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. 2014 Mar 6:4:40.
doi: 10.3389/fonc.2014.00040. eCollection 2014.

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

Martina McDermott  1 , Alex J Eustace  2 , Steven Busschots  3 , Laura Breen  4 , John Crown  5 , Martin Clynes  4 , Norma O'Donovan  4 , Britta Stordal  6
Affiliations

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

Martina McDermott et al. Front Oncol. .

Abstract

The development of a drug-resistant cell line can take from 3 to 18 months VSports手机版. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance. .

Keywords: cancer; cell lines; chemotherapy; drug-resistance; selection strategy V体育安卓版. .

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Figures

Figure 1
Figure 1
Recovery of OVCAR8 and UPN251 from carboplatin or paclitaxel treatment. 1 × 104 cells were treated for 3 days with the indicated doses of either carboplatin or paclitaxel and recovery monitored as described in Section “Methods.”
Figure 2
Figure 2
Comparative selection of drug-resistant cell lines. (A) Plan for selection of two parent cell lines with two different drugs to produce four drug-resistant daughter cell lines. (B) Plan for selection of one parent cell line with two drugs at different doses or treatment intervals, producing four drug-resistant daughter cell lines.
Figure 3
Figure 3
Continuous selection of HCC1954 with lapatinib. (A) Proliferation of HCC1954 cells following a 5-day treatment with lapatinib. (B) HCC1954 cells treated with varying concentrations of lapatinib over a 4-day period. Cell counts were performed using ViaCount reagent and Guava Software and expressed relative to control untreated cells. (C) Images of HCC1954-par and HCC1954-L cells after 3 months of lapatinib conditioning at 100× magnification. (D) After 3 months conditioning with 1 μM lapatinib, the proliferation of HCC1954-par and HCC1954-L cells was measured. (E) After 6 months conditioning with lapatinib, the proliferation of HCC1954-par and HCC1954-L was assessed. (F) Images of HCC1954-par and HCC1954-L cells after 6 months of lapatinib conditioning at 200× magnification. (G) Sensitivity of HCC1954-par and HCC1954-L cells to lapatinib following a freeze-thaw cycle. Growth is expressed relative to untreated control cells. (H) Lapatinib IC50 values for HCC1954-par and HCC1954-L cells following 1, 2, and 3 months growth in the absence of lapatinib. All growth rates and IC50s were calculated following a 5-day lapatinib treatment. Growth is expressed relative to untreated control cells. All error bars represent the standard deviation of triplicate experiments. Student’s t-test was performed to determine significant differences: *p < 0.05; **p < 0.01.
Figure 4
Figure 4
Pulse selection of Malme-3M and HT144 with temozolomide. (A) Selection strategy of Malme-3M and HT144, each treatment of temozolomide was performed in duplicate. Effect of temozolomide in Malme-3M and HT144 and temozolomide “pulse selected” resistant variants. (B) Malme-TMZ(A) and Maleme-TMZ(B) vs. Malme-3M cells (C). HT144-TMZ vs. HT144 cells. Error bars represent the standard deviation of triplicate assays.
Figure 5
Figure 5
Maintaining drug-resistant cell lines in cell culture. (A) Stable cell lines require no drug treatment to maintain their resistant phenotype. (B) Some cell lines are grown continually in the presence of chemotherapy, chemotherapy needs to be removed for one subculture prior to using for experiments. (C) Some models are repeat pulse treated after a certain number of passages or weeks in culture once their resistant phenotype begins to fade. (D) Some models are discarded and new cells grown from master stocks after a certain number of passages or weeks in culture once their resistant phenotype begins to fade.
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