[Three-dimensional reconstruction of solid tumors : Morphological evidence for tumor heterogeneity]

Background: In histopathological routine diagnostics, three-dimensional tissue samples are analyzed histologically and/or immunohistochemically in two-dimensional sectional planes due to the high expenditure of time and the lack of digitization possibilities.

Aim: Here, we demonstrate the application of three-dimensional reconstruction to solid tumors and analyze inter-/intratumoral heterogeneity with respect to epithelial-mesenchymal transition (EMT).

Methods: Tissue samples from pancreatic, lung, colorectal, and breast cancers as well as colorectal liver metastases were serially processed in 4μm sections. For individual analyses, alternating stains (cytokeratin AE1/3, zinc finger E‑box-binding homeobox 1 (ZEB1), eCadherin) were performed. Subsequently, the tumor cells were analyzed for their morphology (epitheloid amoeboid, mesenchymal) and the expression of ZEB1 and eCadherin. For statistical analysis, all tumor cell aggregates were hierarchically annotated and analyzed.

Results: Tumor buds are predominantly associated with the main tumor mass. Furthermore, a shutteling of eCadherin could be observed within tumor cell aggregates smaller than nine cells. ZEB1 is only increasingly expressed in tumor cell groups smaller than five cells.

Conclusions: The initial tumor budding and the subsequent decoupling of the tumor bud from the main tumor mass is most likely a two-part process. However, the EMT is not statistically significantly increased within the tumor bud detached from the main tumor mass. It could be shown that the currently valid and known definition of a tumor bud as a cell cluster of less than or equal to five cells cannot be completely classified in the concept of EMT represented by eCadherin and ZEB1.