Three-dimensional mRNA measurements reveal minimal regional heterogeneity in esophageal squamous cell carcinoma

Abstract

The classic tumor clonal evolution theory postulates that cancers change over time to produce unique molecular subclones within a parent neoplasm, presumably including regional differences in gene expression. More recently, however, this notion has been challenged by studies showing that tumors maintain a relatively stable transcript profile. To examine these competing hypotheses, we microdissected discrete subregions containing approximately 3000 to 8000 cells (500 to 1500 μm in diameter) from ex vivo esophageal squamous cell carcinoma (ESCC) specimens and analyzed transcriptomes throughout three-dimensional tumor space. Overall mRNA profiles were highly similar in all 59 intratumor comparisons, in distinct contrast to the markedly different global expression patterns observed in other dissected cell populations. For example, normal esophageal basal cells contained 1918 and 624 differentially expressed genes at a greater than twofold level (95% confidence level of <5% false positives), compared with normal differentiated esophageal cells and ESCC, respectively. In contrast, intratumor regions had only zero to four gene changes at a greater than twofold level, with most tumor comparisons showing none. The present data indicate that, when analyzed using a standard array-based method at this level of histological resolution, ESCC contains little regional mRNA heterogeneity.

Figure 1

Microdissection design. A: H&E staining showing an ESCC focus before and after dissection of the tumor periphery and center. B: Three-dimensional microdissection design and definition of the tumor cluster and normal area. Dissected areas are outlined in green. C: Histology of normal esophageal epithelium compartment before and after microdissection.

Figure 2

Differential gene expression comparisons of TP/TC (blue/green) versus NB/ND (orange/red) in nine cases by hierarchical cluster analysis. Each bar presents one case.

Figure 3

Differential gene expression comparisons of three-dimensional-microdissected ESCC in five cases by hierarchical cluster analysis. Each bar represents one dissection (including normal epithelium for case 5). Microdissection was performed as shown in Figure 1B.