85622-93-1 br Contents lists available at ScienceDirect br C
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journal homepage: www.elsevier.com/locate/yclim
BCG-induced formation of neutrophil extracellular traps play an important T role in 85622-93-1 cancer treatment
Kangkang Liua,1, Erlin Suna, ,1, Mingde Leia,1, Limin Lib, Jingda Gaoc, Xuewu Niand, Lining Wanga
a Tianjin Institute of Urology, Department of Urology, The Second Hospital of Tianjin Medical University, Tianjin 300211, PR China
b Department of Urology, The 4th People's Hospital of Hengshui, Hengshui 053000, PR China
c Department of Urology, Children's Hospital of Hebei Province, Shijiazhuang 050031, PR China
d Department of Urology, Tianjin Nankai Hospital, Tianjin 300100, PR China
Bacillus Calmette-Guerin (BCG) is one of the most eﬀective treatments for bladder cancer. Little attention has been paid to the possible role of neutrophils in BCG immunotherapy. In this study, we examined neutrophil extracellular traps (NETs) formation induced by BCG stimulation, and found that BCG-induced NETs exerted cytotoxicity, induced apoptosis and cell-cycle arrest, and inhibited migration in bladder tumor cells. BCG-acti-vated tumor cells but not non-activated ones elicited NETs formation, in which IL-8 and TNF-α from activated tumor cells both took eﬀect. Moreover, NETs activated peripheral blood mononuclear cells (PBMCs) exhibited a higher expression of CD4 and Th1 cytokines. Additionally, the role of NETs in vivo contributed to the recruitment of T cells and monocytes-macrophages and tissue damage, thus preventing tumor growth. NETs proteins mainly caused these eﬀects on tumor and cellular immunity. In conclusion, we demonstrated a novel immunoregulatory role for NETs in the early stages of BCG immunotherapy.
Bladder urothelial carcinoma (BC) is one of the most widespread cancers, with 81, 190 new cases and 17,240 deaths estimated in the USA, 2018 . The majority of newly diagnosed patients suﬀer non-muscle invasive BC (NMIBC) and 20–25% of patients have muscle in-vasive BC (MIBC).  Although NMIBC can be successfully identified and removed before becoming invasive, BC still has a high rate of re-currence and progression . Bacillus Calmette-Guerin (BCG) was firstly reported in 1976 to be eﬀective in the prevention and treatment of NMIBC . Intravesical BCG has become the most eﬀective im-munotherapy for NMIBC, and eﬀectively reduces postoperative recur-rence and tumor progression; [5,6] however, the side eﬀects and in-tolerance associated with BCG limit its application . Therefore, understanding the mechanism of action by which intravesical BCG af-fects tumors can provide new strategies for immunotherapy.
Recent studies have revealed that neutrophils are required for the
eﬃcacy of BCG immunotherapy [8–10]. Mycobacteria induce a strong local immune response that is characterized by a secretion of large amounts of cytokines and an inflammatory cellular infiltration . Polymorphonuclear neutrophils (PMN) are the first line of defense in human innate immunity and the main leukocyte subpopulation to ap-pear in the urethra following treatment with intravesical BCG . BCG-induced neutrophils release IL-8, GRO-α, and MIP-1 to promote monocyte migration and exploit the accessory function of immune cells to attract T cells . In a mouse orthotopic model, PMN deficiency directly aﬀects the therapeutic eﬃcacy of BCG and survival of mice . In addition, tumor necrosis factor (TNF)-related apoptosis-inducing li-gand (TRAIL) is induced by BCG treatment, and TRAIL is expressed on PMNs in the urine obtained from patients after intravesical BCG in-stillation . Furthermore, TRAIL has been confirmed to induce apoptosis of tumor cells in vitro and in vivo [14,15].
In addition to the well-known bactericidal eﬀect, neutrophils can generate extracellular traps (NETs) in response to certain stimuli. It was
Abbreviations: BC, Bladder urothelial carcinoma; NMIBC, Non-muscle invasive bladder cancer; BCG, Bacillus Calmette-Guerin; PMN, Polymorphonuclear neu-trophils; NETs, Neutrophils extracellular traps; PMA, phorbol 12-myristate 13-acetate; PBMCs, Peripheral blood mononuclear cells; DC, Dendritic Cells; NE, neu-trophil elastase; MPO, myeloperoxidase; TLR, Toll-like receptor; CLSM, Confocal Laser Scanning Microscope; SEM, scanning electron microscopy; FCM, Flow Cytometry
Corresponding author at: The 2nd Hospital of Tianjin Medical University, No. 23, PingJiang Road, Hexi District, Tianjin 300211, China.