Endocanalicular transendothelial crossing (ETC): A novel intravasation mode used by HEK-EBNA293-VEGF-D cells during the metastatic process in a xenograft model.

Federico Armando,Davide Dallatana,Giacomo Azzali,Attilio Corradi,Luca Ferrari,Rosanna Di Lecce,Maura Ferrari,Paolo Lunghi,Guerino Lombardi,Anna M Cantoni,Maria Luisa Arcari,Matteo Zanfabro,Ewan R Cameron,Benedetta Bussolati

doi:10.1371/journal.pone.0239932

Abstract

In cancer metastasis, intravasation of the invasive tumor cell (TCi) represents one of the most relevant events. During the last years, models regarding cancer cell intravasation have been proposed, such as the “endocanalicular transendothelial crossing” (ETC) theory. This theory describes the interplay between two adjacent endothelial cells and the TCi or a leukocyte during intravasation. Two endothelial cells create a channel with their cell membranes, in which the cell fits in without involving endothelial cell intercellular junctions, reaching the lumen through a transendothelial passage. In the present study, ten SCID mice were subcutaneously xenotransplanted with the HEK-EBNA293-VEGF-D cell line and euthanized after 35 days. Post-mortem examinations were performed and proper specimens from tumors were collected. Routine histology and immunohistochemistry for Ki-67, pAKT, pERK, ZEB-1, TWIST-1, F-actin, E-cadherin and LYVE-1 were performed followed by ultrastructural serial sections analysis. A novel experimental approach involving Computed Tomography (CT) combined with 3D digital model reconstruction was employed. The analysis of activated transcription factors supports that tumor cells at the periphery potentially underwent an epithelial-to-mesenchymal transition (EMT)-like process. Topographical analysis of LYVE-1 immunolabeled lymphatics revealed a peritumoral localisation. TEM investigations of the lymphatic vessels combined with 3D digital modelling enhanced the understanding of the endotheliocytes behavior during TCi intravasation, clarifying the ETC theory. Serial ultrastructural analysis performed within tumor periphery revealed numerous cells during the ETC process. Furthermore, this study demonstrates that ETC is an intravasation mode more frequently used by the TCi than by leukocytes during intravasation in the HEK-EBNA293-VEGF-D xenograft model and lays down the potential basis for promising future studies regarding intravasation blocking therapy.

Highlights

Tumor metastasis is a multi-step process and one of the most relevant events during the neoplastic invasion cascade is the intravasation of the invasive tumor cell (TCi)
The results of the present study, namely the increased number of tumor cells at the tumor periphery expressing pAKT and pERK together with low numbers of E-cadherin expressing cells are in line with the results reported by Saba and colleagues in non-small cell lung cancer cells [38]
The aforementioned study suggested that tumors with increased activated pAKT and pERK together with decreased E-cadherin expression are destined to undergo epithelial-to-mesenchymal transition (EMT) [38]

Summary

Introduction

Tumor metastasis is a multi-step process and one of the most relevant events during the neoplastic invasion cascade is the intravasation of the invasive tumor cell (TCi). In some types of neoplasia, the transendothelial migration of the tumor cell (TC) is assumed to occur via the breakage of the endothelial barrier due to the dissolution of the E-cadherin/β-catenin complex [2] or to apoptosis of the endothelial cells and consequent irreversible retraction of the endothelium [3]. According to Miles and colleagues [6], the MCF-7 cell line of the metastatic mammary adenocarcinoma and other tumoral cell lines induce apoptosis of the endothelial cells and consequent irreversible retraction of the endothelium. Vestweber [11] theorized that, to leukocyte extravasation, the TCi migratory transendothelial process requires the active involvement of the endothelial cell, with the TCi releasing signals responsible for the opening and closure of the interendothelial cell contacts following adhesion to the endothelium. The lack of a dynamic morphological ultrastructural study can be overcome by combining two approaches: transmission electron microscopy (TEM) microphotographs and a novel approach of 3D virtual model editing. 3D models make possible the explicit visualization of this particular process starting from Born’s technique [17]

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Conclusion