Ortega Quesada Braulio Andrés, Cuccia Jonathan, Coates Rachael, Nassar Blake, Littlefield Ethan, Martin Elizabeth C, Melvin Adam T
Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803 USA.
Department of Chemical and Biological Engineering, Clemson University, Clemson, SC 29634 USA.
Microsyst Nanoeng. 2024 Feb 16;10:25. doi: 10.1038/s41378-024-00653-0. eCollection 2024.
Metastatic breast cancer leads to poor prognoses and worse outcomes in patients due to its invasive behavior and poor response to therapy. It is still unclear what biophysical and biochemical factors drive this more aggressive phenotype in metastatic cancer; however recent studies have suggested that exposure to fluid shear stress in the vasculature could cause this. In this study a modular microfluidic platform capable of mimicking the magnitude of fluid shear stress (FSS) found in human vasculature was designed and fabricated. This device provides a platform to evaluate the effects of FSS on MCF-7 cell line, an estrogen receptor positive (ER) breast cancer cell line, during circulation in the vessels. Elucidation of the effects of FSS on MCF-7 cells was carried out utilizing two approaches: single cell analysis and bulk analysis. For single cell analysis, cells were trapped in a microarray after exiting the serpentine channel and followed by immunostaining on the device (on-chip). Bulk analysis was performed after cells were collected in a microtube at the outlet of the microfluidic serpentine channel for western blotting (off-chip). It was found that cells exposed to an FSS magnitude of 10 dyn/cm with a residence time of 60 s enhanced expression of the proliferation marker Ki67 in the MCF-7 cell line at a single cell level. To understand possible mechanisms for enhanced Ki67 expression, on-chip and off-chip analyses were performed for pro-growth and survival pathways ERK, AKT, and JAK/STAT. Results demonstrated that after shearing the cells phosphorylation of p-AKT, p-mTOR, and p-STAT3 were observed. However, there was no change in p-ERK1/2. AKT is a mediator of ER rapid signaling, analysis of phosphorylated ERα was carried out and no significant differences between sheared and non-sheared populations were observed. Taken together these results demonstrate that FSS can increase phosphorylation of proteins associated with a more aggressive phenotype in circulating cancer cells. These findings provide additional information that may help inform why cancer cells located at metastatic sites are usually more aggressive than primary breast cancer cells.
转移性乳腺癌因其侵袭性和对治疗的不良反应，导致患者预后不良和结局较差。目前仍不清楚哪些生物物理和生化因素驱动转移性癌症中这种更具侵袭性的表型；然而，最近的研究表明，血管中的流体剪切应力暴露可能会导致这种情况。在本研究中，设计并制造了一种模块化微流控平台，该平台能够模拟人体血管中发现的流体剪切应力（FSS）大小。该装置提供了一个平台，用于评估FSS对雌激素受体阳性（ER）乳腺癌细胞系MCF-7细胞在血管循环过程中的影响。利用两种方法对FSS对MCF-7细胞的影响进行了阐明：单细胞分析和整体分析。对于单细胞分析，细胞在离开蛇形通道后被困在微阵列中，然后在装置上（芯片上）进行免疫染色。在微流控蛇形通道出口处的微管中收集细胞后，进行整体分析以进行蛋白质印迹（芯片外）。研究发现，在单细胞水平上，暴露于大小为10 dyn/cm且停留时间为60 s的FSS的细胞增强了MCF-7细胞系中增殖标志物Ki67的表达。为了了解Ki67表达增强的可能机制，对促生长和存活途径ERK、AKT和JAK/STAT进行了芯片上和芯片外分析。结果表明，剪切细胞后观察到p-AKT、p-mTOR和p-STAT3的磷酸化。然而，p-ERK1/2没有变化。AKT是ER快速信号传导的介质，对磷酸化ERα进行了分析，未观察到剪切组和未剪切组之间的显著差异。综上所述，这些结果表明FSS可以增加循环癌细胞中与更具侵袭性表型相关蛋白质的磷酸化。这些发现提供了额外的信息，可能有助于解释为什么位于转移部位的癌细胞通常比原发性乳腺癌细胞更具侵袭性。
