A Miniaturized Screening Platform to Identify Novel Regulators of Extracellular Matrix Alignment

Abstract

Extracellular matrix (ECM) alignment contributes to metastasis in a number of cancers and is a known prognostic stromal factor; however, the mechanisms controlling matrix organization remain unclear. Cancer-associated fibroblasts (CAF) play a critical role in this process, particularly via matrix production and modulation of key signaling pathways controlling cell adhesion and contractility. Stroma normalization, as opposed to elimination, is a highly sought strategy, and screening for drugs that effectively alter ECM alignment is a practical way to identify novel CAF-normalizing targets that modulate ECM organization. To meet this need, we developed a novel high-throughput screening platform in which fibroblast-derived matrices were produced in 384-well plates, imaged with automated confocal microscopy, and analyzed using a customized MATLAB script. This platform is a technical advance because it miniaturizes the assay, eliminates costly and time-consuming experimental steps, and streamlines data acquisition and analysis to enable high-throughput screening applications. As a proof of concept, this platform was used to screen a kinase inhibitor library to identify modulators of matrix alignment. A number of novel potential regulators were identified, including several receptor tyrosine kinases [c-MET, tropomyosin receptor kinase 1 (NTRK1), HER2/ERBB2] and the serine/threonine kinases protein kinase A, C, and G. The expression of these regulators was analyzed in publicly available patient datasets to examine the association between stromal gene expression and patient outcomes. Significance: ECM fiber organization and alignment contribute to metastasis in a number of cancers and are a known prognostic stromal factor; however, the mechanisms controlling matrix organization remain unclear. Here, a high-throughput assay was developed to enable discovery-based screening for an in vitro ECM fiber alignment assay. As proof of concept, this platform was used to screen a kinase inhibitor library and identified several novel modulators of matrix alignment.