Fibronectin (FN) is an important adhesion molecule in mediating mammalian cell migration 50

Fibronectin (FN) is an important adhesion molecule in mediating mammalian cell migration 50. shape shows distinct differential migration characteristics of amoeboid and mesenchymal cells. Notably, the fastest moving cells belong to the subpopulation of amoeboid cells. Together, these findings highlight Tegobuvir (GS-9190) the important roles of biophysical forces in modulating tumor cell migration heterogeneity and plasticity, as well as the suitability of microfluidic models in interrogating tumor cell dynamics at single-cell and subpopulation level. Introduction Interstitial flows are ubiquitous in human tissues. They are driven by the hydrostatic and osmotic pressure differences among the arterial, ISG20 venous, and lymphatic vessels 1. In healthy tissue, interstitial flow rates are on the order of a few micrometers per second 2. Within malignant tumor, interstitial flow rates can reach as Tegobuvir (GS-9190) Tegobuvir (GS-9190) high as 10 m/s in animal models 2-4, and up to 55 m/s in human cancer patients 4, 5. A number of dynamically evolving tumor microenvironment factors have been identified to contribute to the elevated interstitial fluid flows, including the continual expansion of tumor mass which builds up the interstitial fluid pressure within the tumor 6, 7, the subsequent abnormal growth of vascular vessels via angiogenesis 8, 9 and/or lymphangiogenesis 10-12, as well as the denser extracellular matrix (ECM) deposited and remodeled by stromal cells with higher hydraulic conductivity 13, 14. Clinically, lymph nodes are known to be the first metastatic sites for many cancer types, including breast 15 and prostate cancers 16. Recognizing that interstitial flows drain towards lymph nodes, an emerging question is: whether and how interstitial flows guide and modulate tumor cell invasion into the lymph nodes 17. Indeed, pioneer work from the Swartz lab has demonstrated that interstitial flows (0.2 and 0.7 m/s) can spatially redistribute chemokine secretions of breast and glioma tumor cells, and direct tumor cells invasion along the flow direction in a chemokine receptor CCR7/CXCR4 dependent manner using a modified Boyden Chamber Tegobuvir (GS-9190) model 17, 18. Tumor cells are known to be heterogeneous (ensemble variability) and plastic (temporal variability) in response to the complex tumor microenvironment 19. In cancer metastasis, only a subpopulation of the tumor cells or rare cells break away from the primary tumor and migrate through the interstitial space, with only a fraction of those eventually establishing a secondary tumor at an ectopic site. Cancer cell heterogeneity and plasticity are also demonstrated through their diverse motility types. Single animal cell migration within a 3D architecture can be broadly categorized into amoeboid and mesenchymal motility phenotypes 20, 21. In amoeboid motility, cells appear rounded in shape, form actin protrusions and dynamically change their shapes to squeeze through pores within the collagen fiber network 22-24. Traction is distributed all around the cell surface through many short-lived adhesive contacts with the ECM 25, 26. In mesenchymal motility, cells appear elongated in shape, climb along the collagen fibers, and proceed by either remodeling or degrading the matrix in an integrin and/or proteolysis dependent manner 27, 28. Traction is exerted through long-lived, polarized and highly localized focal adhesion complexes 29-31. While leukocytes typically exhibit amoeboid motility, and fibroblasts assume mesenchymal motility, cancer cells are known to be able to switch between these two motility types depending on the microenvironment 32, 33. Wolf discovered that fibrosarcoma cells switch from a mesenchymal to amoeboid motility when matrix metalloproteinase (MMPs) was inhibited in both 3D model and mouse model 32. For understanding the heterogeneity and plasticity of tumor cell, there is a need for tools that can interrogate cancer cell invasion at single-cell or subpopulation level, and in real time. Although modified Boyden chamber models have played instrumental roles in revealing effects of interstitial flows on molecular mechanism governing tumor cell invasion 17, 18, 34, these results are limited in endpoints and population levels. Recently, microfluidic models have emerged for studying effects of interstitial flows on tumor cell invasion because of their compatibility with optical microscope, making it possible to follow single-cell dynamics in both time and space 35-37. In addition, microfluidic models have the advantage of providing well controlled microenvironments, such as fluid flows within a 3D ECM 38. Current microfluidic models have revealed that flow-guided cell migration depended on a number of critical parameters within the tumor microenvironment, including chemokine receptors, matrix stiffness, cell density, and flow rates 35, 36, 39. In a recent Tegobuvir (GS-9190) work, Polacheck showed that.