Disruption of 3D tissue integrity facilitates adenovirus infection by deregulating the coxsackievirus and adenovirus receptor

Abstract

The human coxsackievirus and adenovirus receptor (CAR) represents the primary cellular site of adenovirus attachment during infection. An understanding of the mechanisms regulating its expression could contribute to improving efficacy and safety of adenovirus-based therapies. We characterized regulation of CAR expression in a 3D cell culture model of human breast cancer progression, which mimics aspects of the physiological tissue context in vitro. Phenotypically normal breast epithelial cells (S1) and their malignant derivative (T4-2 cells) were grown either on tissue culture plastic (2D) or 3D cultures in basement membrane matrix. S1 cells grown in 3D showed low levels of CAR, which was expressed mainly at cell-cell junctions. In contrast, T4-2 cells expressed high levels of CAR, which was mainly in the cytoplasm. When signaling through the epidermal growth factor receptor was inhibited in T4-2 cells, cells reverted to a normal phenotype, CAR protein expression was significantly reduced, and the protein relocalized to cell-cell junctions. Growth of S1 cells as 2D cultures or in 3D in collagen-I, a nonphysiological microenvironment for these cells, led to up-regulation of CAR to levels similar to those in T4-2 cells, independently of cellular growth rates. Thus, expression of CAR depends on the integrity and polarity of the 3D organization of epithelial cells. Disruption of this organization by changes in the microenvironment, including malignant transformation, leads to up-regulation of CAR, thus enhancing the cell's susceptibility to adenovirus infection.

Figure 1

Analysis of CAR expression in S1 and T4-2 cells. (a) Western blot analysis of expression of CAR, αv-integrin, and β-actin in S1, T4-2, and phenotypically reverted T4-2 (T4-2rev) cells. Cells were grown as monolayer or 3D cultures in basement membrane (BM). Chinese hamster ovary cells (CHO) with or without stable expression of CAR were used as a control. CAR (b) and E-cadherin (c) expression in S1 cells grown in 3D as detected by confocal fluorescence microscopy. (d) The overlay of images b and c. Staining of T4-2 cells grown in 3D for CAR and E-cadherin (e and f) and the overlay image (g) is shown. (Magnification: ×630.) (h) CAR mRNA expression levels in S1 and T4 cells grown in 2D and 3D were measured relative to expression of β-glucuronidase by using a real-time PCR assay.

Figure 2

Assessment of susceptibility to infection with adenovirus of S1 and T4-2 cells grown in basement membrane matrix in 3D. 3D cultures of S1, T4-2, and T4-2rev cells were grown in basement membrane gel and infected with a GFP-expressing, nonreplicating adenovirus (Ad-GFP) at a multiplicity of infection of 10 plaque-forming units per cell. Microscopic images (magnification: ×50) represent overlays of phase-contrast (red, false-color) and fluorescence microscopy (green) after infection. Each equatorial section of S1 cell spheroids contains an average of eight cells (8).

Figure 3

Western blot analysis of CAR protein expression in S1, T4-2, and S2 (clones 1–3) cells grown in basement membrane matrix (BM) or collagen I. Protein levels are also shown for T4-2rev cells after treatment with the anti-EGFR antibody mAb225. E-cadherin expression was shown previously not to change and is used as a control.

Figure 4

Detection of CAR (green) and E-cadherin (red) in normal human mammary tissue by immunofluorescence microscopy. The arrows indicate CAR expression at the apical cell contacts. 4′,6-Diamidino-2-phenylindole staining (blue) was used to visualize DNA in the cell nuclei. (Magnifications: ×400.)

Figure 5

Detection of CAR protein in human tissue samples by immunohistochemistry. (a) Normal breast tissue. (Inset) A representative acinus is shown at higher magnification. (b) DCIS. (c and d) Two cases of invasive ductal carcinomas. Asterisks indicate adjacent or entrapped normal ducts. (Magnifications: ×200.)