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

Brian A. Boone, Pranav Murthy, Jennifer Miller-Ocuin, W. Reed Doerfler, Jarrod T. Ellis, Xiaoyan Liang, Mark A. Ross, Callen T. Wallace, Jason L. Sperry, Michael T. Lotze, Matthew D. Neal, Herbert J. Zeh

Research output: Contribution to journalArticle

16 Citations (Scopus)

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).

Original languageEnglish (US)
Article number678
JournalBMC Cancer
Volume18
Issue number1
DOIs
StatePublished - Jun 22 2018
Externally publishedYes

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Thrombophilia
Chloroquine
Pancreatic Neoplasms
Thromboplastin
Venous Thromboembolism
Platelet Aggregation
Hydroxychloroquine
gemcitabine
Extracellular Traps
Blood Platelets
Neoplasms
Platelet Activation
Electric Impedance
Knockout Mice
Histones
Adenocarcinoma
Neutrophils
Thrombosis
Collagen
Therapeutics

Keywords

  • Autophagy
  • Chloroquine
  • Hypercoagulability
  • Neutrophil extracellular traps (NETs)
  • Venous thromboembolism

ASJC Scopus subject areas

  • Oncology
  • Genetics
  • Cancer Research

Cite this

Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps. / Boone, Brian A.; Murthy, Pranav; Miller-Ocuin, Jennifer; Doerfler, W. Reed; Ellis, Jarrod T.; Liang, Xiaoyan; Ross, Mark A.; Wallace, Callen T.; Sperry, Jason L.; Lotze, Michael T.; Neal, Matthew D.; Zeh, Herbert J.

In: BMC Cancer, Vol. 18, No. 1, 678, 22.06.2018.

Research output: Contribution to journalArticle

Boone, BA, Murthy, P, Miller-Ocuin, J, Doerfler, WR, Ellis, JT, Liang, X, Ross, MA, Wallace, CT, Sperry, JL, Lotze, MT, Neal, MD & Zeh, HJ 2018, 'Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps', BMC Cancer, vol. 18, no. 1, 678. https://doi.org/10.1186/s12885-018-4584-2
Boone, Brian A. ; Murthy, Pranav ; Miller-Ocuin, Jennifer ; Doerfler, W. Reed ; Ellis, Jarrod T. ; Liang, Xiaoyan ; Ross, Mark A. ; Wallace, Callen T. ; Sperry, Jason L. ; Lotze, Michael T. ; Neal, Matthew D. ; Zeh, Herbert J. / Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps. In: BMC Cancer. 2018 ; Vol. 18, No. 1.
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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).",
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AU - Boone, Brian A.

AU - Murthy, Pranav

AU - Miller-Ocuin, Jennifer

AU - Doerfler, W. Reed

AU - Ellis, Jarrod T.

AU - Liang, Xiaoyan

AU - Ross, Mark A.

AU - Wallace, Callen T.

AU - Sperry, Jason L.

AU - Lotze, Michael T.

AU - Neal, Matthew D.

AU - Zeh, Herbert J.

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N2 - 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).

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