Michael Overholtzer, PhD

Entosis: Adherens Junctions Promote Cell-in-Cell Structures

A variety of human tumors exhibit unusual cytological features, noted by pathologists for decades, termed cell-in-cell, where viable tumor cells are internalized inside of neighboring tumor cells. We found that these cell-in-cell structures can form by an adhesion-based mechanism, termed entosis, involving adherens junctions and Rho-dependent contractile force (Figure 1A,B). Adherens junctions normally bind cells together by linking cadherin transmembrane protein complexes of adjacent cells to the contractile machinery of the cytoskeleton. When certain cell types are detached from extracellular matrix (ECM) and have lost integrin-dependent adhesion, cell-cell compaction force can drive cells to push into their neighbors, forming cell-in-cell structures (Figure 1, Movies 1,2).

Cell-in-cell structures can form between 2 cells (Movie 1), or between three or more cells (Movie 2). Note in Movie 2 that as the cell-in-cell structure forms between three cells, the middle cell acts as both an internalizing and an outer host cell at the same time.

• Movie 1
• Movie 2
• Movie 3

	[Enlarge Image] Entosis: Cell-Cell Adhesion Drives the Formation of Cell-in-Cell Structures

(A) Model for entosis. Left image: blue cell pushes into green cell by utilizing Rho-dependent contractile force. Right image: internalized cell undergoes cell death by lysosome-dependent degradation. (B) Stages of cell-in-cell formation: left image, half-way internalized; middle image, almost completely internalized; right image, completely internalized. Red=â-catenin; green=E-cadherin. Arrows: plaques characteristic of adherens junctions. (C) Internalized cells undergo cell death. Left image: Internalized cell is surrounded by lysosomal membrane marked by LAMP1 staining (green). Right image: Internalized cell displays features of nuclear and total cellular degradation (arrow (TEM).

	[Enlarge Image] Cell-in-Cell Structures in Human Breast Tumors Human primary breast tumor sections stained for collagen (red)(left image, cell-in-cell structure circled by dashed line), or Beta-catenin (green)(right image). Note that cell-in-cell structure in left image occurs between cells detached from the extracellular matrix, shown by collagen staining.

Although cells internalized by entosis are initially viable and can actually divide within or be released from the host cell, the majority of internalized cells eventually undergo cell death (Figure 1C, Movie 3). Cell death occurs by a non-apoptotic mechanism involving total cellular degradation inside of a large lysosomal compartment (Figure 1C, Movie 3). Because entosis can lead to the death of matrix-detached cells, and a variety of tumor cells grow away from proper matrix attachment (Figure 2), this mechanism of cell-in-cell formation could suppress primary tumor growth, or could inhibit metastatic lesions from forming outside of their natural matrix environment. Adherens junctions are inhibited in a variety of tumors, either directly by the ablation of genes such as E-cadherin or á-catenin, or indirectly by altered extracellular matrices, which disrupt cell-cell junctions by increasing integrin-dependent adhesion force. The down regulation of adherens junctions promotes cell scattering; thus, adherens junctions are thought to inhibit invasion and metastasis. Through the process of entosis, adherens junctions could also affect cell survival and tumor growth by eliminating cells with weakened adhesion to ECM.