## 4 Discussion
It was indicated that both an endothelial monolayer and soluble fibrin(ogen) affected the local hydrodynamic environment around adherent PMNs more than that around adherent TCs. Since a PMN has a smaller diameter (about 8 \(\mu\)m) than a melanoma cell (16-20 \(\mu\)m), it might be more easily influenced by the external factors. Although we examined an endothelial monolayer and soluble fibrin(ogen) to simulate physiological tumor microenvironment in blood flow in vitro as much as possible, limitations still exist. For example, only one concentration of soluble fibrin(ogen) was considered. The diffusion of soluble fibrin(ogen) might be influenced by different shear conditions, thus the gradient of soluble fibrin(ogen) might affect cell adhesion. Moreover, dynamic wall shear stress distribution might be influenced by viscoelasticity of the extracellular matrix. Another limitation is the use of EI cells to mimic endothelial cells. Although the stable E-selectin and ICAM-1 expression of EI cells were comparable with those of activated ECs [28], other changes were neglected in this study. Shear flow would activate endothelial cells to promote the release of von Willebrand factor (vWF) from flow-stimulated EC [40], and it is reported that vWF could directly bind and immobilize extracellular DNA released from leukocytes [41]. Fibrin also induces release of vWF from endothelial cells [42]. Therefore, vWF released from activated endothelial cells might also act as a bridge for neutrophil adhesion to endothelial cells in the neutrophil-tumor cell-endothelial cell adhesion system with the presence of sFg or sFn. Moreover, highly metastatic melanoma cells could activate endothelial cells and tissue-factor-mediated thrombin formation, promoting the aggregation of platelets on tumor-activated endothelium, and leading to enhanced metastasis of tumor cells [43; 44; 45; 46]. Side-view micro-PIV was a 2D observation and also had its limitations. In order to further reveal the mechanisms behind the \"two-step theory\" of tumor metastasis, there are several improvements that could be made in future studies: (1) 3D micro-PIV technique could be applied to capture the dynamic process of PMN-facilitated tumor cell adhesion on an endothelial monolayer; (2) fluid-structure interaction could be imported to simulate the process of calculating contact area and contact time during the collisions of cells; (3) the impact of protein concentration gradient and chemokine could be involved; (4) endothelial cells could be used rather than EI cells to investigate the impact of vWF released from activated endothelial cells on the neutrophil-tumor cell-endothelial cell adhesion system. [PERSON], [PERSON] ([PERSON]), and [PERSON] developed the initial concept. [PERSON], [PERSON], [PERSON] ([PERSON]), and [PERSON] designed the experiment. [PERSON] performed the experiment and analyzed data. [PERSON] performed simulation work. [PERSON] wrote the manuscript. [PERSON]. ([PERSON]) and [PERSON] gave critical thoughts and revised the manuscript. [PERSON], [PERSON] ([PERSON]), and [PERSON] supervised the study. All authors discussed the results and commented on the manuscript. All authors have read and agreed to the published version of the manuscript. This work is supported by grants from the National Natural Science Foundation of China (11302129, to Y.F. ), the Hundred Teacher Talent Program of Shanghai University of Medicine & Health Sciences (B3-0200-20-311008-23, to Y.F. ), Construction project of Shanghai Key Laboratory of Molecular Imaging(18 DZ2260400), Shanghai Municipal Education Commission (Class II Plateau Disciplinary Construction Program for Medical Technology of SUMHS, 2018-2020), the Key Program of National Natural Science Foundation of China(81830052), the National Institute of Health Grants (CA-125707, to C.D. ), a National Science Foundation Grant (CBET-0729091, to C.D. ), the National Natural Science Foundation of China (11432006 and 31170887, to [PERSON].