Single cell transcriptional signatures of breast cancer cells after completion of chemotherapy: The effects of the TGF-beta pathway inhibition

This work investigates enduring changes in endothelial cells that occur after completion of anthracycline chemotherapy and identifies a central role for canonical TGF-β/activin signaling in driving sustained mesenchymal reprogramming. Motivated by the clinical observation that cardiovascular disease is a leading cause of death among cancer survivors and that anthracyclines such as doxorubicin (Dox) produce progressive vascular and cardiac injury after treatment ends, the authors used a Dox treatment/washout model to probe molecular and cellular events that persist following drug exposure. Using human umbilical vein endothelial cells (HUVEC) in culture and cardiac microvascular endothelial cells in vivo, the study documents accumulation of Dox in endothelial nuclei (including mice treated with a 5 mg/kg intravenous dose and cultured cells treated with 100 nM during uptake experiments and 16 nM in treatment/washout experiments), and follows responses during a defined washout period to reveal lasting signaling and phenotypic reprogramming.

A multi-modal experimental approach linked enhanced canonical TGF-β and activin pathway activity to the post-washout phenotype. ChIP sequencing and transcriptomic analyses demonstrated increased Smad3 binding and upregulation of transcripts characteristic of mesenchymal, fibroblastic, and smooth muscle lineages in endothelial cultures and in cardiac microvascular endothelial cells in vivo. Reporter plasmid assays and direct measurement of Smad2/3 phosphorylation confirmed heightened responsiveness of the Smad2/3 axis: Dox-treated then washed endothelial cultures exhibited increased phospho-Smad2 and phospho-Smad3 in response to low-dose TGF-β2 stimulation. The authors report increased production of ALK4/5 receptor ligands—specifically TGF-β2 and activin—during washout and a heightened Smad2/3 activation response to these ligands.

Functional consequences of this signaling shift included sustained mesenchymal reprogramming with cytoskeletal rearrangements, increased fibronectin production, and loss of endothelial barrier integrity manifested as increased monolayer permeability. To interrogate causality, the team applied SB431542 (SB), a selective ALK4/5/7 receptor kinase inhibitor, during the Dox washout period. Presence of SB blocked the upregulation of mesenchymal transcripts and protein markers, prevented cytoskeletal changes and fibronectin production, and abolished the increased permeability of the endothelial monolayer. These findings indicate that canonical TGF-β/activin signaling is necessary for the sustained endothelial-to-mesenchymal reprogramming and barrier compromise observed after Dox treatment.

Collectively, the study provides mechanistic insight into how anthracycline exposure can produce delayed vascular dysfunction: Dox exposure primes endothelial cells to produce and respond to TGF-β/activin ligands, activating Smad2/3-dependent transcriptional programs that shift endothelial identity toward mesenchymal-like states and weaken barrier function. By showing that pharmacologic inhibition of ALK4/5/7 receptors during washout can prevent these changes, the work points to a potential interventional strategy to mitigate progressive vascular and cardiac injury that emerges after chemotherapy completion.