Consecutive entosis stages in human substrate-dependent cultured cells

Abstract

Entosis, or cell death by invading another cell, is typical for tumor epithelial cells. The formation of cell-in-cell structures is extensively studied in suspension cultures, but remains poorly understood in substrate-dependent cells. Here, we used electron, confocal and time-lapse microscopy in combination with pharmacological inhibition of intracellular components to study the kinetics of entosis using two human substrate-dependent tumor cultures, A431 and MCF7. In total, we identified and characterized five consecutive stages of entosis, which were common for both examined cell lines. We further demonstrated that actin filaments in the entotic as well as invading cells were crucial for entosis. Microtubules and the Golgi apparatus of entotic cells provided membrane expansion required for internalization of the invading cell. Depolymerization of microfilaments and microtubules, and disintegration of the Golgi complex inhibited entosis. We confirmed the presence of adhesive junctions and discovered the formation of desmosomes between the invading and entotic cells. The internalized cell was shown to be degraded due to the lysosomal activation in both cells whereas the disintegration of the Golgi apparatus did not affect the process. Thus, in the substrate-dependent cultures, entosis requires microfilaments, microtubules and the Golgi complex for cell invasion, but not for internalized cell degradation.

Multiple cell death modalities and their key features (Review)

“…Wan et al proposed that the overactivation of myosin or unbalanced myosin activation through regulatory polarity proteins between the contacting cells acted as the driving force for entosis in matrix-attached epithelial cells (30). The engulfment is followed by lysosome-mediated degradation, which differs from autophagic cell death (31). The autophagic protein, microtubule-associated protein light chain 3 (LC3), does not participate to form the autophagosome.…”

Entosis: From Cell Biology to Clinical Cancer Pathology

“…In cancer cell lines, entosis was described almost only in cells, either artificially detached from a substratum by culturing them in nonadhesive conditions (MCF7 cells), after starvation [ 12 ], or due to the effects of anticancer drugs such as paclitaxel [ 12 ], nintedanib [ 24 ], and novel methylselenoesters [ 23 ]. Only non-cancerous HaCaT keratinocytes were shown to undergo entosis as adherent cells, but the rate of entosis was very low (less than 0.05%) [ 37 ]. In contrast, in in vivo samples of tumours, entosis can be found under normal circumstances, without any anticancer treatment, both in primary lesions and in metastases (see Figure 4 ).…”

Biological relevance of cell-in-cell in cancers

“…A low level of entosis is encountered in susceptible cell lines under normal tissue culture conditions, but higher rates are seen when cells are grown in matrix-detached conditions [3–5]. Even in ‘spontaneous’ entosis under normal growth conditions, the invading cell detaches prior to engulfment, suggesting that matrix detachment is an important trigger for entosis [6,7]. Under normal culture conditions, matrix detachment occurs prior to mitosis or apoptosis [6,8].…”