Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Summary

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7_TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

Abstract

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics.

Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged α-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7_CC cells, the microtubule dynamics of docetaxel-resistant MCF-7_TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

Introduction

The leading cause of breast cancer mortality is through metastasis^1,2. Taxanes, such as docetaxel and paclitaxel, are currently used as first-line regimens in the treatment of metastatic breast cancer^2,3,4,5,6. They are part of a group of microtubule-targeting agents (MTAs) that disrupt microtubule dynamics. However, one of the greatest challenges to using taxanes in curative therapy is the development of taxane resistance in cancer cells, which lea....

Protocol

1. Preparing the Cells for Live Imaging

1. Cell culture and seeding
   1. Use MCF-7 breast cancer cells selected for resistance to docetaxel (MCF-7_TXT) and their non-resistant parental cell line (MCF-7_CC). The detailed selection process and the characterization of these selected cell lines were described previously²⁴.
   2. Grow all cells in 10 cm culture dishes at 37 °C in a medium composed 90% of Dulbecco's modified Eagle's medium (DMEM) and 10% of fetal bovine serum (FBS) and supplemented with non-essential amino acids. Maintain the medium in a 5% CO₂ atmosphere. Maintain ....

Results

Using the protocol presented here, we studied the effects of docetaxel on the microtubule dynamics of normal (MCF-7_CC) and docetaxel-resistant (MCF-7_TXT) breast cancer cells. Two sets of images show the effects of docetaxel (0.5 µM) on microtubule growth and shortening in MCF-7_CC and MCF-7_TXT cells (Figure 1A).

We also calculated the rate of microtubule growth or short.......

Discussion

There are two major methods to measure microtubule dynamic instability: in vitro and in vivo. In the in vitro method, purified tubulin is used to measure microtubule dynamic instability with computer-enhanced time-lapse differential interference-contrast microscopy. In the in vivo method, microinjected fluorescent tubulin, or expressed GFP-tubulin, is incorporated into microtubules. The dynamics (growth and shortening) of the microtubules is then recorded by time-lapse using a sensitiv.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle's Medium (DMEM)	Sigma-Aldrich	D5796
Non-essential amino acids	Life Technologies, Invitrogen	11140-050
FBS	Gibco, Invitrogen	12483
Anti-Anti (100x)	Life Technologies, Invitrogen	15240-062
docetaxel	Sigma-Aldrich	01885-5mg-F
DMEM phenol red-free	Gibco, Invitrogen	21063
CellLight Reagent *BacMam 2.0* GFP-tubulin	ThermoFisher Scientific	C10613	Key reagent for expressing GFP tubulin in cells
CellLight Reagent *BacMam 2.0* GFP	ThermoFisher Scientific	B10383	Control
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich+B9:AA9	472301	for dissoving decetaxel
22-mm glass coveslip	Fisher Scientifics	12-545-101
6-well culture plate	Greiner Bio-One International	6 Well Celi Culture Plate
DeltaVision Microscopy Imaging Systems	GE Health		This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.
Trypsin-EDTA (0.25%), phenol red	ThermoFisher Scientific	25200056
Bright-Line Hemacytometer Set, Hausser Scientific	Hausser Scientific, Distributed by VWR	Supplier No.: 1492 VWR No.:15170-172