Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps

Abstract

Background: The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis.

Methods: Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated.

Results: The addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE.

Conclusion: NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer.

Trial registration: This study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).

Keywords: Autophagy; Chloroquine; Hypercoagulability; Neutrophil extracellular traps (NETs); Venous thromboembolism.

Conflict of interest statement

Ethics approval and consent to participate

All experimental animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh (Protocol # 14084123).

Correlative patient samples and data were included from two clinical trial protocols that were approved by the Institutional Review Board for the University of Pittsburgh (Protocol #10010028 and #13080444). All patients signed informed consent prior to participation in these clinical protocols.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

NETs promote hypercoagulability through platelet aggregation. Tumor bearing mice have elevated platelet aggregation compared with sham controls (a, AUC 40.2 ± 5.5 vs. 25.8 ± 1.5, n = 5). Treatment of human (b) and murine (c) blood with NET supernatant led to a dose dependent increase in platelet aggregation compared with treatment with media control. Tumor bearing PAD4 KO mice had decreased platelet aggregation compared to WT (AUC 8.4 ± 2.4 vs. 3.7 ± 1.7, n = 7) with no difference in sham controls (d). *p < 0.05

Fig. 2

NET upregulation of platelet aggregation is mediated by neutrophil DNA and platelet RAGE. Removing DNA from NET supernatant using DNase I treatment prior to exposure to whole blood reversed the treatment effects of NET supernatant on platelet aggregation in human blood (a, 25.9 ± 2.2 vs. 11.35 ± 0.31, n = 4, p < 0.05). In vivo treatment with DNase I resulted in decreased aggregation in tumor bearing mice (b, AUC 22.1 ± 2.3 vs. 38.4 ± 2.1, n = 4, p < 0.05). Tumor bearing RAGE KO mice have decreased platelet aggregation compared to WT mice (c, AUC 30.6 ± 1.5 vs. 40.2 ± 5.5, n = 4, p < 0.05). Blood from RAGE knockout mice had decreased aggregation after treatment with 100 μL of NET supernatant compared with WT (d, AUC 25.5 ± 2.6 vs. 43.3 ± 3.9, n = 4, p < 0.05). *p < 0.05

Fig. 3

NETs promote hypercoagulability in PDA by releasing circulating tissue factor. Tissue factor ELISA was performed on serum from orthotopic mice, demonstrating that tumor burdened mice had elevated levels of circulating tissue factor compared to sham (a, 255 ± 49 vs. 159 ± 26 pg/mL, p < 0.05). Genetic deletion of PAD4, thereby inhibiting NET formation, resulted in a substantial decrease in circulating tissue factor levels in tumor bearing mice (269 ± 26 vs. 202 ± 30 pg/mL, p < 0.05). Blue = WT, Red = PAD4 KO, Circle = Sham, Triangle = Tumor. RAGE knockout tumor bearing mice, who we have previously shown have decreased NET formation, also had lower levels of tissue factor compared to WT controls (b, 331 ± 39 vs. 390 ± 34 pg/mL, p < 0.05). *p < 0.05. Blue = WT, Red = RAGE KO, Circle = Sham, Triangle = Tumor

Fig. 4

CQ inhibition of NETs reverses platelet aggregation and decreases tissue factor. In vitro treatment of whole blood with CQ led to a significant reduction in platelet aggregation in blood harvested from tumor bearing mice (a, AUC 50 ± 2.4 vs. 68.1 ± 8.8, n = 4, p < 0.05). Treatment of mice with CQ led to a decrease in aggregation in tumor bearing animals with no change in sham (b, AUC 52.6 ± 5.3 vs. 68.1 ± 8.8, n = 4, p < 0.05). Importantly, CQ had minimal effects in PAD4KO mice, suggesting that it decreases platelet aggregation through inhibition of NETs (c). CQ treatment led to a decrease in circulating tissue factor in tumor bearing mice (d, 186.9 ± 5.6 vs. 228.2 ± 21 pg/mL, p < 0.05). Hydroxychloroquine treatment resulted in significant reduction in tissue factor levels in patients with elevated preoperative serum tissue factor compared to control, with a mean response to treatment of − 240 ± 120 versus − 8.74 ± 26 pg/mL (p < 0.05, n = 10 gem/nab-paclitaxel, n = 7 HCQ). Waterfall plot demonstrating individual treatment response to gemcitabine/nab-paclitaxel with and without hydroxychloroquine in patients with elevated preoperative levels (e)

Fig. 5

Chloroquine reverses hypercoagulability in pancreatic cancer. Representative TEG curves demonstrating orthotopically injected mice are hypercoagulable compared with sham controls (a). Treatment with CQ reverses the hypercoagulability on TEG as measured by coagulation index (b). The 90 day VTE rate for patients treated with 2 cycles of preoperative gemcitabine/abraxane + HCQ was 9.1% (n = 3 of 33) compared to 30% (n = 9 of 30) in patients treated with gemcitabine/abraxane alone (c, p = 0.053)