A high-throughput mechanofluidic screening platform for investigating tumor cell adhesion during metastasis

Abstract

The metastatic spread of cancer is a major barrier to effective and curative therapies for cancer. During metastasis, tumor cells intravasate into the vascular system, survive in the shear forces and immunological environment of the circulation, and then extravasate into secondary tumor sites. Biophysical forces are potent regulators of cancer biology and are key in many of the steps of metastasis. In particular, the adhesion of circulating cells is highly dependent upon competing forces between cell adhesion receptors and the shear stresses due to fluid flow. Conventional in vitro assays for drug development and the mechanistic study of metastasis are often carried out in the absence of fluidic forces and, consequently, are poorly representative of the true biology of metastasis. Here, we present a novel high-throughput approach to studying cell adhesion under flow that uses a multi-well, mechanofluidic flow system to interrogate adhesion of cancer cell to endothelial cells, extracellular matrix and platelets under physiological shear stresses. We use this system to identify pathways and compounds that can potentially be used to inhibit cancer adhesion under flow by screening anti-inflammatory compounds, integrin inhibitors and a kinase inhibitor library. In particular, we identify several small molecule inhibitors of FLT-3 and AKT that are potent inhibitors of cancer cell adhesion to endothelial cells and platelets under flow. In addition, we found that many kinase inhibitors lead to increased adhesion of cancer cells in flow-based but not static assays. This finding suggests that even compounds that reduce cell proliferation might also enhance cancer cell adhesion during metastasis. Overall, our results validate a novel platform for investigating the mechanisms of cell adhesion under biophysical flow conditions and identify several potential inhibitors of cancer cell adhesion during metastasis.

Biomechanical Regulation of Breast Cancer Metastasis and Progression

“…These included inhibitors to FLT-3, PI-3K, JAK3, EGFR and VEGFR. Consistent with our findings here, our previous study using the high throughput shear stress device also identified FLT-3 inhibitors to be effective in blocking cancer cell adhesion to endothelial cells under shear stress (15). The PI3K inhibitor reduced metastasis to the skull in immune compromised mice for the load conditioned cells, suggesting the strategy of screening for inhibitors of cancer cell to endothelial cell adhesion could be a means to identify therapeutics that protect against metastatic spread of cancer but that multiple pathway targeting may be needed.…”

“…In our past work, we developed a high throughput system for applying flow to cells cultured in 96 well plates (14)(15)(16). The device is composed of a rotational motor that drives a 96 gear box, simultaneously rotating 96 shafts with low angle cone tips, which interface with a standard format 96 well plate.…”

“…Using the high throughput mechanical loading system, we applied mechanical stretch to MDA-MB-231 breast cancer cells over a range from 0 to 17.5% maximal strain for 24 hours. Our group has developed a high throughput flow system that allows the application of controlled flow in a 96 well plate format (14,16,27). The system uses low angle cones rotated near the culture surface of the well to produce shear on the cells.…”

Recent Advances in Microfluidic Platforms Applied in Cancer Metastasis: Circulating Tumor Cells' (CTCs) Isolation and Tumor‐On‐A‐Chip

“…The results revealed that mechanical strain in the vascular microenvironments could accelerate tumor necrosis factor‐related apoptosis inducing ligand (TRAIL)‐induced apoptosis and regulate adhesion of tumor cells to the endothelium. The Baker group developed a high‐throughput mechanofluidic device to investigate the adhesion of tumor cells subjected to physiological shear stress . With the help of the proposed platform, the authors identified FLT‐3 and AKT inhibitors that impeded tumor cell adhesion to platelets and endothelial cells in the presence of fluid flow.…”

“…With the help of the proposed platform, the authors identified FLT‐3 and AKT inhibitors that impeded tumor cell adhesion to platelets and endothelial cells in the presence of fluid flow. Moreover, kinase inhibitors were observed to promote tumor cell adhesion in flow‐based, other than, static assays . Additionally, Garcia et al designed a robust microfluidic chip to establish stable orthogonal gradients of ECM stiffness and chemical concentration to study Madin–Darby canine kidney (MDCK) canine cell migration .…”