Design and implementation of a microfluidic device capable of temporal growth factor delivery reveal filtering capabilities of the EGFR/ERK pathway.
Design and implementation of a microfluidic device capable of temporal growth factor delivery reveal filtering capabilities of the EGFR/ERK pathway.
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具有时间生长因子递送的微流体设备的设计和实现揭示了EGFR/ERK途径的过滤功能。
DOI：
10.1063/5.0059011
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发表时间：
2021-12
影响因子：
6
通讯作者：
Kreeger PK
中科院分区：
文献类型：
--
作者：
Krause HB;Bondarowicz H;Karls AL;McClean MN;Kreeger PK
利用微流体来模拟体内生长因子和细胞因子浓度的动态时间变化，极大地增加了我们对信号转导通路如何被构造成编码细胞外刺激的理解。迄今为止，这些装置集中于递送不同频率的脉冲，并且用于递送细胞可能在体内经历的缓慢增加浓度的刺激的细胞培养模型有限。为了检查这种设置，我们开发并验证了一种微流体装置，该装置可以在6至24小时的时间内提供浓度不断增加的生长因子。使用该装置和细胞外调节激酶（ERK）活性的荧光生物传感器，我们向人乳腺上皮细胞递送了缓慢增加浓度的表皮生长因子（EGF），并且令人惊讶地观察到最小的ERK激活，即使在团注递送中刺激强烈活性的浓度下。细胞对随后的EGF挑战仍然没有反应，免疫细胞化学表明表皮生长因子受体的丢失是原因。然后用更快的EGF变化速率挑战细胞，揭示了ERK活性的增加作为变化速率的函数。具体而言，响应的细胞比例和ERK激活时间的长度都随着变化率的增加而增加。这种微流控装置填补了目前体外微流控装置中的空白，并表明生长因子呈递中更慢、更生理的变化可以揭示信号转导途径如何编码细胞外生长因子环境变化的新调控机制。
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.
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影响因子：
64.5
作者：
Regot S;Hughey JJ;Bajar BT;Carrasco S;Covert MW
通讯作者：
Covert MW
影响因子：
12.3
作者：
Carpenter AE;Jones TR;Lamprecht MR;Clarke C;Kang IH;Friman O;Guertin DA;Chang JH;Lindquist RA;Moffat J;Golland P;Sabatini DM
通讯作者：
Sabatini DM
影响因子：
9.7
作者：
Puccinelli, Tracy J.;Bertics, Paul J.;Masters, Kristyn S.
通讯作者：
Masters, Kristyn S.
影响因子：
6.1
作者：
Lin, F;Saadi, W;Jeon, NL
通讯作者：
Jeon, NL
影响因子：
9.3
作者：
Goglia AG;Wilson MZ;Jena SG;Silbert J;Basta LP;Devenport D;Toettcher JE
通讯作者：
Toettcher JE