将操纵的肿瘤相关中性粒细胞转移到肿瘤携带小鼠中,以研究其血管生成潜力在Vivo
Summary
在这里,我们展示了抗血管性肿瘤相关中性粒细胞转移到肿瘤小鼠后的治疗潜力。该协议可用于操纵体内的嗜中性粒细胞活性,并随后评估其在体内的肿瘤发展功能。它是研究潜在的中微粒细胞免疫疗法的适当模型。
Abstract
中性粒细胞对肿瘤发生调节的贡献越来越受到重视。这些细胞是异质的,根据肿瘤环境可以具有亲或抗肿瘤的能力。调节肿瘤环境中中性粒细胞功能的重要细胞因子之一是I型干扰素。在干扰素的存在下,嗜中性粒细胞获得抗肿瘤的特性,包括细胞毒性或免疫系统的刺激。相反,缺乏干扰素信号导致突出的亲肿瘤活性,其特征是肿瘤血管生成的强烈刺激。最近,我们可以证明,嗜中性粒细胞的亲血管生成特性取决于这些细胞中烟酰胺磷酸二酯转移酶(NAMPT)信号通路的激活。抑制肿瘤相关中性粒细胞的这一途径导致其强大的抗血管生成表型。在这里 , 我们演示了我们新建立的模型 , 允许在体内评估纵的肿瘤相关中性粒细胞 ( TAN ) 的肿瘤生成潜力。不久,亲血管性肿瘤相关中性粒细胞可以从肿瘤产生干扰素缺乏的小鼠中分离出来,并通过阻断NAMPT信号,重新极化为抗血管生成表型。这些细胞的血管生成活性随后可以使用主动脉环测定进行评估。抗血管生成性TAN可转移到肿瘤携带野生型受体中,肿瘤生长应监测14天。在第一天,14只小鼠被牺牲,肿瘤被切除,并经过血管化评估。总体而言,我们的方案提供了一种新的工具,用于在体内评估原发细胞(如肿瘤相关中性粒细胞)的血管生成能力,而无需使用人工中性粒细胞系模型。Vc
Introduction
I型干扰素(IFN)在刺激宿主对肿瘤反应方面起着重要作用,因为I型IFN信号的缺乏导致肿瘤生长显著升高1。这个过程所涉及的机制之一是肿瘤相关嗜中性粒细胞的肿瘤原生活性的调节,由菌群刺激因子3受体(CSF3R)下游信号2控制。结肠刺激因子3(CSF3),或粒细胞位细胞刺激因子,被证明激活涉及烟酰胺磷酸酯转移酶(NAMPT)3,4的信号。NAMPT是一种控制速率的酶,用于烟酰胺腺苷二核苷酸合成,它增强糖解,调节DNA修复、基因表达和应激反应,促进癌细胞生存和增殖5。NAMPT在多种癌症类型中过度表达,包括结肠直肠癌、卵巢癌、乳腺癌、胃癌、前列腺癌和胶质瘤6。NAMPT不仅对肿瘤细胞至关重要,而且对肿瘤中存在的多种其他细胞类型(如骨髓细胞)也至关重要,它驱动其分化4,抑制凋亡,刺激多种细胞因子的表达,或巨噬细胞中的基质降解酶7。
肿瘤相关中性粒细胞是肿瘤生长的重要调节剂。TAN功能严重依赖I型IFN可用性,因为这些细胞因子主要抗中性粒细胞的抗肿瘤活性。相反,IFN的缺乏支持这些细胞的肿瘤激活,尤其是其亲血管生成特性。与这一点一致,缺乏IFN的小鼠会发展出明显更大和更好的血管化肿瘤,这些肿瘤被强效与亲肿瘤/亲血管性中性粒细胞1,2,8, 9,10.重要的是,这种亲血管化的TAN显示NAMPT的活性升高,表明它在中性粒细胞的亲肿瘤极化中的重要作用。
使用Ly6G抗体消耗中性粒细胞或抑制其迁移(CXCR2抗体)会导致肿瘤血管生成、生长和转移减少1,8。然而,产生的单克隆抗体是免疫原性的,其管理与一系列危及生命的副作用11相关。使用小分子(如NAMPT抑制剂FK866)进行调节中性粒细胞肿瘤原性的治疗,可以帮助避免此类并发症。不幸的是,NAMPT的药理系统抑制,旁边其对肿瘤生长的治疗作用,导致严重的副作用,包括胃肠道毒性和血小板减少症。因此,NAMPT抑制剂的系统应用是不可行的12,13,14。
因此,我们在此建议一种协议,其中 NAMPT 活动直接在隔离的 AN 中被阻止。这种抗肿瘤中性粒细胞然后被收养转移到肿瘤宿主。该协议将有助于避免化合物的系统性毒性副作用,同时其对靶细胞的影响将持续下去。
Protocol
所有程序,包括动物主体都已得到监管机构的批准:LANUV(Landesamt für Natur,Umwelt und Verbraucherschutz NRW)和德国的雷吉隆斯普雷西迪·蒂宾根。所有操作应在无菌条件下(在层流罩下)使用无菌试剂和仪器(注射器、剪刀、钳子、一次性手术刀、培养皿)进行。 注:协议的总体方案如图1所示。 1. 制备B16F10黑色素瘤细胞系 在完整的Iscove的改性…
Representative Results
使用此处描述的程序,Ifnar1 -/-嗜中性粒细胞从肿瘤中分离出来,并使用NAMPT抑制剂FK866治疗2小时。 使用主动脉环测定来评估治疗效果,这反映了血管生成(基质退化、迁移、扩散、重组)所涉及的关键步骤。我们可以证明,与未经处理的细胞相比,FK866治疗的嗜中性粒细胞刺激主动脉分支形成的能力显著下降(图3A,3B)。</stron…
Discussion
尽管在外科和药理癌症治疗方面取得了进展,但成功的治疗仍然是一个挑战。由于免疫细胞在调节肿瘤生长方面起着重要作用,因此应建立抑制肿瘤原性的新方法。在这里,我们演示了一种通过抗血管性肿瘤相关中性粒细胞的接受转移来抑制肿瘤生长的新方法。使用FK866抑制剂,选择性地靶向TAN中的亲血管性NAMPT信号,可防止副作用,在全身FK866治疗时观察到副作用。
该协议最关键的?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们的工作得到了德国克雷布西尔夫的资助,赠款编号:111647,德国研究理事会(DFG),赠款编号:JA 2461/2-1。
Materials
| 15 ml tubes | Sarstedt AG & Co., Nümbrecht, Germany | 62,554,502 | |
| 50 ml tubes | Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany | 227261 | |
| 5ml / 10ml / 25ml sterile tipps for the automatic pipette | Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany | 6006180 / 607180 / 760180 | |
| 6 well flat-bottom cell culture plates | Sarstedt AG & Co., Nümbrecht, Germany | 833,920 | |
| 96 well flat-bottom cell culture plates | Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany | 655180 | |
| 96 well U-bottom cell culture plates | Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany | 65018 | |
| AMG EVOS fl digital inverted microscope | AMG, Bothel, U.S. | ||
| anti-mouse CD11b | BD Pharmigen, Becton Dickinson, Franklin Lakes, U.S. | 553312 | clone M1/70, APC-conjugated, 0.2mg/mL |
| anti-mouse Ly6G | BioLegend, California, U.S. | 127608 | clone 1A8, PE-conjugated, 0.2mg/mL |
| BD FACS AriaII | BD Biosciences, Becton Dickinson, Franklin Lakes, U.S. | cell sorter | |
| Caliper | Vogel Germany, Kevelaer, Germany | ||
| Casy cell counter | Innovatis, Roche Innovatis AG, Bielefeld, Germany | ||
| Cell Trics 50µm / 100 µm sterile filters | Sysmex Partec GmbH, Goerlitz, Germany | 04-004-2327 / 04-004-2328 | |
| Centrifuge Rotina 420 R | Andreas Hettich, Tuttlingen, Germany | 4706 | |
| Collagenase D | Sigma-Aldrich/Merck, Darmstadt, Germany | 11088858001 | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) | BioLegend, California, U.S. | 422801 | Stock: 5mg/ml |
| Dispase I | Sigma-Aldrich/Merck, Darmstadt, Germany | D4818-2MG | |
| DMEM | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | 41966-029 | DMEM complete: DMEM + 10% FBS + 1% penicillin-streptomycin |
| DMSO (Dimethylsufoxide) | WAK-Chemie Medical GmbH, Steinbach, Germany | WAK-DMSO-10 | CryoSure-DMSO |
| DNase I | Sigma-Aldrich/Merck, Darmstadt, Germany | DN25-100MG | |
| DPBS | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | 14190-094 | |
| Endothelial cell growth medium | PromoCell, Heidelberg, Germany | c-22010 | |
| FBS (Fetal Bovine Serum) | Biochrom, Berlin, Germany | S0115 | |
| Fc-block (Anti-mouse CD16/32) | BD Pharmingen, Becton Dickinson,Becton Dickinson, Franklin Lakes, U.S. | 553142 | clone 2.4G2, Stock: 0.5mg/mL |
| FK 866 hydrochloride | Axon Medchem, Groningen, Netherlands | Axon 1546 | Stock: 100 mM |
| Goat Anti-Rabbit IgG H&L | Abcam, Cambridge, U.K. | ab97075 | Cy3-conjugated, Stock: 0.5 mg/mL |
| Heracell 240i CO2 Incubator | Thermo Fisher Scientific, Waltham, U.S. | 51026334 | |
| IMDM | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | 12440-053 | IMDM complete: IMDM + 10% FBS + 1% penicillin-streptomycin |
| Isis GT420 shaver | B. Braun Asculap, Suhl, Germany | 90200714 | |
| Matrigel Matrix basement membrane | Corning Life Sciences, Amsterdam, Netherlands | 7205011 | |
| Microtome Cryostat Microm HM 505 N | Microm International GmbH, Walldorf, Germany | ||
| Monoclonal Anti-Actin, α-Smooth Muscle | Sigma-Aldrich/Merck, Darmstadt, Germany | F3777 | FITC-conjugated, no information about stock concentration |
| Needles 0.4 mm x 16 mm | BD Microlance, Becton Dicson, Becton Dickinson, Franklin Lakes, U.S. | 302200 | |
| Neomount | Merck, Darmstadt, Germany | HX67590916 | |
| Normal goat serum | Jackson ImmunoResearch Laboratories, West Grove, U.S. | 005-000-121 | |
| Penicillin Streptomycin | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | 15140-122 | |
| Pipetus automatic pipette | Hirschmann Laborgeräte, Eberstadt, Germany | 9907200 | |
| ProLong Gold Antifade Mountant with DAPI | Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S. | P36935 | |
| rabbit anti mouse Laminin gamma 1 chain | Immundiagnostik, Bensheim, Germany | AP1001.1 | No information about stock concentration |
| StemPro Accutase | Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S. | A1105-01 | |
| Sterile disposal scalpel (no. 15) | MedWare, Naples, U.S. | 120920 | |
| Syringes 1 ml | BD Plastipak, Becton Dickinson, Franklin Lakes, U.S. | 303172 | |
| Syringes 10 ml | BD Discardit II, Becton Dickinson, Franklin Lakes, U.S. | 309110 | |
| T75 sterile cell culture flasks | Sarstedt AG & Co., Nümbrecht, Germany | 833,911,302 | |
| Tissue-Tek O.C.T. Compound | Sakura Finetek, Torrance, U.S. | 4583 | |
| Zeiss AxioObserver.Z1 Inverted Microscope with ApoTome Optical Sectioning | Carl Zeiss, Oberkochen, Germany |
References
- Jablonska, J., Leschner, S., Westphal, K., Lienenklaus, S., Weiss, S. Neutrophils responsive to endogenous IFN-beta regulate tumor angiogenesis and growth in a mouse tumor model. Journal of Clinical Investigation. 120 (4), 1151-1164 (2010).
- Andzinski, L., Wu, C. F., Lienenklaus, S., Kröger, A., Weiss, S., Jablonska, J. Delayed apoptosis of tumor associated neutrophils in the absence of endogenous IFN-β. International Journal of Cancer. 136, 572-583 (2015).
- Rongvaux, A., et al. Pre-B-cell colony-enhancing factor, whose expression is upregulated in activated lymphocytes, is a nicotinamide phosphoribosyltransferase, a cytosolic enzyme involved in NAD biosynthesis. European Journal of Immunology. 32, 3225-3234 (2002).
- Skokowa, J., et al. NAMPT is essential for the G-CSF-induced myeloid differentiation via a NAD(+)-sirtuin-1-dependent pathway. Nature Medicine. 15, 151-158 (2009).
- Yaku, K., Okabe, K., Hikosaka, K., Nakagawa, T. NAD Metabolism in Cancer Therapeutics. Frontiers in Oncology. 8, 622 (2018).
- Audrito, V., et al. Extracellular nicotinamide phosphoribosyltransferase (NAMPT) promotes M2 macrophage polarization in chronic lymphocytic leukemia. Blood. 125, 111-123 (2015).
- Brentano, F., et al. Pre-B cell colony-enhancing factor/visfatin, a new marker of inflammation in rheumatoid arthritis with proinflammatory and matrix degrading activities. Arthritis & Rheumatology. 56, 2829-2839 (2007).
- Jablonska, J., Wu, C. -. F., Andzinski, L., Leschner, S., Weiss, S. CXCR2-mediated tumor associated neutrophilrecruitment is regulated by IFN-beta. International Journal of Cancer. 134, 1346-1358 (2014).
- Andzinski, L., et al. Type I IFNs induce anti-tumor polarization of tumor associated neutrophils in mice and human. International Journal of Cancer. 138, 1982-1993 (2016).
- Wu, C. -. F., et al. The lack of type I interferon induces neutrophil-mediated pre-metastatic niche formation in the mouse lung. International Journal of Cancer. 137, 837-847 (2015).
- Hansel, T. T., Kropshofer, H., Singer, T., Mitchell, J. A., George, A. J. The safety and side effects of monoclonal antibodies. Nature Reviews Drug Discovery. 9 (4), 325-338 (2010).
- Hasmann, M., Schemainda, I. FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis. Recherche en cancérologie. 63, 7436-7442 (2003).
- Holen, K., Saltz, L. B., Hollywood, E., Burk, K., Hanauske, A. R. The pharmacokinetics, toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor. Investigational New Drugs. 26, 45-51 (2008).
- von Heideman, A., Berglund, A., Larsson, R., Nygren, P. Safety and efficacy of NAD depleting cancer drugs: results of a phase I clinical trial of CHS 828 and overview of published data. Cancer Cancer Chemotherapy and Pharmacology. 65, 1165-1172 (2010).
- De Rossi, G., Scotland, R., Whiteford, J. Critical Factors in Measuring Angiogenesis Using the Aortic Ring Model. Journal of Genetic Syndromes and Gene Therapy. 4 (5), (2013).
- Pylaeva, E., et al. NAMPT signaling is critical for the proangiogenic activity of tumor-associated neutrophils. International Journal of Cancer. 144 (1), 136-149 (2019).
Citer Cet Article
Pylaeva, E., Spyra, I., Bordbari, S., Lang, S., Jablonska, J. Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo. J. Vis. Exp. (149), e59807, doi:10.3791/59807 (2019).