	Corning	353003
15 mL conical tube	Corning	352097
2.0 mL Cryogenic Vials	Corning	431386
96 well cell culture plate	Corning	353072
Cell counting chamber slide	Invitrogen	C10228
Cell Proliferation Reagent WST-1	Sigma	5015944001
Centrifuge 5702 R	eppendorf
countless II	Invitrogen		Cell counter
Dimethyl sulfoxide	Sigma	D2650
DPBS	Gibco	D4W2J
DU145	ATCC	HTB-81	Prostate cancer cell line
EVOS FLc	Thermo Fisher Scientific		Digital Inverted Microscope
FBS	Gibco	A5670701
Paclitaxel	Selleck	S1150
Penicillin–streptomycin	Invitrogen	15140-122
RPMI 1640	Gibco	11875-093
Synergy H1	BioTek		Microplate Reader
Trypan Blue solution 0.4%	Invitrogen	15250061