An Intravital Multiphoton Microscopy Model for visualization of tumor cell dissemination and lymphatic vasculature

Katharina Eder,Steffen Dietzel,Brigitte Mack,Manuel Guhlich,Olivier Gires,Alexander Berghaus,Carola Eggert,Martin Canis,Ulrich Harréus

doi:10.17980/2015.200

Abstract

Tumor metastasis and its impact on the organism are the main causes of death in patients with squamous cell carcinoma of the head and neck region. The presence of metastasis at the time of diagnosis represents a negative prognostic parameter of overall survival and correlates with a 50 % mean reduction of life expectancy, independent of tumor size. The potential for metastasis depends on factors such as the proliferation rate, neoangiogenesis and invasive capacity in intact tissue structures. Many aspects of the molecular mechanisms and supportive factors are not understood. To allow the investigation of such factors, we developed an intravital mouse model for the visualization of tumor cell proliferation, dissemination and lymphatic vasculature by multiphoton microscopy. This technology offers deep tissue penetration, low phototoxicity, superior image contrast, and four-dimensional resolution for investigations at the single cell level in a physiological setting. The human oral cancer cell line OSC-19 was transfected with turbo-red fluorescent protein and implanted in murine pinna. Lymphatic structures were labeled with a deep red-labeled antibody specific for lymphatic vessel endothelial receptor-1. In future research, this model might serve as a tool to gain deeper insights in surface molecules involved in lymphatic metastasis as well as other molecular mechanisms of metastasis and tumor cell migration.

Highlights

Evidence of lymphatic spread is one of the most important prognostic factors for patients with head and neck squamous cell carcinoma (HNSCC) because overall survival can be reduced by more than 50 % given the presence of lymph node metastases, independently of primary tumor size [1, 2]
The lymphatic system plays a major role as a route of circulation and dissemination, enabling cancer cells to circulate and metastasize into potentially more than 400 lymph nodes in the cervical region [3]
All experimental procedures were performed in compliance with the German legislation for animal experimentation and approved by the local government authorities (Regierung von Oberbayern; animal license no.: 55.2-1-54-2531-172-09). This projection of 41 images with an axial Zstep size of 1 μm shows the lymphatic vasculature labeled with a subcutaneous injection of anti-mouse lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) antibody coupled to eFluor® 660 in red and blood vessels labeled by intravenous injection of high-molecular Fluorescein isothiocyanate (FITC)-dextran in green

Summary

Introduction

Evidence of lymphatic spread is one of the most important prognostic factors for patients with head and neck squamous cell carcinoma (HNSCC) because overall survival can be reduced by more than 50 % given the presence of lymph node metastases, independently of primary tumor size [1, 2]. The lymphatic system plays a major role as a route of circulation and dissemination, enabling cancer cells to circulate and metastasize into potentially more than 400 lymph nodes in the cervical region [3]. Most cells provided a red fluorescent signal (b) In a Scratch Wound Healing Assay a monolayer of OSC-19 cells was scratched with a sterile 1 ml pipette tip and observed every 4 hours.

Objectives

Methods

Results