1(b)]. however, to date single-cell morphological changes have not been quantified to support this observation. Furthermore, the methodology of previous studies entails inducing FSS by flowing cells through the tubing, which lacks a precise and tunable control of FSS. Here, a microfluidic approach is used for isolating and characterizing the biophysical response of single breast malignancy cells to conditions experienced in the circulatory system during metastasis. To evaluate the single-cell response of MK-7246 multiple breast malignancy types, two model circulating tumor cell lines, MDA-MB-231 and MCF7, were challenged with FSS at precise magnitudes and durations. As expected, both MDA-MB-231 and MCF7 cells exhibited greater deformability due to increasing period and magnitudes of FSS. However, wide variations in single-cell responses were observed. MCF7 cells were found to rapidly deform but reach a threshold value after 5?min of FSS, while MDA-MB-231 cells were observed to INSL4 antibody deform at a slower rate but with a larger threshold of deformation. This behavioral diversity suggests the presence of unique cell subpopulations with different phenotypes. I.?INTRODUCTION Metastatic malignancy is the leading cause of cancer related deaths.1 As such, an understanding of the biophysics of metastasizing malignancy cells is necessary to develop new treatment strategies and therapies against secondary metastasis. Tumor cells can metastasize via several different routes, such as lymphatic and hematogenous spread. Hematogenous spreading can be simplified into a coordinated series of actions: detachment from a primary tumor, intravasation into the circulatory system, movement of the cells through the circulatory system, adhesion and extravasation, and, finally, growth of a secondary tumor.2 While inside the circulatory system, the malignancy cells are called circulating tumor cells (CTCs). The role of CTCs is particularly important in malignancy metastasis as they constitute the intermediate state between the main and secondary tumors.3,4 One unique aspect of hematogenous spread is that the fluid flow in the circulatory system constantly exerts fluid shear stress (FSS) around the cells. In the human circulatory system, FSS can range from as low as 0.1?dyn/cm2 in interstitial circulation to as high as 3000?dyn/cm2 round the heart.5 However, the average FSS that CTCs experience ranges from 0.5 to 30?dyn/cm2 for several minutes, until their arrest in the vasculature.6,7 Despite the importance of CTCs, the effect of FSS on CTC phenotype is not fully understood. Previous studies have tried to replicate the hemodynamic environment of the circulatory system by injecting cells through the tubing and utilizing the Hagen-Poiseuille Equation to approximate shear MK-7246 stress magnitudes.5,8,9 These studies suggested that FSS elicits a strong response in CTCs, such as an acquisition of cancer stem cell (CSC)-like properties.9 CSCs are a specialized sub-population of tumors that have the ability to self-renew and differentiate much like healthy stem cells and progenitor cells, but also more poignantly, have tumor initiating characteristics.2,10 Therefore, they are thought to be the root cause of tumor relapse, especially in patients with metastatic tumors. In breast tumors, the CSC populace can be prospectively isolated using CD44+/CD24? surface markers and/or by aldehyde dehydrogenase (ALDH) activity, and CTCs MK-7246 of metastatic breast malignancy patients were previously observed to have a correlated overexpression of stem cell markers.11C13 CTCs are also commonly characterized by their expression of the epithelial cell adhesion molecule (EpCAM). EpCAM is an important marker as it is usually generally used in CTC detection devices.14C16 EpCAM is also a marker for epithelial to mesenchymal transition (EMT), which allows for cell motility and detachment from the primary tumor. Prior studies have shown that FSS can induce EMT-like characteristics under constant circulation.17 During EMT, epithelial marker expression can be downregulated, which is a challenge for CTC detection devices reliant on EPCAM.9 As a result, physical properties such as morphology and stiffness are also used as a CTC phenotype. For example, EMT causes morphological changes resulting in a cellular phenotype that is more elongated, less stiff, and highly motile..