Fig. 2

Ovarian cancer ascites is chemoattractive to neutrophils and induces NETs and suppressive neutrophils. a–h Resected tumours were collected from patients with newly diagnosed advanced EOC (n = 5). Tumours were evaluated for the presence of (a–d) intact neutrophils (PMNs, white arrows), identified by a hypersegmented nucleus (DNA, blue; histone H1, red), and cytoplasmic NE (green), and (e–h) NETs, identified by co-localised extracellular DNA, histone H1, and NE (white boxes). PMNs within tumours were rare. NETs were visualised in 4/5 tumours evaluated. i Ascites were collected from patients with newly diagnosed HGSOC (n = 9). >90% of the cells are CD45⁺ leukocytes and on average 15–20% are granulocytic (blue). j–m Ascites supernatants (ASC) were evaluated for pathways of PMN activation. PMNs were isolated from peripheral blood of healthy donors. j Donor PMN chemotaxis was measured in response to mtDAMPs (positive control) and ascites supernatants (n = 2). Sphingosine kinase inhibitor (SKI) was added to both mtDAMPs and ascites supernatants as a negative control to inhibit chemotaxis (**p < 0.01). k Donor PMNs were treated for 1 h with ascites supernatants and evaluated for NET generation (white boxes) by immunofluorescent confocal microscopy. Exposure of neutrophils to 3 of 4 ascites supernatants resulted in NETs. l Donor PMNs were treated for 1 h with media, 100 nM PMA (positive control), ascites supernatants (n = 4), DNase I-pre-treated ascites supernatants, or heat-inactivated ascites supernatants, and evaluated for degranulation by NE ELISA. Ascites-induced neutrophil degranulation, which was partially reversed with heat-inactivation (HI-ASC) or DNase I pre-treatment. m Autologous donor PMNs, CD4⁺, and CD8⁺ T cells were isolated and used in co-culture. Neither PMNs nor ascites supernatants alone suppress anti-CD3/CD28-stimulated T cell proliferation, however, the majority of ascites supernatants from patients with newly diagnosed HGSOC (n = 17/22) induced PMNs to suppress T cell proliferation. ASC-1 is an example of an ascites sample that induced the neutrophil suppressor phenotype, and ASC-2 is an example of an ascites sample that did not induce the neutrophil suppressor phenotype. Data are from ≥3 independent experiments