Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Summary

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Abstract

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Introduction

Live-cell microscopy and longitudinal tracking of single cells is not a new technique. From the earliest microscopes, enthusiasts and scientists have observed and studied single cells and organisms, their behaviors, and development^1-3. A famous example from the late David Rogers at Vanderbilt University in the 1950s shows a human neutrophil in a blood smear chasing a Staphylococcus aureus bacterium and eventually the process of phagocytosis⁴. This live-cell movie is an excellent illustration of how multiple processes can be observed and correlated in a single experiment: sensing of a chemical gradient, mechanics and speed of cell motilit....

Protocol

 The following protocol uses parameters defined by the experiments in Figures 4 and 6 regarding acquisition settings and experimental conditions. Many of these parameters can be modified to fit other experiments (i.e., exposure times, binning, fluorescent channels, etc.). All procedures must adhere to institutional guidelines and regulations and be approved by the institutional biosafety committee. Microscope manufacturer websites contain excellent information for live cell imaging.

1. Microscopes and Imaging Software

1. Perform live cell imaging on a wide variety of inverted....

Results

Long-term time-lapse microscopy and direct longitudinal tracking allows for the study of many anti-cancer effects during drug response. Following the general outline in Figure 1, multiple examples of cells are shown expressing validated fluorescent reporters that treated with anti-cancer drugs, tracked, and analyzed using different approaches.

Phase contrast microscopy alone is very informative and robustly repo.......

Discussion

Advantages of Time-lapse Microscopy and Longitudinal Tracking

The microscope is an ideal instrument for longitudinal studies of drug response as it allows investigators to track individual cells and their fates as well as the entire population. Variability in drug response within a population of cells is a major issue for anti-cancer therapeutic design. Longitudinal tracking of single cells allows investigators to observe this variability and begin to understand the underlying.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Taxol (paclitaxel)	Sigma	T7191	microtubule stabilizing drug
Etoposide	Selleckchem	S1225	topoisomerase II inhibitor
Selinexor	Karyopharm Therapeutics	na	XPO1/CRM1 inhibitor, gift
Kinesin-5 inhibitor	Merck Serono	na	gift, also available from American Custom Chemicals Corporation. CAS 858668-07-2
Cell growth medium	HyClone (Fisher) or Mediatech		many companies available
5% CO2/balance air, certified	Airgas	Z03NI7222004379
35 mm Dish, 20 mm glass bottom	Cellvis	D35-20-1.5-N	many companies available
35 mm 4-well Dish, 20 mm glass bottom	Cellvis	D35C4-20-1.5-N	many companies available
35 mm Dish, gridded glass bottom	MatTek	P35G-2-14-CGRD	many companies available
Multi-well, glass bottom	Cellvis	P12-1.5H-N	many companies available
Olympus IX81 inverted epifluorescence microscope	Olympus
Olympus IX2-UCB controller	Olympus
PRIOR LumenPro200	Prior Scientific	Lumen200PRO
PRIOR Proscan III motorized stage	Prio Scientific	H117
STEV chamber	InVivo Scientific	STEV.ECU.HC5 STAGE TOP
Environmental Controller Unit	InVivo Scientific	STEV.ECU.HC5 STAGE TOP
Hamamatsu ORCA R2 CCD with controller	Hamamatsu	C10600
Nikon Eclipse Ti	Nikon
Nikon laser launch	Nikon
SOLA light engine	lumencor
iXon Ultra 897 EM-CCD	ANDOR
TOKAI HIT inclubation chamber	TOKAI HIT	TIZSH