Background

Entosis is a form of cell–cell interaction in which a living cell (inner cell) orchestrates its invasion into another cell (outer cell) by a Rho/ROCK signaling–dependent mechanism (Overholtzer et al., 2007). Entosis results in the formation of a unique morphological microscopic structure, also known as cell-in-cell structure or bird’s eye structure, which is characterized by evidence of an inner cell, a visible entotic vacuole between inner and outer cells, and an outer cell with a crescent-shaped nucleus as it is pushed towards the periphery (Fais and Overholtzer, 2018). Entosis spontaneously—but rarely—occurs in cultured cells in vitro; nevertheless, it has important consequences for both inner and outer cells. From one perspective, entosis seems to be an assisted-suicide mechanism for the inner cells, as the vast majority of these undergo lysosomal degradation by a mechanism known as entotic cell death (Florey et al., 2011). However, inner cells can also survive and be released, and even complete full cell division inside outer cells. Intriguingly, outer cells also benefit from this invasion as they gain survival advantage under stress conditions (Hamann et al., 2017) or become resistant to apoptosis (Bozkurt et al., 2021). Entosis-like structures are more frequently observed in cancers compared to normal tissues, and the presence of these structures is associated with poor outcome and disease recurrence in cancer (Mackay et al., 2018; Bozkurt et al., 2021).

Quantification of entosis relies on morphological detection of the events using microscopy images. Currently, it is not possible to automatically quantify entosis events when both inner and outer cells are alive. Thus, events are first detected with the aid of nuclear, cytoplasmic, and/or membrane staining, and then manually quantified (Overholtzer et al., 2007). When inner cells undergo entotic cell death, however, quantifying entosis events is relatively easy due to lysosomal acidification of inner cells. Lysosomal marker Lamp1 or autophagic marker LC3 can be fluorescently expressed in the cells to quantify the dynamics of entotic cell death (Florey et al., 2011; Overholtzer et al., 2007). However, overexpression of artificially produced proteins might also affect the rate of entosis events. Probably the most practical method to label entotic cell death is using lysosomal dyes such as LysoTracker, which freely passes the cell membrane and is sequestered inside acidic organelles. Alternatively, fluorogenic cathepsin substrates or acridine orange have been used to detect entosis and entotic cell death (Overholtzer et al., 2007; Garanina et al., 2017).

Recently, we reported a novel alternative approach for detecting entosis events and measuring the dynamics of entotic cell death by using tetramethylrhodamine methyl ester (TMRM) (Bozkurt et al., 2021). This cationic cell-permeable dye is sequestered by active mitochondria and has been widely used to analyze real-time changes in mitochondrial events such as loss of mitochondrial membrane potential during apoptosis as well as fusion/fission (Dußmann et al., 2003; Cho et al., 2019). Here, we provide a detailed protocol for performing time-lapse microscopy in any cell line to detect entosis events and entotic cell death by using TMRM staining. Our approach allows simultaneous detection of mitochondrial and entotic events as well as quantification of the real-time kinetics in living cells at the single-cell level.