A High-Content Screen Identifies Drugs That Restrict Tumor Cell Extravasation across the Endothelial Barrier

Abstract

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell–matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor–endothelial cell interactions. Significance: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor–endothelial cell interactions.

Development of a cancer metastasis-on-chip assay for high throughput drug screening

“…Therefore, we tested whether Imatinib prevents endothelial cell monolayer disruption by seeding MDA-MB-231 tumor cells into the microvessel perfusion channel with a confluent monolayer of HUVECs in OrganoPlate ® . Tumor cells adhered to the endothelium and progressively disrupted the endothelial barrier by dislodging endothelial cells and cell expansion over 72 hours, as previously described (46). We found that the endothelial cell number and endothelium area were preserved more in the Imatinib-treated chips than in controls (Figure 6B), and many tumor cells remained on the endothelium surface in Imatinibtreated chips.…”

Enhancing PKA-dependent mesothelial barrier integrity reduces ovarian cancer transmesothelial migration via inhibition of contractility

“… 39 In addition, forskolin treatment of endothelial barriers rescued the tumor cell-induced hyperpermeability and decreased the transendothelial migration of pancreatic cancer cells. 40 Forskolin is a widely used inducer of cyclic adenosine monophosphate (cAMP)-driven PKA activation. 35 PKA activity in mesothelial cells regulates inflammatory response as evidenced by a previous study that demonstrated PKA-dependent shedding of tumor necrosis factor receptors upon stimulation with interleukin-1-α.…”

High-content screening of active components of Traditional Chinese Medicine inhibiting TGF-β-induced cell EMT

“…It allows comprehensive recording of phenotypic changes and quantifies different morphological parameters by segmenting cell images using intelligent algorithms. In recent years, HCS has been used for drug screening, virus infection, toxicology and tumor migration studies [ 28 , 29 , 30 , 31 , 32 ]. Therefore, HCS is an appropriate system for the quantification of cell morphological changes during the EMT allowing distinction of the inhibitory effects of different compounds on this process.…”