Category: Micro- and Nanotechnologies
Mechanical forces produced by cells play key roles within many physiological systems, including active functions such as vasgoregulation, cardiac output, and intestinal propulsion, as well as more passive but critical structural functions. Additionally, cell-generated forces are vital to cellular-level processes such as phagocytosis of pathogens, cytotoxic killing, division, and remodeling of extracellular matrix (ECM) fibers.
Accordingly, disruptions in proper cellular force generation give rise to a plethora of disease including chronic conditions such as asthma, hypertension, and bowel disease. Such disorders place a significant burden on patient health, most severely impacting the treatment-resistant patient subpopulations. These patient groups are in dire need of new, orthogonally-acting treatment options, for which development has stalled arguably due to a lack of adequate tools for evaluating cellular force in early drug discovery.
We previously introduced a high-throughput phenotypic screening platform that met this need by directly measuring the contractile forces generated by single-cells and verified its feasibility as a drug discovery tool. This platform, based on fluorescently-labeled elastomeric contractible surfaces (FLECS), enables image-based evaluation of cellular contractility based on the cell-induced displacements of precise fluorescent and adhesive micropatterns.
We implemented this phenotypic assay in a 384-wellplate format and integrated these FLECS wellplates with lab automation workflows to perform automated drug screens using primary human cell types comprising three distinct drug discovery programs:
Our first drug discovery program targets asthmatic hypercontractility. Here, we screened compound libraries using primary human airway smooth muscle cells, and obtained a collection of hits that relaxed these cells comprising both known classes of force-generation effectors as well as surprising new compounds. We followed up these screens using fatal-asthmatic patient-derived smooth muscle cells, comparing their relative responsiveness to these compound libraries.
Our second program targets hypertension, and aims to discover compounds that potently and specifically relax vascular smooth muscle leading to vasodilation. Here, we screened compound libraries using primary human vascular smooth muscle cells.
Currently, we are optimizing a third program targeting myofibroblast contractility, which contributes to fibrosis, and are screening using primary human fibroblasts as well as differentiated myofibroblasts.
In this presentation, I will give a detailed overview of FLECS technology and present the results of these screens, discussing both overlapping and unique hits across these three programs, and give an update on the progress of our other programs.
I will also describe a novel cell-multiplexing strategy we are developing that enables simultaneous screening of up to 8 cell types in the same FLECS wellplates without a reduction in compound-per-plate throughput.
The success of these screens in identifying modulators of cellular force underscores the usefulness and efficiency of this phenotypic screening methodology and creates an optimistic outlook concerning the its full-scale application to drug development.
Ivan Pushkarsky– Postdoctoral Researcher, UCLA, Los Angeles, CA