Tunneling Nanotubes between Cells Migrating in ECM Mimicking Fibrous Environments

SIMPLE SUMMARY: Tumor cells grow, spread, and invade in a three-dimensional manner, but most experimental approaches in cancer cell biology focus on the behavior of cells in two-dimensional spaces. Patterns of cell invasion and spread in 2D may not accurately depict cell behavior in 3D tumors. We cultivated and investigated malignant mesothelioma cells on a 3D scaffold composed of nanofibers positioned in parallel and crosshatched network configurations to overcome this barrier. We focused on studying long extensions called tunneling nanotubes (TNTs) protruding from cells and connecting with other cells, which have been shown to transmit signals from cell to cell and extend into the tumor microenvironments in a 3D manner. This manuscript describes the biophysics of the formation and function of cancer cell TNTs. These findings will impact the research community by accurately assessing how TNTs affect cancer cell invasion and migration in their natural 3D microenvironment using a novel bioengineered platform. ABSTRACT: Tunneling nanotubes (TNTs) comprise a unique class of actin-rich nanoscale membranous protrusions. They enable long-distance intercellular communication and may play an integral role in tumor formation, progression, and drug resistance. TNTs are three-dimensional, but nearly all studies have investigated them using two-dimensional cell culture models. Here, we applied a unique 3D culture platform consisting of crosshatched and aligned fibers to fabricate synthetic suspended scaffolds that mimic the native fibrillar architecture of tumoral extracellular matrix (ECM) to characterize TNT formation and function in its native state. TNTs are upregulated in malignant mesothelioma; we used this model to analyze the biophysical properties of TNTs in this 3D setting, including cell migration in relation to TNT dynamics, rate of TNT-mediated intercellular transport of cargo, and conformation of TNT-forming cells. We found that highly migratory elongated cells on aligned fibers formed significantly longer but fewer TNTs than uniformly spread cells on crossing fibers. We developed new quantitative metrics for the classification of TNT morphologies based on shape and cytoskeletal content using confocal microscopy. In sum, our strategy for culturing cells in ECM-mimicking bioengineered scaffolds provides a new approach for accurate biophysical and biologic assessment of TNT formation and structure in native fibrous microenvironments.