Abstract
Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells in tumor microenvironments. These cells strongly support tumor progression and are considered to be potent therapeutic targets. Therefore, drugs targeting CAFs have been developed, but most of them have failed in clinical trials. The discovery of additional drugs to inactivate or eliminate CAFs is thus essential. In this study, we developed a high-throughput screening system to find anti-CAF drugs using reporter cells that express Twist1 promoter-GFP. This screening system uses the activity of the Twist1 promoter as an indicator of CAF activation because Twist1 is known to be a central player in CAF activation. Using this screening system, we found that dihydrorotenone (DHR), an inhibitor of electron transfer chain complex 1 in mitochondria, can effectively deactivate CAFs. DHR-treated CAFs exhibited reduced expression of CAF-enriched markers, decreased capability of collagen gel contraction, and impaired ability to engage in tumor-promoting activities, such as facilitating the proliferation and colonization of cancer cells. Furthermore, conditioned media from DHR-treated CAFs attenuated tumor progression in mice grafted with MNK28 cells. In conclusion, DHR can be considered as a candidate drug targeting CAFs.
Highlights
Fibroblasts play a key role in maintaining tissue homeostasis by contributing to tissue repair during wound healing
Because we found Twist[1] to be a novel marker that distinguishes Cancer-associated fibroblasts (CAFs) from normal fibroblasts (NFs), we developed a novel drug screening system using Twist[1] as a marker to search for new candidate drugs that deactivate CAFs
To determine the mRNA transcript expression level of the CAF-enriched markers, SCAF#36 cells were treated with DHR for three days, and RNA was extracted for a qPCR analysis
Summary
Fibroblasts play a key role in maintaining tissue homeostasis by contributing to tissue repair during wound healing In this process, quiescent fibroblasts become activated to support tissue repair, and they return to their initial state after tissue recovery[1,2]. A large proportion of CAFs are activated fibroblasts that express fibroblast activation markers such as FSP1, FAP, PDGFR, and α-SMA1,4 They acquire proliferation capacity and synthesize abundant paracrine factors and extracellular matrix (ECM) components[1]. Despite growing evidence for the central role of CAFs in promoting tumor progression, very little is known about the regulating factors responsible for the differentiation of NFs into CAFs. Recent studies have indicated that Twist[1] is a key regulator of CAF activation[7,8,9]. Our data demonstrate that DHR treatment inactivated CAFs and caused them to lose their tumor-promoting activity both in vitro and in vivo
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