Neutrophil extracellular traps promote liver micrometastasis in pancreatic ductal adenocarcinoma via the activation of cancer‑associated fibroblasts

Takesue,Shin; Ohuchida,Kenoki; Shinkawa,Tomohiko; Otsubo,Yoshiki; Matsumoto,Sokichi; Sagara,Akiko; Yonenaga,Akiko; Ando,Yohei; Kibe,Shin; Nakayama,Hiromichi; Iwamoto,Chika; Shindo,Koji; Moriyama,Taiki; Nakata,Kohei; Miyasaka,Yoshihiro; Ohtsuka,Takao; Toma,Hiroki; Tominaga,Yohei; Mizumoto,Kazuhiro; Hashizume,Makoto; Nakamura,Masafumi

doi:10.3892/ijo.2019.4951

Neutrophil extracellular traps promote liver micrometastasis in pancreatic ductal adenocarcinoma via the activation of cancer‑associated fibroblasts

Authors:
- Shin Takesue
- Kenoki Ohuchida
- Tomohiko Shinkawa
- Yoshiki Otsubo
- Sokichi Matsumoto
- Akiko Sagara
- Akiko Yonenaga
- Yohei Ando
- Shin Kibe
- Hiromichi Nakayama
- Chika Iwamoto
- Koji Shindo
- Taiki Moriyama
- Kohei Nakata
- Yoshihiro Miyasaka
- Takao Ohtsuka
- Hiroki Toma
- Yohei Tominaga
- Kazuhiro Mizumoto
- Makoto Hashizume
- Masafumi Nakamura
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Affiliations: Department of Surgery and Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812‑8582, Japan, Department of Advanced Medical Initiatives, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812‑8582, Japan, Department of Endoscopic Diagnostics and Therapeutics, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812‑8582, Japan
Published online on: December 24, 2019 https://doi.org/10.3892/ijo.2019.4951
Pages: 596-605

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Abstract

Cancer‑associated fibroblasts (CAFs) promote the progression of pancreatic ductal adenocarcinoma (PDAC) via tumor‑stromal interactions. Neutrophil extracellular traps (NETs) are extracellular DNA meshworks released from neutrophils together with proteolytic enzymes against foreign pathogens. Emerging studies suggest their contribution to liver metastasis in several types of cancer. Herein, in order to investigate the role of NETs in liver metastasis in PDAC, the effects of NET inhibitors on spontaneous PDAC mouse models were evaluated. It was demonstrated that DNase I, a NET inhibitor, suppressed liver metastasis. For further investigation, further attention was paid to liver micrometastasis and an experimental liver metastasis mouse model was used that was generated by intrasplenic tumor injection. Furthermore, DNase I also suppressed liver micrometastasis and notably, CAFs accumulated in metastatic foci were significantly decreased in number. In vitro experiments revealed that pancreatic cancer cells induced NET formation and consequently NETs enhanced the migration of hepatic stellate cells, which was the possible origin of CAFs in liver metastasis. On the whole, these results suggest that NETs promote liver micrometastasis in PDAC via the activation of CAFs.

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1	Siegel RL, Miller KD and Jemal A: Cancer statistics, 2016. CA Cancer J Clin. 66:7–30. 2016. View Article : Google Scholar : PubMed/NCBI
2	McAllister SS and Weinberg RA: The tumour-induced systemic environment as a critical regulator of cancer progression and metastasis. Nat Cell Biol. 16:717–727. 2014. View Article : Google Scholar : PubMed/NCBI
3	Malanchi I, Santamaria-Martínez A, Susanto E, Peng H, Lehr HA, Delaloye JF and Huelsken J: Interactions between cancer stem cells and their niche govern metastatic colonization. Nature. 481:85–89. 2011. View Article : Google Scholar : PubMed/NCBI
4	Quail DF and Joyce JA: Microenvironmental regulation of tumor progression and metastasis. Nat Med. 19:1423–1437. 2013. View Article : Google Scholar : PubMed/NCBI
5	Erler JT, Bennewith KL, Cox TR, Lang G, Bird D, Koong A, Le QT and Giaccia AJ: Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche. Cancer Cell. 15:35–44. 2009. View Article : Google Scholar
6	Schmidt H, Bastholt L, Geertsen P, Christensen IJ, Larsen S, Gehl J and von der Maase H: Elevated neutrophil and monocyte counts in peripheral blood are associated with poor survival in patients with metastatic melanoma: A prognostic model. Br J Cancer. 93:273–278. 2005. View Article : Google Scholar : PubMed/NCBI
7	Tibaldi C, Vasile E, Bernardini I, Orlandini C, Andreuccetti M and Falcone A: Baseline elevated leukocyte count in peripheral blood is associated with poor survival in patients with advanced non-small cell lung cancer: A prognostic model. J Cancer Res Clin Oncol. 134:1143–1149. 2008. View Article : Google Scholar : PubMed/NCBI
8	Schmidt H, Suciu S, Punt CJA, Gore M, Kruit W, Patel P, Lienard D, von der Maase H and Eggermont AM: Pretreatment levels of peripheral neutrophils and leukocytes as independent predictors of overall survival in patients with American Joint Committee on Cancer Stage IV Melanoma: Results of the EORTC 18951 Biochemotherapy Trial. J Clin Oncol. 25:1562–1569. 2007. View Article : Google Scholar : PubMed/NCBI
9	Teramukai S, Kitano T, Kishida Y, Kawahara M, Kubota K, Komuta K, Minato K, Mio T, Fujita Y, Yonei T, et al: Pretreatment neutrophil count as an independent prognostic factor in advanced non-small-cell lung cancer: An analysis of Japan Multinational Trial Organisation LC00-03. Eur J Cancer. 45:1950–1958. 2009. View Article : Google Scholar : PubMed/NCBI
10	Halazun KJ, Aldoori A, Malik HZ, Al-Mukhtar A, Prasad KR, Toogood GJ and Lodge JPA: Elevated preoperative neutrophil to lymphocyte ratio predicts survival following hepatic resection for colorectal liver metastases. Eur J Surg Oncol. 34:55–60. 2008. View Article : Google Scholar
11	Walsh SR, Cook EJ, Goulder F, Justin TA and Keeling NJ: Neutrophil-lymphocyte ratio as a prognostic factor in colorectal cancer. J Surg Oncol. 91:181–184. 2005. View Article : Google Scholar : PubMed/NCBI
12	Liu H, Liu G, Bao Q, Sun W, Bao H, Bi L, Wen W, Liu Y, Wang Z, Yin X, et al: The baseline ratio of neutrophils to lymphocytes is associated with patient prognosis in rectal carcinoma. J Gastrointest Cancer. 41:116–120. 2010. View Article : Google Scholar : PubMed/NCBI
13	Kim IY, You SH and Kim YW: Neutrophil-lymphocyte ratio predicts pathologic tumor response and survival after preoperative chemoradiation for rectal cancer. BMC Surg. 14:942014. View Article : Google Scholar : PubMed/NCBI
14	Liao W, Zhang J, Zhu Q, Qin L, Yao W, Lei B, Shi W, Yuan S, Tahir SA, Jin J, et al: Preoperative neutrophil-to-lymphocyte ratio as a new prognostic marker in hepatocellular carcinoma after curative resection. Transl Oncol. 7:248–255. 2014. View Article : Google Scholar : PubMed/NCBI
15	Mishalian I, Bayuh R, Eruslanov E, Michaeli J, Levy L, Zolotarov L, Singhal S, Albelda SM, Granot Z and Fridlender ZG: Neutrophils recruit regulatory T-cells into tumors via secretion of CCL17 - A new mechanism of impaired antitumor immunity. Int J Cancer. 135:1178–1186. 2014. View Article : Google Scholar : PubMed/NCBI
16	Gordon-Weeks AN, Lim SY, Yuzhalin AE, Jones K, Markelc B, Kim KJ, Buzzelli JN, Fokas E, Cao Y, Smart S, et al: Neutrophils promote hepatic metastasis growth through fibroblast growth factor 2-dependent angiogenesis in mice. Hepatology. 65:1920–1935. 2017. View Article : Google Scholar : PubMed/NCBI
17	Wculek SK and Malanchi I: Neutrophils support lung colonization of metastasis-initiating breast cancer cells. Nature. 528:413–417. 2015. View Article : Google Scholar : PubMed/NCBI
18	Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y and Zychlinsky A: Neutrophil extracellular traps kill bacteria. Science. 303:1532–1535. 2004. View Article : Google Scholar : PubMed/NCBI
19	Gupta AK, Hasler P, Holzgreve W, Gebhardt S and Hahn S: Induction of neutrophil extracellular DNA lattices by placental microparticles and IL-8 and their presence in preeclampsia. Hum Immunol. 66:1146–1154. 2005. View Article : Google Scholar
20	Sur Chowdhury C, Giaglis S, Walker UA, Buser A, Hahn S and Hasler P: Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: Analysis of underlying signal transduction pathways and potential diagnostic utility. Arthritis Res Ther. 16:R1222014. View Article : Google Scholar : PubMed/NCBI
21	Brill A, Fuchs TA, Savchenko AS, Thomas GM, Martinod K, De Meyer SF, Bhandari AA and Wagner DD: Neutrophil extracellular traps promote deep vein thrombosis in mice. J Thromb Haemost. 10:136–144. 2012. View Article : Google Scholar :
22	Wada H, Matsumoto T, Aota T, Yamashita Y, Suzuki K and Katayama N: Management of cancer-associated disseminated intravascular coagulation: guidance from the SSC of the ISTH: comment. J Thromb Haemost. 14:1314–1315. 2016.comment. View Article : Google Scholar : PubMed/NCBI
23	Demers M, Krause DS, Schatzberg D, Martinod K, Voorhees JR, Fuchs TA, Scadden DT and Wagner DD: Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proc Natl Acad Sci USA. 109:13076–13081. 2012. View Article : Google Scholar : PubMed/NCBI
24	Chrysanthopoulou A, Mitroulis I, Apostolidou E, Arelaki S, Mikroulis D, Konstantinidis T, Sivridis E, Koffa M, Giatromanolaki A, Boumpas DT, et al: Neutrophil extracellular traps promote differentiation and function of fibroblasts. J Pathol. 233:294–307. 2014. View Article : Google Scholar : PubMed/NCBI
25	Park J, Wysocki RW, Amoozgar Z, Maiorino L, Fein MR, Jorns J, Schott AF, Kinugasa-Katayama Y, Lee Y, Won NH, et al: Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps. Sci Transl Med. 8:361ra1382016. View Article : Google Scholar : PubMed/NCBI
26	Tohme S, Yazdani HO, Al-Khafaji AB, Chidi AP, Loughran P, Mowen K, Wang Y, Simmons RL, Huang H and Tsung A: Neutrophil extracellular traps promote the development and progression of liver metastases after surgical stress. Cancer Res. 76:1367–1380. 2016. View Article : Google Scholar : PubMed/NCBI
27	Kozono S, Ohuchida K, Eguchi D, Ikenaga N, Fujiwara K, Cui L, Mizumoto K and Tanaka M: Pirfenidone inhibits pancreatic cancer desmoplasia by regulating stellate cells. Cancer Res. 73:2345–2356. 2013. View Article : Google Scholar : PubMed/NCBI
28	Mederacke I, Dapito DH, Affò S, Uchinami H and Schwabe RF: High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers. Nat Protoc. 10:305–315. 2015. View Article : Google Scholar : PubMed/NCBI
29	Park J, Wysocki RW, Amoozgar Z, et al: Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps. Sci Transl Med. 8:361ra1382016. View Article : Google Scholar : PubMed/NCBI
30	Ikenaga N, Ohuchida K, Mizumoto K, Cui L, Kayashima T, Morimatsu K, Moriyama T, Nakata K, Fujita H and Tanaka M: CD10+ pancreatic stellate cells enhance the progression of pancreatic cancer. Gastroenterology. 139:1041–1051. 1051.e1–1051.e8. 2010. View Article : Google Scholar : PubMed/NCBI
31	Endo S, Nakata K, Ohuchida K, Takesue S, Nakayama H, Abe T, Koikawa K, Okumura T, Sada M, Horioka K, et al: Autophagy Is Required for Activation of Pancreatic Stellate Cells, Associated With Pancreatic Cancer Progression and Promotes Growth of Pancreatic Tumors in Mice. Gastroenterology. 152:1492–1506.e24. 2017. View Article : Google Scholar : PubMed/NCBI
32	Livak KJ and Schmittgen TD: Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar
33	Najmeh S, Cools-Lartigue J, Rayes RF, Gowing S, Vourtzoumis P, Bourdeau F, Giannias B, Berube J, Rousseau S, Ferri LE, et al: Neutrophil extracellular traps sequester circulating tumor cells via β1-integrin mediated interactions. Int J Cancer. 140:2321–2330. 2017. View Article : Google Scholar : PubMed/NCBI
34	Nielsen SR, Quaranta V, Linford A, Emeagi P, Rainer C, Santos A, Ireland L, Sakai T, Sakai K, Kim YS, et al: Macrophage-secreted granulin supports pancreatic cancer metastasis by inducing liver fibrosis. Nat Cell Biol. 18:549–560. 2016. View Article : Google Scholar : PubMed/NCBI
35	Costa-silva B, Aiello NM, Ocean AJ, Singh S, Zhang H, Thakur BK, Becker A, Hoshino A, Mark MT, Molina H, et al: Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol. 17:816–826. 2015. View Article : Google Scholar : PubMed/NCBI
36	Patutina O, Mironova N, Ryabchikova E, Popova N, Nikolin V, Kaledin V, Vlassov V and Zenkova M: Inhibition of metastasis development by daily administration of ultralow doses of RNase A and DNase I. Biochimie. 93:689–696. 2011. View Article : Google Scholar : PubMed/NCBI
37	Salganik RI, Martynova RP, Matien ko NA and Ronichevskaya GM: Effect of deoxyribonuclease on the course of lymphatic leukaemia in AKR mice. Nature. 214:100–102. 1967. View Article : Google Scholar : PubMed/NCBI
38	Sugihara S, Yamamoto T, Tanaka H, Kambara T, Hiraoka T and Miyauchi Y: Deoxyribonuclease treatment prevents blood-borne liver metastasis of cutaneously transplanted tumour cells in mice. Br J Cancer. 67:66–70. 1993. View Article : Google Scholar : PubMed/NCBI
39	Cools-Lartigue J, Spicer J, McDonald B, Gowing S, Chow S, Giannias B, Bourdeau F, Kubes P and Ferri L: Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest. 123:3446–3458. 2013. View Article : Google Scholar :
40	O’Connell JT, Sugimoto H, Cooke VG, MacDonald BA, Mehta AI, LeBleu VS, Dewar R, Rocha RM, Brentani RR, Resnick MB, et al: VEGF-A and Tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization. Proc Natl Acad Sci USA. 108:16002–16007. 2011. View Article : Google Scholar
41	Conti J and Thomas G: The role of tumour stroma in colorectal cancer invasion and metastasis. Cancers (Basel). 3:2160–2168. 2011. View Article : Google Scholar
42	Sousa S, Brion R, Lintunen M, Kronqvist P, Sandholm J, Mönkkönen J, Kellokumpu-Lehtinen PL, Lauttia S, Tynninen O, Joensuu H, et al: Human breast cancer cells educate macrophages toward the M2 activation status. Breast Cancer Res. 17:1012015. View Article : Google Scholar : PubMed/NCBI
43	Penheiter AR, Erdogan S, Murphy SJ, Hart SN, Lima JF, Rohakhtar FR, O’Brien DR, Bamlet WR, Wuertz RE, Smyrk TC, et al: Transcriptomic and Immunohistochemical Profiling of SLC6A14 in Pancreatic Ductal Adenocarcinoma. Biomed Res Int. 2015:5935722015. View Article : Google Scholar : PubMed/NCBI