Neutrophil extracellular traps promote cancer-associated inflammation and myocardial stress

Figure 1.. NET formation and elevated coagulation in mice with mammary carcinoma. (a) ELISA for H3Cit-DNA complexes in mouse plasma (Healthy n = 4; MMTV-PyMT n = 6; MMTV-PyMT+DNase I n = 4; MMTV-PyMT+Gp1b n = 3, Healthy vs. MMTV-PyMT p = .0095; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .0381; MMTV-PyMT vs. MMTV-PyMT+Gp1b p = 0,0476). (b) TAT levels analyzed in mouse plasma (Healthy n = 8; MMTV-PyMT n = 12; MMTV-PyMT+DNase I n = 3, Healthy vs. MMTV-PyMT p = .0168; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .0308). Statistical test used: non-parametric two-tailed Mann-Whitney.

Figure 2.. Mice with mammary carcinoma display cardiac inflammation. Heart-derived RNA was analyzed by qPCR for (a) TNFα (Healthy n = 12; MMTV-PyMT n = 16; MMTV-PyMT+DNase I n = 8, Healthy vs. MMTV-PyMT p = .0340; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .0091) and (b) IL-1β (Healthy n = 13; MMTV-PyMT n = 16; MMTV-PyMT+DNase I n = 8, Healthy vs. MMTV-PyMT p = .0037; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .0926). (c) ELISA for sTNFR1 in plasma (Healthy n = 5; MMTV-PyMT n = 18; MMTV-PyMT+DNase I n = 5, Healthy vs. MMTV-PyMT p < .0001; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .3630). (d) Correlation between plasma sTNFR1 levels and RNA expression levels of TNFα in heart tissue (n = 5, p = .0292, r = 0.8208). RNA expression is presented as relative expression to HPRT. Heart tissue sections were immunostained for (e) CD45 (Healthy n = 5; MMTV-PyMT n = 10; MMTV-PyMT+DNase I n = 6, Healthy vs. MMTV-PyMT p = .0013; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .0030) and (f) CD68 (Healthy n = 5; MMTV-PyMT n = 10; MMTV-PyMT+DNase I n = 6, Healthy vs. MMTV-PyMT p = .0043; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .0146). Scale bars indicate 100 μm. Statistical test used: non-parametric two-tailed Mann-Whitney and Spearman…

Figure 3.. Endothelial proliferation and expression of biomarkers for myocardial strain in PyMT+ mice. (a) Proliferation of cells in heart tissue sections was analyzed by immunostaining for Ki67 (Healthy n = 4; MMTV-PyMT n = 6; MMTV-PyMT+DNase I n = 4, Healthy vs. MMTV-PyMT p = .0190; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .0286). Scale bar indicates 100 μm. (b) Representative image showing proliferating (Ki67+) endothelial (CD31+) cells. RNA from heart tissue was analyzed by qPCR for expression of (c) BNP (Healthy n = 17; MMTV-PyMT n = 18; MMTV-PyMT+DNase I n = 7, Healthy vs. MMTV-PyMT p = .0126; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .0023) and (d) GDF-15 (Healthy n = 9; MMTV-PyMT n = 16; MMTV-PyMT+DNase I n = 8, Healthy vs. MMTV-PyMT p = .0002; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .0448). RNA expression is presented as relative expression to HPRT. (e) ELISA for GDF-15 in serum (Healthy n = 14; MMTV-PyMT n = 15; MMTV-PyMT+DNase I n = 8, Healthy vs. MMTV-PyMT p = .0008; MMTV-PyMT vs. MMTV-PyMT+DNase I p = .3833). RNA from heart tissue was analyzed by qPCR for expression of (f) cTnI (Healthy n = 11; MMTV-PyMT n = 17; MMTV-PyMT+DNase I n = 8, Healthy vs. MMTV-PyMT p = .0109; Healthy vs.…

Figure 4.. NET formation and inflammation in patients with malignancies. Plasma from patients with various types of malignant disorders were analyzed by ELISA for (a) H3Cit-DNA complexes (Healthy n = 33; Cancer patients n = 33, p = .0002), (b) MPO-DNA complexes (Healthy n = 22; Cancer patients n = 33, p < .0001) and (c) neutrophil elastase (NE) (Healthy n = 22; Cancer patients n = 33, p < .0001). (d) CRP concentration in plasma (Healthy n = 33; Cancer patients n = 32, p = .0002). (e) Correlation between H3Cit-DNA complexes and CRP in cancer patient plasma (n = 32, p = .0229, r = 0.1296). (f) Correlation between MPO-DNA complexes and CRP in cancer patient plasma (n = 32, p = .0105, r = 0.4462). (g) Correlation between NE and CRP in cancer patient plasma (n = 32, p < .0001, r = 0.7445). Statistical test used: non-parametric two-tailed Mann-Whitney and Spearman correlation test.

Figure 5.. Patients with malignancies have elevated levels of biomarkers for cardiovascular disease. Plasma analysis of (a) GDF-15 (Healthy n = 33; Cancer patients n = 33, p = .0020), (b) sTNFR1 (Healthy n = 33; Cancer patients n = 33, p = .0004), (c) sTNFR2 (Healthy n = 33; Cancer patients n = 33, p = .0274), (d) NT-proBNP (Healthy n = 32; Cancer patients n = 32, p = .0004) and (e) cTnI (Healthy n = 33; Cancer patients n = 33, p = .0584). Analysis of correlation between (f) H3Cit-DNA and NT-proBNP (n = 32, p = .0003, r = 0.3394), and (g) H3Cit-DNA and sTNFR1 (n = 33, p = .0203, r = 0.5318). Statistical test used: non-parametric two-tailed Mann-Whitney and Spearman correlation test.