E-cadherin suppression directs cytoskeletal rearrangement and intraepithelial tumor cell migration in 3D human skin equivalents

Abstract

The link between loss of cell-cell adhesion, the activation of cell migration, and the behavior of intraepithelial (IE) tumor cells during the early stages of skin cancer progression is not well understood. The current study characterized the migratory behavior of a squamous cell carcinoma cell line (HaCaT-II-4) upon E-cadherin suppression in both 2D, monolayer cultures and within human skin equivalents that mimic premalignant disease. The migratory behavior of tumor cells was first analyzed in 3D tissue context by developing a model that mimics transepithelial tumor cell migration. We show that loss of cell adhesion enabled migration of single, IE tumor cells between normal keratinocytes as a prerequisite for stromal invasion. To further understand this migratory behavior, E-cadherin-deficient cells were analyzed in 2D, monolayer cultures and displayed altered cytoarchitecture and enhanced membrane protrusive activity that was associated with circumferential actin organization and induction of the nonmuscle, beta actin isoform. These features were associated with increased motility and random, individual cell migration in response to scrape-wounding. Thus, loss of E-cadherin-mediated adhesion led to the acquisition of phenotypic properties that augmented cell motility and directed the transition from the precancer to cancer in skin-like tissues.

Figure 1. Abrogation of cell–cell adhesion results in transepithelial, individual migration of E-cadherin-deficient cells in 3D tissues

Morphology analysis of HEK following 2 days of growth showed formation of continuous cell layers on (a) contracted collagen gel or on (d) AlloDerm. Tissues generated by seeding β-Gal-positive, E-cadherin-competent pBabe-II-4 cells on top of HEK layers on (b) collagen or (e) AlloDerm showed organized architecture of basal and suprabasal layers and clusters of cells with aberrant morphology (arrows). Tissues generated by seeding β-Gal-positive, E-cadherin-deficient H-2K^d-Ecad-II-4 cells on top of HEK layers on (c) collagen or (f) AlloDerm demonstrated cells that had migrated into (c, arrows) the collagen or (f, arrows) the AlloDerm. Dashed line indicates the interface between the epithelium and the underlying matrix. Bar, 50 µm. Tissue sections were double-immunostained with anti-β-Gal (green) and anti-β-catenin (red) antibodies. (g, h) pBabe-II-4 cells were confined to superficial layers of the epithelium (arrows) and β-catenin was localized to cell–cell borders of HEK and pBabe-II-4 cells in tissues grown on (g) collagen or (h) AlloDerm. (i, j) H-2K^d-Ecad-II-4 cells had migrated between HEK layers (arrowheads) and invaded into (i, arrows) the collagen or (j, arrows) the AlloDerm. Whereas β-catenin was restricted to cell–cell borders of HEK, cytoplasmic colocalization of β-Gal and β-catenin was seen in H-2K^d-Ecad-II-4 cells (yellow). Dashed line indicates the interface between the epithelium and the underlying matrix. Bar, 50 µm.

Figure 2. Loss of E-cadherin function is associated with single cell scattering

Phase-contrast images of (a) E-cadherin-competent pBabe, (b) H-2K^d-EcadC25-II-4, and (c) E-cadherin-deficient H-2K^d-Ecad-II-4 cultures. Note the fan-shape lamellipodia of pBabe- and H-2K^d-EcadC25-II-4 cells at the periphery of well-organized colonies (long arrows and insets) and the scattered H-2K^d-Ecad-II-4 cells that displayed extensive membrane ruffles, filopodia, and retraction fibers (short arrows and inset). Representative images are shown. Bar, 50 µm.

Figure 3. E-cadherin suppression is linked to accelerated wound closure in response to scrape-wounding in vitro

Phase contrast images of scrape-wounded (a–c) E-cadherin-competent pBabe and (d–f) H-2K^d-EcadC25-II-4 cultures, (g–i) and E-cadhrein-deficient H-2K^d-Ecad-II-4 culture at 0, 24, and 48 hours after wounding. Dashed lines indicate the edges of the wound gaps and demonstrate accelerated wound closure in H-2K^d-Ecad-II-4 culture. Bar, 200 µm. (j) The percentage of wound closure in pBabe (white), H-2K^d-EcadDC25 (gray) and H-2K^d-Ecad-II-4 (black) cultures at 24 and 48 hours was calculated relatively to the initial width of the wound gaps in the cultures. The mean ± SD of the width of the open wound gap was calculated in each of the indicated time points.

Figure 4. E-cadherin suppression is associated with independent, disoriented, and rapid cell migration

E-cadherin-competent pBabe- and E-cadherin-deficient H-2K^d-Ecad-II-4 cultures were grown on coverslides, and the cellular response to scrape-wounding injury was analyzed by time-lapse video microscopy, using phase-contrast microscope-imaging workstation. Representative video frames at the indicated time points are shown. Bar, 25 µm. (a) pBabe-II-4 cells at the wound edge displayed polarized morphology, developed lamellipodia at their leading edge (arrows), and demonstrated collective migration while maintaining their position within the monolayer. (b) Highly motile H-2K^d-Ecad-II-4 cells migrated randomly as single cells into the wound gap and displayed increased membrane protrusions and retraction fibers (arrows). (c) Graphic representation of cell tracking measurements show cell position and migration paths of four individual cells at the wound edge, marked by colored asterisks (marked cells in panels a and b), over the experiment time course. The position of the asterisk shows directional migration of pBabe-II-4 cells and indicates random migration of H-2K^d-Ecad-II-4 cells.

Figure 5. Loss of E-cadherin function is coordinated with actin cytoskeleton rearrangement and β actin induction

E-cadherin-competent- and E-cadherin-deficient cells were grown on coverslides, fixed and visualized by phalloidin. Colonies of (a) pBabe- and (b) H-2K^d-EcadDC25-II-4 cells showed an abundance of actin stress fibers between adjacent cells (arrowheads) and polarized lamellipodia (arrows), whereas (c) H-2K^d-Ecad-II-4 cells showed circumferential actin (arrows) and actin in filopodia and spikes (arrowheads). Representative micrographs are shown. Bar, 50 µm. Cultures were fixed 7 hours after scrape-wounding and double-stained with phalloidin (red) and anti-β actin antibodies (green). (d–f) Phase-contrast images of cells at the wound edge and (g–i) the respective immunofluorescence images of the same cells are shown. (d and g) pBabe- and (e and h) H-2K^d-EcadDC25-II-4 cells displayed β actin in lamellipodia (arrows) and in a rim of short filopodia (insets). (f and i) H-2K^d-Ecad-II-4 cells demonstrated intense β-actin staining throughout the cells, in membrane protrusions, elongated filopodia, and spikes (arrows and inset). Bar, 25 µm. (j) Western blot analysis of β actin level in equal protein samples of lysates from pBabe-, H-2K^d-EcadDC25-, and H-2K^d-Ecad-II-4 cell cultures (IB: β-actin). E-cadherin-deficient cells demonstrated a threefold increase in β-actin expression in comparison with E-cadherin-competent cells. Densitometry of the relative intensity of β actin in the presented immunoblot is shown. GAPDH blotting verified equal protein loading (IB: GAPDH). Representative data from five different experiments are shown.

Figure 6. Transepithelial migration model of E-cadherin-deficient tumor cells in in vivo-like, 3D tissues during the transition from precancer to carcinoma

The schematic model proposes the occurrence of distinct stages of transmigration of E-cadherin-deficient tumor cells in the epithelium during the transition to early cancer cell invasion. (a) In this transepithelial migration model, E-cadherin-competent- or E-cadherin-deficient cells (designated in red) are seeded onto layers of stratified HEK. (b) When grown at an air–liquid interface, E-cadherin-competent cells are maintained in a dormant, suppressed state within the upper layers of the epithelium. (c) E-cadherin-deficient tumor cells separate from adjacent normal keratinocytes and undergo transepithelial, individual cell migration within the tissue, and attached to the BM, creating conditions that are permissive for invasion into the underlying stroma.