Viability Assessment Following Anticancer Treatment Requires Single-Cell Visualization.

Razmik Mirzayans,David Murray,Bonnie Andrais

doi:10.3390/cancers10080255

Abstract

A subset of cells within solid tumors become highly enlarged and enter a state of dormancy (sustained proliferation arrest) in response to anticancer treatment. Although dormant cancer cells might be scored as “dead” in conventional preclinical assays, they remain viable, secrete growth-promoting factors, and can give rise to progeny with stem cell-like properties. Furthermore, cancer cells exhibiting features of apoptosis (e.g., caspase-3 activation) following genotoxic stress can undergo a reversal process called anastasis and survive. Consistent with these observations, single-cell analysis of adherent cultures (solid tumor-derived cell lines with differing p53 status) has demonstrated that virtually all cells—irrespective of their size and morphology—that remain adherent to the culture dish for a long time (weeks) after treatment with anticancer agents exhibit the ability to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide (MTT). The purpose of this commentary is to briefly review these findings and discuss the significance of single-cell (versus population averaged) observation methods for assessment of cancer cell viability and metabolic activity.

Highlights

Cell-based assays are indispensable for preclinical evaluation of agents with potential anticancer properties
In a typical anticancer drug discovery approach, the test compound is first evaluated in a short-term, high throughput colorimetric/fluorimetric assay in a multiwell plate format to determine the IC50 value
Apoptosis derives from the Greek for “dropping off”, and refers to a highly complex and sophisticated programmed cell death process involving an energy-dependent cascade of molecular events

Summary

Introduction

Cell-based assays are indispensable for preclinical evaluation of agents with potential anticancer properties. All of the aforementioned short-term assays depend on using large numbers of cells and averaging the measured response parameters Despite their relative ease of performance and high throughput (e.g., using a plate reader to generate “viability” data for a large number of samples simultaneously), all global analyses. The assayby optimized us is typically completed in 4 days from the seeding post-treatment direct cellby counting proved to be most informative and versatile [3,4,5].of cells [3,4,5] Despite it generates radiosensitivity/chemosensitivity results that are similar to. We have compared these two assays for radiosensitivity assessment in twelve solid tumor-derived and lines the chemotherapeutic drug2), oxaliplatin in the HCT116 colon carcinoma andand its p53 cell [3,4] ( see Figure and for sensitivity to 254-nm ultraviolet cell lightline the knockout derivative (data to be published elsewhere). Obtained from Mirzayans et al [3,4]

Single-Cell

Surviving

Apoptotic and Non-Apoptotic Functions of Caspase-3

Reversibility of Apoptosis in Solid Tumor-Derived Cell Lines

The Single-Cell MTT Viability Assay

Conclusions