Histological, immunohistochemical and transcriptomic characterization of human tracheoesophageal fistulas

Abstract

Esophageal atresia (EA) and tracheoesophageal fistula (TEF) are relatively frequently occurring foregut malformations. EA/TEF is thought to have a strong genetic component. Not much is known regarding the biological processes disturbed or which cell type is affected in patients. This hampers the detection of the responsible culprits (genetic or environmental) for the origin of these congenital anatomical malformations. Therefore, we examined gene expression patterns in the TEF and compared them to the patterns in esophageal, tracheal and lung control samples. We studied tissue organization and key proteins using immunohistochemistry. There were clear differences between TEF and control samples. Based on the number of differentially expressed genes as well as histological characteristics, TEFs were most similar to normal esophagus. The BMP-signaling pathway, actin cytoskeleton and extracellular matrix pathways are downregulated in TEF. Genes involved in smooth muscle contraction are overexpressed in TEF compared to esophagus as well as trachea. These enriched pathways indicate myofibroblast activated fibrosis. TEF represents a specific tissue type with large contributions of intestinal smooth muscle cells and neurons. All major cell types present in esophagus are present-albeit often structurally disorganized-in TEF, indicating that its etiology should not be sought in cell fate specification.

Fig 1

A-E. Hematoxylin and eosin (HE) stained sections of normal esophagus and trachea. A. Cross section of normal esophagus (E) and trachea (T) with surrounding cartilage (C) at 1.25 x magnification. B-C. Overview and detail of normal trachea with multilayered cylindrical epithelium (ep), underlying lamina propria (lp), muscular layer (mm) and seromucinous glands (smg); at the bottom of the image, the circular muscular layer (cml) and underlying cartilage can be seen. (at 10x (left) and 20x (right) magnification) C-D. Overview and detail of normal trachea covered with multilayered cylindrical epithelium (ep), with underlying glands, circular (cml) and longitudinal muscle layer (lml), both next to cartilage. (at 10x (left) and 20x (right) magnification) F-G and H-I. Normal esophagus covered by multilayered squamous epithelium (ep). (at 10x (left) and 20x (right) magnification) J and K. Elastic stain with elastic fibers in black (EF), collagen in pink (COL) and muscle (M) in yellow. In J an overview of normal esophagus is seen, with normal trachea in K. (at 4x magnification).

Fig 2

A-D. Overview and details of TEF. TEF walls covered by squamous epithelium (ep), with underlying lamina propria (lp) and muscular layer (m). The muscle layer appears irregular and fragmented in the overview of A-C and D, E and F. In the detail images a mild chronic inflammatory infiltrate can be appreciated. (at 4x, 10x and 40x magnification respectivly) G. Elastic and corresponding HE stain (H) of fistula covered with squamous epithelium, showing disorganized muscle bundles and glandular structures. (at 10x magnification).

Fig 3. Immunostaining of esophagus, trachea and TEF.

The first panel shows presence of bcl2 staining in the basal epithelial cells in both esophagus and trachea, but not in TEF. The second panel shows faint cytoplasmic BMP2 staining in esophagus and trachea, but not in TEF. The third panel shows cytoplasmic retinoic acid receptor alpha (RARα) staining in the upper part of the squamous epithelium of the esophagus and TEF, but not in the cylindrical epithelium of the trachea. In the fourth panel retinoic acid receptor beta (RARβ) shows similar cytoplasmic staining in the lower portion of the epithelium in all three structures. Interestingly, in the lower panel SOX2 shows cytoplasmic staining in esophagus and trachea, while there is evident nuclear labeling of epithelial cells in TEF. BLC2 esophagus and trachea at 10x magnification, TEF at 20x magnification. BMP2 and SOX2 at 40x magnification and RAR-α and RAR-β at 10x magnification.