Design and implementation of a microfluidic device capable of temporal growth factor delivery reveal filtering capabilities of the EGFR/ERK pathway.

Hanna Bondarowicz,Alexis L Karls,Harris B Krause,Pamela K Kreeger,Megan N Mcclean

doi:10.1063/5.0059011

Abstract

Utilizing microfluidics to mimic the dynamic temporal changes of growth factor and cytokine concentrations in vivo has greatly increased our understanding of how signal transduction pathways are structured to encode extracellular stimuli. To date, these devices have focused on delivering pulses of varying frequency, and there are limited cell culture models for delivering slowly increasing concentrations of stimuli that cells may experience in vivo. To examine this setting, we developed and validated a microfluidic device that can deliver increasing concentrations of growth factor over periods ranging from 6 to 24 h. Using this device and a fluorescent biosensor of extracellular-regulated kinase (ERK) activity, we delivered a slowly increasing concentration of epidermal growth factor (EGF) to human mammary epithelial cells and surprisingly observed minimal ERK activation, even at concentrations that stimulate robust activity in bolus delivery. The cells remained unresponsive to subsequent challenges with EGF, and immunocytochemistry suggested that the loss of an epidermal growth factor receptor was responsible. Cells were then challenged with faster rates of change of EGF, revealing an increased ERK activity as a function of rate of change. Specifically, both the fraction of cells that responded and the length of ERK activation time increased with the rate of change. This microfluidic device fills a gap in the current repertoire of in vitro microfluidic devices and demonstrates that slower, more physiological changes in growth factor presentation can reveal new regulatory mechanisms for how signal transduction pathways encode changes in the extracellular growth factor milieu.

Highlights

The cellular microenvironment is dynamic with changes in the production, transport, and degradation of growth factors and cytokines, leading to complex presentation of these stimuli over time
A set concentration of epidermal growth factor (EGF) is applied to cells, extracellular-regulated kinase (ERK) is quickly phosphorylated, and the amount of phosphorylated ERK shows a dose-dependency with respect to the initial EGF concentration
The device consists of two inputs [serum free media (SFM) and Serum-free HMEC media (SFM) þ growth factor (GF)] driven by programmable syringe pumps that flow through a resistance feature to minimize flow variation [Figs. 1(a) and S1]

Summary

Introduction

The cellular microenvironment is dynamic with changes in the production, transport, and degradation of growth factors and cytokines, leading to complex presentation of these stimuli over time These temporal patterns are sensed by cellular receptors and encoded to signaling hubs, such as extracellular-regulated kinase (ERK), a serine/threonine protein kinase that plays a major role in human development and disease by regulating fundamental cellular processes such as apoptosis, proliferation, and collective cell migration.. Despite the dynamic nature of the in vivo microenvironment, the majority of studies of EGFR/ERK have utilized tissue culture plates with static wells In this setting, a set concentration of EGF is applied to cells, ERK is quickly phosphorylated, and the amount of phosphorylated ERK shows a dose-dependency with respect to the initial EGF concentration.. A set concentration of EGF is applied to cells, ERK is quickly phosphorylated, and the amount of phosphorylated ERK shows a dose-dependency with respect to the initial EGF concentration. While often interpreted as a constant concentration of EGF throughout the experiment, ligand depletion in this paradigm will lead to a decrease in EGF concentration over time. To overcome this complication, cells have been plated as a small island in a well plate, such that ligand depletion relative to the initial EGF concentration was neglible. With this modification, it was observed that as EGF concentration increased, ERK pulsed on and off in individual cells with a greater frequency

Methods

Results

Conclusion