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

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.

Rate thresholds in cell signaling have functional and phenotypic consequences in non-linear time-dependent environments

“…However, fluctuations with intermediate durations resulted in a fourfold reduction in yeast growth rate, showing that environmental fluctuations have phenotypic consequences ( Mitchell et al, 2015 ). Several labs developed novel cell culture assays to expose cells to non-acute environmental changes, followed by quantitative experiments to investigate molecular mechanisms in non-acute environmental conditions ( Figures 3C, D ) ( Block et al, 1983 ; Muzzey et al, 2009 ; Fujita et al, 2010 ; Shimizu et al, 2010 ; Pelet et al, 2011 ; Kubota et al, 2012 ; 2018 ; Wang et al, 2012 ; Noguchi et al, 2013 ; Sgro et al, 2015 ; Mokashi et al, 2019 ; Thiemicke et al, 2019 ; Krause et al, 2021 ). The novel experimental setup developed by Thiemicke et al recently investigated how Hog1 signaling and cell viability are impacted by different rates of osmolyte gradients ( Figures 3C–H ) ( Johnson et al, 2021 ).…”

A guide to ERK dynamics, part 1: mechanisms and models

“…However, under more atypical experimental conditions, the regulation of EGFR internalization can result in surprising behavior. Under conditions in which EGF is slowly ramped to high concentrations, receptors become down-regulated and fail to activate ERK [ 84 ]. This adaptation persists for hours, and even withdrawal of EGF for several hours and subsequent re-stimulation does not elicit ERK activation.…”