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

Abstract

Extracellular matrix 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 extracellular matrix (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 (PKA, PKC, and PKG) . The expression of these regulators was analyzed in publicly available patient datasets to examine the association between stromal gene expression and patient outcomes.

Transcriptomics reveals the effects of NTRK1 on endoplasmic reticulum stress response-associated genes in human neuronal cell lines

“…It was reported that p53 was regulated through the TrkA and p75 neurotrophin receptors in neonatal sympathetic neurons ( Aloyz et al, 1998 ). As for ECM organization in the previous study, the researchers developed a high-throughput screening platform using fibroblast-derived matrices imaged with automated confocal microscopy in 384-well plates to explore mechanisms controlling matrix organization ( Jones et al, 2022 ). It was identified that NTRK1 was one of the modulators of matrix alignment.…”

maGENEgerZ: An Efficient Artificial Intelligence-Based Framework Can Extract More Expressed Genes and Biological Insights Underlying Breast Cancer Drug Response Mechanism

“…The top-enriched term is the vitamin D receptor pathway, which ameliorates breast cancer by contributing to the growth regulation of breast cancer cells [54,55]. Other enriched terms, such as NABA MATRISOME ASSOCIATED, mammary gland development, and extracellular matrix organization, have been reported to play a key role in various biological processes related to breast cancer progression and treatment [56][57][58][59][60]. In Supplementary Enirchment_Dataset1, we list Metascape enrichment analysis results related to Dataset1.…”

maGENEgerZ: An Efficient AI-Based Framework Can Extract More Expressed Genes and Biological Insights Underlying Breast Cancer Drug Response Mechanism

“…We had 11 treatments in which the 736 samples were distributed accodingly as follows. Paclitaxel (169), Paclitaxel + ABT 888 + Carboplatin (63) (49). The gene expression data was performed using Reverse Phase Protein Array (RPPA) microarray at George Mason University.…”