Extracellular matrix and morphology assessment method on live 3D spheroids of thyroid carcinoma.

Abstract

In vitro tumor models might be useful to study tumor growth, invasion and therapy resistance, but also extracellular matrix (ECM) remodeling. This protocol will provide information for the generation of a 3D tumor cell cultures for high content analysis and describes a novel method to monitor the ECM morphology and remodeling as well as therapy responses using live 3D tumor cell spheroids. Using 3D spheroids of thyroid carcinoma (TC) cells as a model, our method involves the treatment of TC cells with inhibitory agents followed by subsequent analysis of ECM components to assess the influence of these drugs on the structural integrity of the EMT using fluorescence labeled antibodies (Ab) and confocal microscopy. Employing this method, the morphology of the formed spheroids under different conditions and co-cultures as well as the distribution of ECM components can be assessed, such as e.g. fibronectin 1 (FN1). The results will also provide valuable insights into the tumor microenvironment (TME) and potential interactions of viable spheroids with the components of the TME during the ECM remodeling process. The implementation of 3D spheroids for studying EMT in TC as a model has been shown to provide more accurate and representative results compared to traditional 2D monolayer cell cultures.