Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis

Abstract

Graphical AbstractHighlights d Demonstrated implementation of high-throughput, cellbased residence time probe d Elucidated multidimensional cell molecular and adhesive profile relationships d Reconciled disparities between in vitro and in vivo adhesion and metastasis models SUMMARY Hematogenous metastasis is a multistep, selectinregulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in in vitro and in vivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.E.E.E. conducted experiments, performed data analysis, and wrote the manuscript. K.G.B., M.J.O., and J.O. assisted in experimentation. S.N.T. conceptualized the framework and contributed to the planning and design of the project, analysis, and writing. All authors discussed the results and commented on the manuscript.

Lymph Node Subcapsular Sinus Microenvironment-On-A-Chip Modeling Shear Flow Relevant to Lymphatic Metastasis and Immune Cell Homing

“…The device, which is easy and rapid to fabricate, is constructed from a sheet of polydimethylsiloxane and a polystyrene tissue culture plate between which is a 125 μm adhesive gasket. High-speed videomicroscopy enabled visualization of cell adhesion of an infused cell pulse along the length of the divergent portion of the channel ( Figure 3 B), which was functionalized with adhesion receptors ( Birmingham et al., 2019 ; 2020 ; Edwards et al., 2018 ; Oh et al., 2015 ) to recapitulate their expression on the lymphatic endothelial cell-lined SCS floor ( Figure 3 C). The channel was also designed with a 10-cm long unfunctionalized portion ( Figures 3 A and 3B) sufficient in length to allow perfused cells to be uniformly in contact with the substrate, enabling all cells used in this work to settle to the channel bottom prior to reaching the functionalized section ( Birmingham et al., 2019 ).…”

“…Adhesion by LS174T colon carcinoma cells, which are well studied for their adhesive properties within flow fields ( Birmingham et al., 2019 ; 2020 ; Erin E. Edwards et al., 2018 ; Oh et al., 2015 ) and express a variety of adhesion ligands for selectins ( Dallas et al., 2012 ; Hanley et al., 2006 ) and other CAMs ( Paschos et al., 2010 ), were evaluated ( Birmingham et al., 2019 , 2020 ; Dallas et al., 2012 ; Edwards et al., 2017 , 2018 ; Hanley et al., 2006 ; Thomas et al., 2008 ). Consistent with prior reports ( Fujisaki et al., 1999 ), LS174T cells did not adhere in flow to ICAM or VCAM ( Figures 4 D, 4H, and 4L) alone under any tested configuration of flow but interacted extensively with E-selectin ( Figures 4 A and 4C) at rolling velocities unchanged by flow condition ( Figure 4 B).…”

“…Such microfluidic systems offer the advantage of enabling high-throughput experimentation under defined molecular, cellular, and/or biophysical conditions, thus substantially increasing the number of experimental conditions that can be explored ( Edwards et al., 2017 ; Hanley et al., 2006 ; Thomas et al., 2008 ). Furthermore, coupling these microfluidic devices with high-speed videomicroscopy permits rapid and facile visualization and quantification of the adhesive behavior of thousands of cells in a single experiment to increase statistical robustness ( Birmingham et al., 2020 ; Edwards et al., 2017 , 2018 ; Oh et al., 2015 ). Using this “LN sinus-on-a-chip” adhesive microfluidic platform, we explored the effects of wall shear stress (WSS) magnitude and dissipation, which were modeled to occur within the LN SCS, on adhesion by cell types that disseminate to LNs via the lymphatic vasculature, including human metastatic colon and pancreatic carcinoma and monocytic cell lines.…”

Patient-Derived In Vitro Models for Drug Discovery in Colorectal Carcinoma

“…This 3D microfluidic cell culture seems to be an extremely useful tool in the study of various phenomena, such as vascularization and oncogenesis under dynamic conditions. In the development of CRC, or its dissemination during the metastasis, the organ-on-chip-like microfluidic device has been developed [100]. This device, which merges microfluidics and photoconvertible protein technology, enables tracing the velocity of the circulating cells in the selectin-regulated process of adhesion and metastasis in a spatiotemporal manner.…”

The Biophysics of Lymphatic Transport: Engineering Tools and Immunological Consequences

“…As examples of the former, microfluidic chips with tightly controlled patterning of adhesion molecules have been implemented to evaluate bond strength and cooperative effects of receptor-ligand interactions on cell adhesion in the context of hemodynamic forces using videomicroscopy (Tong et al., 2012, Edwards and Thomas, 2017) (Figure 2). A velocimetric photoconversion technique (Edwards et al., 2018) was also recently described, for use in conjunction with microfluidic devices, that model circulating cell adhesion in the context of fluid flow. In this approach, cells expressing a photoconvertible fluorophore, which irreversibly alters its spectral properties after light exposure, are perfused through an engineered microfluidic system illuminated with photoconverting light and fabricated to present vascular endothelium-expressed adhesion receptors (Figure 2).…”