Viability Assessment Following Anticancer Treatment Requires Single-Cell Visualization

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.

Intratumor Heterogeneity and Therapy Resistance: Contributions of Dormancy, Apoptosis Reversal (Anastasis) and Cell Fusion to Disease Recurrence

“…The formation of PGCCs following treatment with anticancer agents is not an infrequent response [25][26][27][28][29][30], and their frequency can be influenced by heterogeneous environment and chemotherapeutic drug gradient, increasing remarkably at high drug concentrations [25]. Given that PGCCs cease to proliferate or proliferate at a very slow rate, they are often overlooked or misrepresented as "dead" in the conventional pre-clinical assays that are performed with cultured cells or animal models (reviewed in [31,32]). However, over 60 years ago Puck and Marcus [33] reported that PGCCs (originally called giant cells), arising in cervical carcinoma (HeLa) cell cultures following exposure to ionizing radiation, remain viable and secrete growth-promoting factors.…”

“…A step towards reaching this goal will be to understand not only the bright sides of different stress-induced responses (e.g., senescence) that might impact on the initial tumor control, but also their dark sides that might contribute to tumor repopulation. In addition, as cautioned by the Nomenclature Committee on Cell Death [155] and others [32,156,157], it is crucial to use the available tools (e.g., cell-based assays) in the right contexts to avoid generating misleading information. To this end, as discussed below, the colony formation and multiwell plate colorimetric/fluorometric assays predominantly determine the ability of a genotoxic agent to convert "dangerous" (proliferating) cancer cells to "even more dangerous" (e.g., dormant) cancer cells, rather than dead cancer cells.…”

“…MTT is a yellow tetrazolium salt which is metabolized by viable cells to a purple water-insoluble formazan. MTT metabolites can be visualized as intracellular granules and crystals under a light microscope [32] (also see Figure 2). Crystal violet (gentian violet) is a blue, aniline-derived dye that binds to cellular macromolecules.…”

Loss of p53 Sensitizes Cells to Palmitic Acid-Induced Apoptosis by Reactive Oxygen Species Accumulation

“…In the current work we used standard molecular and cellular features of apoptosis. However, it is now well known that cells triggered to undergo apoptosis can recover from brink of death through a process called anastasis [59]. Therefore, to what extent the responses reported herein reflect death remains to be determined.…”

The Cancer Aneuploidy Paradox: In the Light of Evolution

“…They represent rare single cells undergoing reprogramming and complex multi-step, reversible ploidy cycles, which are experimentally observed over a period of weeks. Although these rare "statistical outliers" present serious methodical difficulties for following and fate-mapping, making studies of their nature and genesis complex, they are apparently the major mediators of anti-cancer therapy resistance responsible for relapse [85].…”