腹腔灌洗液中低密度中性粒细胞分泌的中性粒细胞胞外陷阱介导肿瘤细胞生长与附着
Summary
在这里, 我们提出一种方法, 其中人类低密度中性粒细胞 (LDN), 从术后腹腔灌洗液中恢复, 产生大量的中性粒细胞外圈闭 (网), 并有效地诱捕后生长的游离肿瘤细胞。
Abstract
活化中性粒细胞释放中性粒细胞胞外陷阱 (网), 可以捕获和破坏微生物。最近的研究表明蚊帐参与了各种疾病过程, 如自身免疫性疾病、血栓形成和肿瘤转移。在这里, 我们展示了一个详细的体外技术, 以检测在游离肿瘤细胞的诱捕过程中的净活动, 它生长后, 附着在网。首先, 我们收集了 laparotomies 患者术后腹腔灌洗液中的低密度中性粒细胞 (LDN)。LDN 的短期培养导致了大量的网状形成, 可见绿色荧光核和染色体 counterstain。在人类胃癌细胞系 MKN45、OCUM-1 和 NUGC-4 与网的共同孵化后, 许多肿瘤细胞被网所困住。随后, 由于网 DNase 的降解, 附着物完全被取消了. 时间推移视频显示, 网陷的肿瘤细胞没有死亡, 而是在持续的文化中大力生长。这些方法可用于检测网与各种类型的细胞和材料之间的粘接作用。
Introduction
循环血中变形核中性粒细胞通常通过密度梯度制备方法与单个核干细胞分离。然而, 一些被称为低密度中性粒细胞的中性粒细胞 (LDN), CD11b (+), CD15 (+), CD16 (+) 和 CD14 (-) 表型, 与单个核细胞共纯化。LDN 的相对数量在各种病理条件下显著增加, 包括自身免疫性疾病1、2、脓毒症3和癌症4、5。以前的研究表明, LDN 是一种表型和功能上明显的中性粒细胞6。应注意的是, 循环血液中的 LDN 比正常密度的中性粒细胞2,7更容易产生中性粒细胞胞外陷阱 (蚊帐)。网是由核酸、组蛋白、蛋白酶、颗粒和胞浆蛋白组成的网状结构, 可以有效地诱捕和破坏病原体8。
最近, 蚊帐已被证明不仅捕获微生物, 而且还有血小板和循环肿瘤细胞, 可以帮助血栓形成9和肿瘤转移10,11。然而, 网络与血小板或肿瘤细胞之间的粘接作用背后的分子机制还不清楚。最近,体外黏附检测发现髓细胞白血病 (K56212) 和肺癌细胞 (A54913) 附着在网通过β1和β3整合。作者利用从中性粒细胞中分离的净存量, 用佛波 12-酯 13-醋酸酯 (PMA) 活化的网状物作为粘附基底14。虽然这种检测可以检测到在无中性粒细胞的情况下与净成分的实际相互作用, 但是, 高速离心分离的 “无细胞网” 是否保留了与所生产的网相同的分子结构体内。最近, 我们发现腹部手术后腹腔灌洗液含有许多成熟的 LDN, 产生大量的网状物, 附着于肿瘤细胞引起的腹膜转移15。在这项研究中, 我们成功地检查了肿瘤细胞对完整网的黏附力, 没有任何物理操作。在这里, 我们展示了一种检测网络与游离肿瘤细胞之间粘接作用的技术的细节。
Protocol
LDN 是由接受本研究的病人获得的, 并获 Jichi 医科大学机构评审委员会批准。 1. 腹腔 Lavages LDN 的分离和净检测 样品采集 将1000毫升的正常无菌生理盐水直接注入腹腔内, 经胃肠道恶性肿瘤腹部手术的病人在伤口闭合前, 将其注射至腹腔内。注: 样本是从病人接受胃切除, 结肠切除, 或食管切除没有偏见的基础上的年龄或性别。盐水被转移到一个容器里, 一分钟内?…
Representative Results
在2小时的文化中, 从腹膜灌洗液中提取的 CD66b (+) LDN 显示了用绿色荧光染料染色核和染色体的弦结构 (图 1B), 而 CD66b (-) 单个核细胞没有 (图 1C)。然而, 当 LDN 培养的 100 U/毫升 DNase I, 特征结构被破坏 (图 1D), 表明它们是由细胞外 DNA 排出的中性粒细胞。当 LDN 在没有涂层的塑料板上进行培养时, 大量的网状簇?…
Discussion
以前的研究报告说, 循环肿瘤细胞可以被网络基质所困在体内10,11。转移性乳腺癌细胞已被证明是刺激中性粒细胞和诱导形成的蚊帐, 这有助于肿瘤细胞生长的目标器官17。此外, 我们发现, 从术后灌洗液中 LDN 的短期培养可以有效地诱捕肿瘤细胞, 而无需进一步刺激15。这些观察表明, 在转移过程中, 网的机械参与?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 j. 筱女士和我 Nieda 的技术和文书工作。此外, 我们感谢 Drs, Hidenori Haruta, 宪太郎 Kurashina 和葛屋高桥为他们合作的样品采集在手术室。这项工作得到了日本教育、科学、体育和文化部和日本促进科学学会 (17K10606) 的科学研究资助。
Materials
| Ficoll-Paque PLUS | GE Healthcare, SWEDEN | 17-1440-02 | |
| StraightFrom™ Whole Blood CD66b MicroBeads | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-104-913 | |
| Fc block | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-059-901 | |
| MACS Rinsing Solution | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-091-222 | |
| MACS BSA Stock Solution | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-091-376 | |
| LS Columns | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-041-306 | |
| MACS Magnetic Separator | Miltenyi Biotec, Bergisch Gladbach, Germany | 130-042-501 | |
| SYTOX green nucleic acid stain 5mM solution in DMSO | Thermo Fisher Scientific, Waltham, MA, USA | S7020 | |
| PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling | Sigma-Aldrich, St Louis, MO, USA | P9691 | |
| Diluent C | Sigma-Aldrich, St Louis, MO, USA | CGLDIL | |
| RPMI1640 Medium | Sigma-Aldrich, St Louis, MO, USA | R8758 | |
| Dulbecco’s Modified Eagle Medium-high glucose (DMEM) | Sigma-Aldrich, St Louis, MO, USA | D5796 | |
| Dulbecco’s Phosphate Buffered Saline (DPBS) | Sigma-Aldrich, St Louis, MO, USA | D8537 | |
| 0.5mol/l-EDTA Solution (pH 8.0) | nacalai tesque, Japan | 06894-14 | |
| Fetal Bovine Serum, qualified, USDA-approved regions | gibco by life technologies, Mexico | 10437-028 | |
| Bovine Serum Albumin lyophilized powder, ≥96% (agarose gel electrophoresis) | Sigma-Aldrich, St Louis, MO, USA | A2153 | |
| Penicillin Streptomycin | Life Technologies Japan | 15140-122 | |
| Plasmocin Prophylactic | InvivoGen, San Diego, CA-USA | ant-mpp | |
| DNase I | Worthington, Lakewood NJ) | LS002138 | |
| Poly-L-Lysine-Coated MICROPLATE 6Well | IWAKI, Japan | 4810-040 | |
| Poly-L-Lysine-Coated MICROPLATE 24Well | IWAKI, Japan | 4820-040 | |
| fluorescein stereomicroscope | BX8000, Keyence, Osaka Japan | BZ-X710 | |
| Whole view cell observation system | Nikon, Kanagawa, Japan | BioStudio (BS-M10) | |
| MKN45 human gastric cancer cell line | Riken, Tukuba Japan | N/A | |
| NUGC-4 human gastric cancer cell line | Riken, Tukuba Japan | N/A | |
| OCUM-1 human gastric cancer cell line | Osaka City University, Japan | N/A | Gift from Dr. M.Yashiro |
References
- Hacbarth, E., Kajdacsy-Balla, A. Low density neutrophils in patients with systemic lupus erythematosus, rheumatoid arthritis, and acute rheumatic fever. Arthritis and Rheumatology. 29 (11), 1334-1342 (1986).
- Denny, M. F., et al. A distinct subset of proinflammatory neutrophils isolated from patients with systemic lupus erythematosus induces vascular damage and synthesizes type I IFNs. The Journal of Immunology. 184 (6), 3284-3297 (2010).
- Morisaki, T., Goya, T., Ishimitsu, T., Torisu, M. The increase of low density subpopulations and CD10 (CALLA) negative neutrophils in severely infected patients. Surgery Today. 22 (4), 322-327 (1992).
- Schmielau, J., Finn, O. J. Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients. Cancer Research. 61 (12), 4756-4760 (2001).
- Brandau, S., et al. Myeloid-derived suppressor cells in the peripheral blood of cancer patients contain a subset of immature neutrophils with impaired migratory properties. Journal of Leukocyte Biology. 89 (2), 311-317 (2011).
- Carmona-Rivera, C., Kaplan, M. J. Low-density granulocytes: a distinct class of neutrophils in systemic autoimmunity. Seminars in Immunopathology. 35 (4), 455-463 (2013).
- Villanueva, E., et al. Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus. The Journal of Immunology. 187 (1), 538-552 (2011).
- Brinkmann, V., et al. Neutrophil extracellular traps kill bacteria. Science. 303 (5663), 1532-1535 (2004).
- Demers, M., et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proceedings of the National Academy of Sciences of the United States of America. 109 (32), 13076-13081 (2012).
- Cools-Lartigue, J., et al. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. Journal of Clinical Investigation. , (2013).
- Tohme, S., et al. Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress. Cancer Research. 76 (6), 1367-1380 (2016).
- Monti, M., et al. Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells. Public Library of Science One. 12 (2), e0171362 (2017).
- Najmeh, S., et al. Neutrophil extracellular traps sequester circulating tumor cells via beta1-integrin mediated interactions. International Journal of Cancer. 140 (10), 2321-2330 (2017).
- Najmeh, S., Cools-Lartigue, J., Giannias, B., Spicer, J., Ferri, L. E. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. Journal of Visualized Experiments. (98), (2015).
- Kanamaru, R., et al. Low density neutrophils (LDN) in postoperative abdominal cavity assist the peritoneal recurrence through the production of neutrophil extracellular traps (NETs). Scientific Reports. 8 (1), 632 (2018).
- Eades-Perner, A. M., Thompson, J., van der Putten, H., Zimmermann, W. Mice transgenic for the human CGM6 gene express its product, the granulocyte marker CD66b, exclusively in granulocytes. Blood. 91 (2), 663-672 (1998).
- Park, J., et al. Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps. Science Translational Medicine. 8 (361), 361ra138 (2016).
Cite This Article
Kanamaru, R., Ohzawa, H., Miyato, H., Yamaguchi, H., Hosoya, Y., Lefor, A. K., Sata, N., Kitayama, J. Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment. J. Vis. Exp. (138), e58201, doi:10.3791/58201 (2018).