Neutrophil extracellular traps drive epithelial–mesenchymal transition of human colon cancer

Abstract

Neutrophil extracellular traps (NETs) are extracellular structures, composed of nuclear DNA and various proteins released from neutrophils. Evidence is growing that NETs exert manifold functions in infection, immunity and cancer. Recently, NETs have been detected in colorectal cancer (CRC) tissues, but their association with disease progression and putative functional impact on tumourigenesis remained elusive. Using high-resolution stimulated emission depletion (STED) microscopy, we showed that citrullinated histone H3 (H3cit) is sufficient to specifically detect citrullinated NETs in colon cancer tissues. Among other evidence, this was supported by the close association of H3cit with de-condensed extracellular DNA, the hallmark of NETs. Extracellular DNA was reliably differentiated from nuclear condensed DNA by staining with an anti-DNA antibody, providing a novel and valuable tool to detect NETs in formalin-fixed paraffin-embedded tissues. Using these markers, the clinical association of NETs was investigated in a cohort of 85 patients with colon cancer. NETs were frequently detected (37/85, 44%) in colon cancer tissue sections and preferentially localised either only in the tumour centre or both in the tumour centre and the invasive front. Of note, citrullinated NETs were significantly associated with high histopathological tumour grades and lymph node metastasis. In vitro, purified NETs induced filopodia formation and cell motility in CRC cell lines. This was associated with increased expression of mesenchymal marker mRNAs (vimentin [VIM], fibronectin [FN1]) and epithelialmesenchymal transition promoting transcription factors (ZEB1, Slug [SNAI2]), as well as decreased expression of the epithelial markers E-cadherin (CDH1) and epithelial cell adhesion molecule (EPCAM). These findings indicated that NETs activate an epithelial-mesenchymal transition-like process in CRC cells and may contribute to the metastatic progression of CRC.

Moonlighting chromatin: when DNA escapes nuclear control

“…High resolution stimulated emission depletion (STED) microscopy was used to detect citrullinated NETs frequently recurring in tissue biopsies from patients with colon cancer. Using anti-DNA antibodies directed to extracellular chromatin, the authors distinguished between condensed and compacted chromatin inside neutrophils with a healthy appearance and ejected decondensed DNA displaying the typical characteristic of NETs [ 106 ].…”

Inflammation-related citrullination of matrisome proteins in human cancer

“…Interestingly, NETs have been detected in e.g. , colon cancer and they may participate in processes such as mesenchymal-epithelial transition ( 59 ). Our conclusion that the citrullination of matrisome proteins in cancer is mainly related to inflammation is based on the following facts: Firstly, analysis of cancer proteomics data sets revealed that many of the most frequently citrullinated matrisome proteins, such as fibrinogen and fibronectin, are also abundantly citrullinated in e.g.…”

Neutrophil extracellular traps and pulmonary fibrosis: an update

“…Other pathways besides NETs-DNA may also damage lung epithelial cells, such as NETs mediating inflammasome activation and IL-1β secretion from monocytes and causing airway epithelial cell injury, and NETs modify and regulate lung injury by activating ferroptosis in alveolar epithelial [65][66][67]. Additionally, NETs are associated with the development of lung fibrosis after COVID-19, because NETs induce the downregulation of the epithelial marker E-cadherin and the upregulation of the mesenchymal marker α-SMA in pulmonary epithelial cells [68][69][70][71]. Exposure to butyl benzyl phthalate (BBP), a widely used plasticizer, has been linked to asthma and impaired lung functions [72,73].…”