ContactBlot: Microfluidic Control and Measurement of the Cell–Cell Contact State to Assess Contact-Inhibited ERK Signaling,Analytical Chemistry

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ContactBlot: Microfluidic Control and Measurement of the Cell–Cell Contact State to Assess Contact-Inhibited ERK Signaling
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-09-10 , DOI: 10.1021/acs.analchem.4c02936
Yizhe Zhang ₁ , Isao Naguro _{2,

3} , Hiroki Ryuno ₂ , Amy E Herr ₁

Affiliation

Extracellular signal-regulated kinase (ERK) signaling is essential to regulated cell behaviors, including cell proliferation, differentiation, and apoptosis. The influence of cell–cell contacts on ERK signaling is central to epithelial cells, yet few studies have sought to understand the same in cancer cells, particularly with single-cell resolution. To acquire same-cell measurements of both phenotypic (cell-contact state) and targeted-protein (ERK phosphorylation) profiles, we prepend high-content, whole-cell imaging prior to end-point cellular-resolution Western blot analyses for each of hundreds of individual HeLa cancer cells cultured on that same chip, which we call contactBlot. By indexing the phosphorylation level of ERK in each cell or cell cluster to the imaged cell-contact state, we compare the ERK signaling between isolated and in-contact cells. We observe attenuated (∼2×) ERK signaling in HeLa cells that are in-contact versus isolated. Attenuation is sustained when the HeLa cells are challenged with hyperosmotic stress. Our findings show the impact of cell–cell contacts on ERK activation with isolated and in-contact cells while introducing a multi-omics tool for control and scrutiny of cell–cell interactions.

中文翻译：

ContactBlot：细胞与细胞接触状态的微流体控制和测量，以评估接触抑制的 ERK 信号传导

细胞外信号调节激酶 (ERK) 信号传导对于调节细胞行为至关重要，包括细胞增殖、分化和凋亡。细胞间接触对 ERK 信号传导的影响对于上皮细胞至关重要，但很少有研究试图了解癌细胞中的相同情况，特别是在单细胞分辨率方面。为了获得表型（细胞接触状态）和目标蛋白（ERK 磷酸化）图谱的同细胞测量，我们在终点细胞分辨率蛋白质印迹分析之前对数百个样本进行高内涵全细胞成像。在同一芯片上培养的单个 HeLa 癌细胞，我们称之为接触印迹。通过将每个细胞或细胞簇中 ERK 的磷酸化水平与成像的细胞接触状态进行索引，我们比较了分离细胞和接触细胞之间的 ERK 信号传导。我们观察到接触的 HeLa 细胞与隔离的 HeLa 细胞中 ERK 信号减弱（~2×）。当 HeLa 细胞受到高渗应激的挑战时，衰减会持续。我们的研究结果显示了细胞与细胞接触对分离和接触细胞的 ERK 激活的影响，同时引入了用于控制和审查细胞与细胞相互作用的多组学工具。

更新日期：2024-09-10

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