Introduction: Post-operative infectious complications in cancer patients confer an increased risk of death from distant metastasis, and activated neutrophils appear to be central cellular players in this phenomenon. We have previously demonstrated that Neutrophil extracellular traps (NETs), composed of extracellular neutrophil-derived DNA webs, are intricately implicated in this process by directly binding and capturing circulating tumor cells. However, the mechanisms mediating this interaction remain completely unexplored. We hypothesized that tumor cells adhere to NETs specifically through interactions between neoplastic and neutrophil-derived β 1 integrins.
Methods: In vitro, NETs isolated from phorbol myristate acetate (PMA) activated neutrophils from healthy volunteers were plated on cover slips and human lung or colorectal cancer (A549 or HT29) adhesion assessed. In some experiments, cancer cell adhesion was assessed in the presence of DNAse, RGD peptide, or anti- β 1 integrin antibody (4B4). In vivo, cecal ligation and puncture (CLP) was used to induce NET formation and A549 cells (siRNA anti- β 1 integrin transfected or control) were injected via the intrasplenic route to assess real time single cell migration by hepatic intravital microscopy.
Results: Adhesion of cancer cells to NET isolates was decreased 2-3 fold in the presence of DNAse and proteinase K, indicating the possibility of protein-protein interactions. Pretreatment of malignant cells with RGD peptide, known to inhibit integrin mediated adhesion, similarly resulted in a 2-3 fold decrease in tumor cell adhesion compared to isolated NETs alone. Adhesion of A549 and HT29 cells to NET isolates after pretreatment with function blocking anti- β 1 integrin antibody was 1/3 that of untreated cells. Following PMA stimulation, β 1 integrin staining co-localized within extracellular DNA but was absent in unstimulated neutrophils. Adhesion in vivo was reduced 2 fold after siRNA knockdown of β 1 integrin in A549 cells 24 hours following CLP compared to untreated cells. Hepatic intravital microscopy after CLP demonstrates widespread NET deposition demonstrated by DNA and histone H2A.X deposition. NETs were found to stain positive for β 1 integrin. Beta 1 integrin staining was increased after CLP compared to sham, and was abrogated by neutrophil depletion.
Conclusions: This study is the first to implicate β 1 integrin mediated interactions as a mechanism for tumor-NET adhesion. Cell surface expression of β 1 integrins on tumor cells was necessary for tumor cell adhesion to NETs both in vitro and in vivo. Analysis of NETs both in vitro and in vivo demonstrated β 1 integrin expression, implicating integrin hetero-dimerization as a possible adhesive mechanism.
Citation Format: Sara Najmeh, Jonathan Cools-Lartigue, Stephen Gowing, Jonahan Spicer, Braedon McDonald, Betty Giannias, France Bourdeau, Paul Kubes, Lorenzo Edwin Ferri. Neutrophil extracellular traps sequester circulating tumor cells via beta 1 integrin mediated interactions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-258. doi:10.1158/1538-7445.AM2014-LB-258