Name: transfer of manipulated tumor-associated neutrophils into tumor-bearing mice to study their angiogenic potential in vivo
Uploaded: 2019-07-20T13:00:00
Description: Watch this Scientific Journal Video about Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo at JoVE.com

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。

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
<ol><li>在完整的Iscove的改性...

<ol>
	<li>Jablonska, J., Leschner, S., Westphal, K., Lienenklaus, S., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+responsive+to+endogenous+IFN-beta+regulate+tumor+angiogenesis+and+growth+in+a+mouse+tumor+model.">Neutrophils responsive to endogenous IFN-beta regulate tumor angiogenesis and growth in a mouse tumor model.</a> Journal of Clinical Investigation. 120 (4), 1151-1164 (2010).</li><li>Andzinski, L., Wu, C. F., Lienenklaus, S., Kr&#246;ger, A., Weiss, S., Jablonska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+apoptosis+of+tumor+associated+neutrophils+in+the+absence+of+endogenous+IFN-&#946;.">Delayed apoptosis of tumor associated neutrophils in the absence of endogenous IFN-&#946;.</a> International Journal of Cancer. 136, 572-583 (2015).</li><li>Rongvaux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-B-cell+colony-enhancing+factor,+whose+expression+is+upregulated+in+activated+lymphocytes,+is+a+nicotinamide+phosphoribosyltransferase,+a+cytosolic+enzyme+involved+in+NAD+biosynthesis.">Pre-B-cell colony-enhancing factor, whose expression is upregulated in activated lymphocytes, is a nicotinamide phosphoribosyltransferase, a cytosolic enzyme involved in NAD biosynthesis.</a> European Journal of Immunology. 32, 3225-3234 (2002).</li><li>Skokowa, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAMPT+is+essential+for+the+G-CSF-induced+myeloid+differentiation+via+a+NAD(+)-sirtuin-1-dependent+pathway.">NAMPT is essential for the G-CSF-induced myeloid differentiation via a NAD(+)-sirtuin-1-dependent pathway.</a> Nature Medicine. 15, 151-158 (2009).</li><li>Yaku, K., Okabe, K., Hikosaka, K., Nakagawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAD+Metabolism+in+Cancer+Therapeutics.">NAD Metabolism in Cancer Therapeutics.</a> Frontiers in Oncology. 8, 622 (2018).</li><li>Audrito, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+nicotinamide+phosphoribosyltransferase+(NAMPT)+promotes+M2+macrophage+polarization+in+chronic+lymphocytic+leukemia.">Extracellular nicotinamide phosphoribosyltransferase (NAMPT) promotes M2 macrophage polarization in chronic lymphocytic leukemia.</a> Blood. 125, 111-123 (2015).</li><li>Brentano, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-B+cell+colony-enhancing+factor/visfatin,+a+new+marker+of+inflammation+in+rheumatoid+arthritis+with+proinflammatory+and+matrix+degrading+activities.">Pre-B cell colony-enhancing factor/visfatin, a new marker of inflammation in rheumatoid arthritis with proinflammatory and matrix degrading activities.</a> Arthritis &amp; Rheumatology. 56, 2829-2839 (2007).</li><li>Jablonska, J., Wu, C. -. F., Andzinski, L., Leschner, S., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR2-mediated+tumor+associated+neutrophilrecruitment+is+regulated+by+IFN-beta.">CXCR2-mediated tumor associated neutrophilrecruitment is regulated by IFN-beta.</a> International Journal of Cancer. 134, 1346-1358 (2014).</li><li>Andzinski, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+I+IFNs+induce+anti-tumor+polarization+of+tumor+associated+neutrophils+in+mice+and+human.">Type I IFNs induce anti-tumor polarization of tumor associated neutrophils in mice and human.</a> International Journal of Cancer. 138, 1982-1993 (2016).</li><li>Wu, C. -. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lack+of+type+I+interferon+induces+neutrophil-mediated+pre-metastatic+niche+formation+in+the+mouse+lung.">The lack of type I interferon induces neutrophil-mediated pre-metastatic niche formation in the mouse lung.</a> International Journal of Cancer. 137, 837-847 (2015).</li><li>Hansel, T. T., Kropshofer, H., Singer, T., Mitchell, J. A., George, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+safety+and+side+effects+of+monoclonal+antibodies.">The safety and side effects of monoclonal antibodies.</a> Nature Reviews Drug Discovery. 9 (4), 325-338 (2010).</li><li>Hasmann, M., Schemainda, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FK866,+a+highly+specific+noncompetitive+inhibitor+of+nicotinamide+phosphoribosyltransferase,+represents+a+novel+mechanism+for+induction+of+tumor+cell+apoptosis.">FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis.</a> Cancer Research. 63, 7436-7442 (2003).</li><li>Holen, K., Saltz, L. B., Hollywood, E., Burk, K., Hanauske, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharmacokinetics,+toxicities,+and+biologic+effects+of+FK866,+a+nicotinamide+adenine+dinucleotide+biosynthesis+inhibitor.">The pharmacokinetics, toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor.</a> Investigational New Drugs. 26, 45-51 (2008).</li><li>von Heideman, A., Berglund, A., Larsson, R., Nygren, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+NAD+depleting+cancer+drugs:+results+of+a+phase+I+clinical+trial+of+CHS+828+and+overview+of+published+data.">Safety and efficacy of NAD depleting cancer drugs: results of a phase I clinical trial of CHS 828 and overview of published data.</a> Cancer Cancer Chemotherapy and Pharmacology. 65, 1165-1172 (2010).</li><li>De Rossi, G., Scotland, R., Whiteford, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Factors+in+Measuring+Angiogenesis+Using+the+Aortic+Ring+Model.">Critical Factors in Measuring Angiogenesis Using the Aortic Ring Model.</a> Journal of Genetic Syndromes and Gene Therapy. 4 (5), (2013).</li><li>Pylaeva, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAMPT+signaling+is+critical+for+the+proangiogenic+activity+of+tumor-associated+neutrophils.">NAMPT signaling is critical for the proangiogenic activity of tumor-associated neutrophils.</a> International Journal of Cancer. 144 (1), 136-149 (2019).</li></ol>

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?...

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。

<table>
	<tbody>
		<tr>
			<td>15 ml tubes</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
			<td>62,554,502</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml / 10ml / 25ml sterile tipps for the automatic pipette</td>
			<td>Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany</td>
			<td>6006180 / 607180 / 760180</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well flat-bottom cell culture plates</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
			<td>833,920</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well flat-bottom cell culture plates</td>
			<td>Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well U-bottom cell culture plates</td>
			<td>Cellstar, Greiner Bio One International GmbH, Frickenhausen, Germany</td>
			<td>65018</td>
			<td></td>
		</tr>
		<tr>
			<td>AMG EVOS fl digital inverted microscope</td>
			<td>AMG, Bothel, U.S.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD11b</td>
			<td>BD Pharmigen, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>553312</td>
			<td>clone M1/70, APC-conjugated, 0.2mg/mL</td>
		</tr>
		<tr>
			<td>anti-mouse Ly6G</td>
			<td>BioLegend, California, U.S.</td>
			<td>127608</td>
			<td>clone 1A8, PE-conjugated, 0.2mg/mL</td>
		</tr>
		<tr>
			<td>BD FACS AriaII</td>
			<td>BD Biosciences, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td></td>
			<td>cell sorter</td>
		</tr>
		<tr>
			<td>Caliper</td>
			<td>Vogel Germany, Kevelaer, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Casy cell counter</td>
			<td>Innovatis, Roche Innovatis AG, Bielefeld, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Trics 50&#181;m / 100 &#181;m sterile filters</td>
			<td>Sysmex Partec GmbH, Goerlitz, Germany</td>
			<td>04-004-2327 / 04-004-2328</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Rotina 420 R</td>
			<td>Andreas Hettich, Tuttlingen, Germany</td>
			<td>4706</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Sigma-Aldrich/Merck, Darmstadt, Germany</td>
			<td>11088858001</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dilactate)</td>
			<td>BioLegend, California, U.S.</td>
			<td>422801</td>
			<td>Stock: 5mg/ml</td>
		</tr>
		<tr>
			<td>Dispase I</td>
			<td>Sigma-Aldrich/Merck, Darmstadt, Germany</td>
			<td>D4818-2MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>41966-029</td>
			<td>DMEM complete: DMEM + 10% FBS + 1% penicillin-streptomycin</td>
		</tr>
		<tr>
			<td>DMSO (Dimethylsufoxide)</td>
			<td>WAK-Chemie Medical GmbH, Steinbach, Germany</td>
			<td>WAK-DMSO-10</td>
			<td>CryoSure-DMSO</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich/Merck, Darmstadt, Germany</td>
			<td>DN25-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cell growth medium</td>
			<td>PromoCell, Heidelberg, Germany</td>
			<td>c-22010</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>S0115</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block (Anti-mouse CD16/32)</td>
			<td>BD Pharmingen, Becton Dickinson,Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>553142</td>
			<td>clone 2.4G2, Stock: 0.5mg/mL</td>
		</tr>
		<tr>
			<td>FK 866 hydrochloride</td>
			<td>Axon Medchem, Groningen, Netherlands</td>
			<td>Axon 1546</td>
			<td>Stock: 100 mM</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L</td>
			<td>Abcam, Cambridge, U.K.</td>
			<td>ab97075</td>
			<td>Cy3-conjugated, Stock: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Heracell 240i CO2 Incubator</td>
			<td>Thermo Fisher Scientific, Waltham, U.S.</td>
			<td>51026334</td>
			<td></td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>12440-053</td>
			<td>IMDM complete: IMDM + 10% FBS + 1% penicillin-streptomycin</td>
		</tr>
		<tr>
			<td>Isis GT420 shaver</td>
			<td>B. Braun Asculap, Suhl, Germany</td>
			<td>90200714</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix basement membrane</td>
			<td>Corning Life Sciences, Amsterdam, Netherlands</td>
			<td>7205011</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome Cryostat Microm HM 505 N</td>
			<td>Microm International GmbH, Walldorf, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-Actin, &#945;-Smooth Muscle</td>
			<td>Sigma-Aldrich/Merck, Darmstadt, Germany</td>
			<td>F3777</td>
			<td>FITC-conjugated, no information about stock concentration</td>
		</tr>
		<tr>
			<td>Needles 0.4 mm x 16 mm</td>
			<td>BD Microlance, Becton Dicson, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>302200</td>
			<td></td>
		</tr>
		<tr>
			<td>Neomount</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>HX67590916</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson ImmunoResearch Laboratories, West Grove, U.S.</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetus automatic pipette</td>
			<td>Hirschmann Laborger&#228;te, Eberstadt, Germany</td>
			<td>9907200</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Invitrogen, Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit anti mouse Laminin gamma 1 chain</td>
			<td>Immundiagnostik, Bensheim, Germany</td>
			<td>AP1001.1</td>
			<td>No information about stock concentration</td>
		</tr>
		<tr>
			<td>StemPro Accutase</td>
			<td>Gibco, Life Technologies/Thermo Fisher Scientific, Massachusetts, U.S.</td>
			<td>A1105-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile disposal scalpel (no. 15)</td>
			<td>MedWare, Naples, U.S.</td>
			<td>120920</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes 1 ml</td>
			<td>BD Plastipak, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>303172</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes 10 ml</td>
			<td>BD Discardit II, Becton Dickinson, Franklin Lakes, U.S.</td>
			<td>309110</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 sterile cell culture flasks</td>
			<td>Sarstedt AG &#38; Co., N&#252;mbrecht, Germany</td>
			<td>833,911,302</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura Finetek, Torrance, U.S.</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss AxioObserver.Z1 Inverted Microscope with ApoTome Optical Sectioning</td>
			<td>Carl Zeiss, Oberkochen, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

transfer of manipulated tumor-associated neutrophils into tumor-bearing mice to study their angiogenic potential in vivo

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

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...

Watch this Scientific Journal Video about Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo at JoVE.com

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

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。

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

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

我们的协议为评估体内肿瘤相关原发性嗜中性粒细胞的血管生成潜力提供了一种新颖的工具，而无需使用人工嗜中性粒细胞系模型。直接抑制烟酰胺磷脂基转酶， 不久 NAMPT， 在肿瘤相关的嗜中性粒细胞与随后的收养转移到肿瘤轴承动物允许操作嗜中性粒细胞没有系统性的毒性副作用.我们将展示纵的抗血管原性肿瘤相关嗜中性粒细胞在转移到肿瘤承载宿主后的治疗潜力，以研究不同癌症模型中潜在的中性粒细胞基免疫疗法。要建立同源肿瘤小鼠模型，剃光10个8至12周大雌性干扰素α和β受体亚单位1淘汰小鼠的皮肤用电剃须刀，用70%乙醇消毒皮肤， 然后将每毫升 PBS 的 6 个 B16F10 黑色素瘤细胞加载三倍至 6 个 B16F10 PBS，用 0.4 倍 19 毫米针装入一毫升注射器中，并在后侧翼下皮上注入 100 微升悬浮液，每次剃光动物。对于嗜中性粒细胞的收养转移，将B16F10黑色素瘤细胞稀释至PBS中每毫升浓度6倍至第六细胞，稀释使用NAMPT抑制剂FK866治疗的嗜中性粒细胞，将PBS浓度每毫升的6倍10至第5细胞稀释。然后，以1：10的中性粒细胞与肿瘤比将未经治疗或抑制剂治疗的嗜中性粒细胞与黑色素瘤细胞混合，皮下将100微升细胞注射到每组多达5只小鼠体内。注射后，将多达5只注射的雌性小鼠放在一个笼中，并在14天内每天使用卡钳测量肿瘤的长度、宽度和深度。注射后第14天，用70%乙醇对每个注射动物的皮肤进行消毒，用剪刀和钳子将肿瘤收获到含有冰上完整介质的50毫升锥形管中。将肿瘤分段进行组织分析，将肿瘤淹没在最佳切温化合物中，将样品冷冻在液氮中，储存在零下80摄氏度。在分切当天，将样品解冻至零下20摄氏度，然后使用低温仪获得5微米分段。固定非特异性结合后，用感兴趣的主要抗体在20摄氏度下染色一小时，随后在PBS中洗涤三次。最后一次洗涤后，在DAPI等核染色染料中用适当的荧光结合二次抗体染色细胞，在20摄氏度的温度下进行一小时。在孵化结束时，在20摄氏度下干燥滑梯20分钟，然后用无水安装介质安装样品，然后用盖板盖住每个幻灯片，让安装介质在37摄氏度下干燥1小时，然后用荧光显微镜对组织进行成像。对于肿瘤相关的嗜中性粒细胞分离，将每口5个肿瘤放入无菌六井板的单个孔中，然后用无菌剪刀将肿瘤切成两到三毫米。接下来，在37摄氏度下消化肿瘤片段，用1毫升的脱发胶酶d dnase 1溶液在5%的二氧化碳中，在37摄氏度下45分钟，每15分钟用10毫升注射器混合样品。在孵育结束时，通过100微米滤株将细胞过滤成每井一个15毫升的管子，以去除未消化的纤维，并将PBS添加到15毫升的最终体积中。通过离心收集分离的细胞，用每管一毫升的解压缓冲液来解压红血球。混合后，将每个管中的内容物组合成一个15毫升的管子，两分钟后停止反应，用11毫升的完全4摄氏度的中等温度。离心后，将颗粒重新在PBS的一毫升中重新下水，并阻断任何非特异性结合，与三微升的Fc-block抗体在冰上15分钟。在孵化结束时，用抗CD11b和Ly6G抗体和任何其他感兴趣的抗体，加上DAPI，在防光的冰上进行30分钟的孵育。在孵育结束时，用14毫升PBS洗涤细胞，然后以每毫升新鲜完全中等浓度的10倍重新将颗粒重新给第七个细胞， 然后根据标准分拣协议对荧光激活细胞分拣器上的 CD11b 阳性、Ly6G、高 DAPI 阴性嗜中性粒细胞进行排序，在该排序结束时检查流式细胞仪上分离的中性粒细胞的纯度。对于体外肿瘤相关的嗜中性粒细胞抑制，种子1.5倍10至第五排序的嗜中性粒细胞进入两个孔的96孔U底板和添加FK866到最终浓度100纳米摩尔在完整的介质到一个井和相等体积的完整介质单独到另一个井。在细胞培养培养箱中两小时后，用PBS清洗细胞两次，并在PBS中以适当的浓度重新注入颗粒，以便随后注射。与未经治疗的细胞相比，NAMPT抑制剂治疗的嗜中性粒细胞具有显著降低的刺激大院分支形成的能力。此外，与注射未经治疗的干扰素α和β受体亚单位1淘汰嗜中性粒细胞的小鼠相比，抑制剂治疗的抗血管性嗜中性粒细胞的皮下注射会导致肿瘤生长的显著损伤。对提取的肿瘤进行组织学检查，进一步证实，与注射未经治疗的核缔中性粒细胞相比，从接受抑制剂治疗的肿瘤相关嗜中性粒细胞治疗的小鼠中分离出的肿瘤中，血管发生作用显著。为了评估FK866治疗的肿瘤相关嗜中性粒细胞的特性，可以进行定量PCR和西叶虫研究与中性粒细胞极化有关的细胞内通路活化。由于嗜中性粒细胞是短暂的，因此有必要尽快执行所有步骤，并在整个过程中保持嗜中性粒细胞的寒冷。该技术为研究体内肿瘤相关嗜中性粒细胞的血管生成和肿瘤生成特性的细胞内机制和因素的研究铺平了道路。

Research

JoVE Journal

Cancer Research

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

科研

癌症研究

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

结肠-26癌荷瘤小鼠为癌症恶病质的研究模型

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

转移因子的鉴定和表征转移到新的RIP 标签;tva小鼠模型

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

线粒体膜电位检测 CLIC4 击倒诱导 HN4 细胞凋亡的实验研究

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

体内在肿瘤微环境中 pH 值、 pO2、氧化还原状态、磷酸盐和谷胱甘肽浓度的 EPR 评价

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

在微RNA的Vivo抑制下，降低小鼠的肿瘤生长

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

在斑马鱼肿瘤异种移植物中宿主血管反应的Vivo成像和定量

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...